Microbiology August 2017

MERP Medical Immunology
Course: Microbiology and Immunology

Instructor: Inna Lindner, Ph.D. ilindner@devrymedical.org
Textbook: Recommended: 1) The Immune System 4rd Edition, by Peter Parham ISBN: 978-0-8153-4466-7
2) Medical Microbiology 7th Edition by Patrick Murray, Ken Rosenthal, Michael Pfaller ISBN: 987-
0-323-05470-6
LECTURES
Unit I: Immunology
Lecture 1: Introduction to Immunology
Lectures 2 and 3: B cell differentiation
Lecture 4: Antigen Processing, MHC Presentation, and T cell Development
Lectures 5 and 6: Activation of Immune Response
Lecture 7: Functions of antibodies, Complement, Natural Killer Cells, Blood Types, Antibody Profile in Infants
Lecture 8: Hypersensitivity Reactions
Unit 2: Virology
Lectures 9 and 10: Introduction to Virology I
Lecture 11: DNA Viruses: Poxviridae and Parvoviridae
Lecture 12: DNA Viruses: Papillomaviridae, Polyomaviridae and Adenoviridae
Lecture 13: DNA Viruses: Herpesviridae
Lecture 14: DNA Viruses: Hepadnaviridae
Lecture 15: RNA Viruses: Picornaviridae and Orthomyxoviridae
Lecture 16: RNA Viruses: Paramyxoviridae, Togaviridae and Flaviviridae
Lecture 17: RNA Viruses: Rhabdoviridae and Retroviridae
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Unit 3 Bacteriology
Lecture 18: Introduction to Bacteriology
Lecture 19: Staphylococcus aureus
Lectures 20: Streptococci
Lectures 21: Bacilli and Clostridia
Lectures 22: E coli, Shigella, Salmonella, Vibrio
Lecture 23:
Lecture 24: Corynebacterium, Bordetella, Haemophilus. Neisseria
Lecture 25: Mycobacterium tuberculosis
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IMMUNOLOGY MODULE
LECTURE 1: INTRODUCTION TO IMMUNOLOGY

Learning Objectives:
* List the cells of the immune system and their origin

* Describe innate barriers of the immune system
* Describe phagocytosis and inflammation
* Describe the basic differences between B cells and T cells
* Describe the differences between antigen recognition of cells in the adaptive response and cells in the
innate response
* Describe the function of the primary and secondary lymphoid organs
* Describe and diagram the trafficking of lymphocytes and antigens in the body.
* List the major structures of the lymph node
* Describe the components of and timing of adaptive immunity in both primary and secondary response
* List the advantages and disadvantages of passive versus active immunity
All cellular elements of blood (and of innate and adaptive immune system) arise from
hematopoietic multipotent
stem cells in the bone marrow.
These multipotent cells divide
to produce more specialized
progenitor cells called;
lymphoid and myeloid stem
cells. In the hematopoietic
system, 'leukocyte' is the term
given to any white blood cell.
HSC There are many different types
of leukocytes in the body; for
example neutrophils are
leukocytes. Lymphocytes are
specialized leukocytes that are
derived from lymphoid stem
cells. The two most important
lymphocytes in the adaptive
immune system are T cells
and B cells. Red blood cells
and megakaryocytes are not
considered leukocytes.
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Protection against Infectious Disease
Antigen (Ag) is the name for any molecule that stimulates an immune response. Most antigens are
pathogen proteins or carbohydrates that are "foreign" or "non-self" to the host. Both immune and non-
immune defenses protect us.
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Innate pathogen non-immune barriers are first defense:
1. Physical Barriers to pathogen entry include skin and mucous membranes which have tight
junctions between cells.
2. Mechanical responses include the movement of cilia during blinking, the process of
sneezing, coughing, vomiting – all working to remove antigen.
3. Chemical agents include fatty acids on the skin, HCl in the stomach, lysozyme and other
digestive enzymes in tears and mucus.
Innate immune response are second defense:
Even simple multi-celled animals like sea squirts and starfish, as well as many plants, have
defense systems that can recognize a generic "danger" signal and respond to antigens by engulfing
or walling off foreign organisms.
Humans have a corresponding innate immune response that begins immediately in response
to tissue damage. This innate response is mainly mediated by phagocytes. Phagocytes (such as
macrophages and neutrophils) are attracted to the site of infection or tissue damage and engulf and
destroy pathogens, as well as our own dead or damaged cells. In some instances, innate immune
responses alone are effective at eliminating antigen from the body. Some players of the innate
immune system include:
1. Phagocytes such as neutrophils and monocyte/macrophages patrol the blood and many
body tissues.
2. Inflammatory responses attract leukocytes to the infection site. A broad spectrum of events
occur during inflammation:
Cytokines are released: Inflammation is part of the complex biological response of

vascular tissues to harmful stimuli. This response is mediated mainly by small proteins
called cytokines. There are several roles for cytokines:
i. Cytokines signal the blood vessel endothelial cells to express more adhesion
molecules, so that leukocytes can stick and move between the endothelial cells to enter
the tissues.
ii. Certain cytokines called chemokines attract leukocytes to the site of infection.
Neutrophils and monocytes are attracted to the site of infection in a process known as
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chemotaxis. Chemokines and other cytokines are "locally" produced in tissues

throughout your body.
iii. Cytokines can also increase the amount of fluid that can leave the circulation, so that
antibacterial molecules enter the infection site. They do so by:
a. dilating smooth muscle around the vasculature which increased blood flow and
b. causing contraction of endothelial cells increasing the gaps between them.
iv. Cytokines also signal the bone marrow to produce more leukocytes.
Below is an example of inflammatory response mediated by cytokines
Complement and chemical agents participate in inflammation. Some chemical agents

such as defensins and complement promote phagocytosis and pathogen destruction. Other
antiviral proteins called interferons block virus replication in host cells.
Adaptive Immunity
Although not antigen-specific, inflammation (via phagocyte recruitment and flushing of tissue
with increased flow) is often enough to eliminate small numbers of bacterial pathogens from the body.
However, if antigen is not eliminated, adaptive immunity, which includes lymphocytes and secreted
molecules called antibodies, can also participate in antigen destruction. There are several
characteristics of adaptive immunity that set it apart from innate immunity.
Adaptive immunity:
1. is found only in vertebrates

2. is antigen-specific
3. responds more quickly and efficiently to a repeat infection (immune memory),
often so efficient that no symptoms develop
4. can be long-lasting but is usually not permanent.
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Adaptive immune responses is mediated by lymphocytes and includes:
1. B cells - synthesis of antibody to bind antigen and promote its elimination is a job of a B cell
2. T cell-
a. Some T cells are responsible for killing of virus-infected cells. These are called CD8 T
cells.
b. Other T cells mediate activation of macrophages to destroy phagocytosed pathogens
that resist phagocytosis. These are called CD4 T cells.
Characteristics of Lymphocytes:
Remember that lymphocytes are specialized types of

leukocytes that are derived from lymphoid stem cells.
Lymphocytes are small, round cells with a large nucleus.
Below is a description of lymphocytes.
1. They are antigen-specific leukocytes.

2. Lymphocytes of adaptive immunity are called B cells
and T cells.
a. Each lymphocyte recognizes one specific
antigen with its receptor.
b. The antigen receptor on B lymphocytes is called an immunoglobulin (antibody) or
BCR (B Cell Receptor).
c. The antigen receptor on T lymphocytes is called the T Cell Receptor (TCR).
d. Each lymphocyte has about 100,000 identical copies of its antigen receptors on its
membrane.
Differences between antibodies of B cells and T cell receptors of T cells
• Antibodies are specific for and are able to bind either protein or non-protein antigens.
Antibodies bind antigen in its native conformation. Antibodies can be secreted, T cell
receptors cannot.
• The T cell receptors of T cells (TCRs) cannot bind antigen directly. To be activated,
antigen (Ag) must first be processed (cut into peptides) and presented on major
histocompatibility complex (MHC) molecules expressed by antigen presenting cells
(APC). TCRs only recognize peptides in the context of MHC.
• Professional APCs are: Macrophages, Dendritic cells, B cells.
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Similarities between antibodies and T cell receptors
• Both T cells and B cells undergo a process called recombination in order to have each cell
express a unique receptor that distinguishes it from another cell.
o Recombination occurs randomly, so the millions of lymphocytes produced each day
collectively have millions of different antigen specificities.
o The large number of different specificities that are produced is called the immune
repertoire.
Innate and adaptive immune responses collaborate to fight off infection.
Innate immune mechanisms including inflammation and

phagocytosis are essential for the efficient functioning of the
adaptive immune system. As the diagram below shows, lacking
innate immunity results in a rapid growth of microorganisms.
Lacking adaptive immunity also results in prolonged infection
that usually cannot be cleared, however, the organisms are at
least partially controlled by innate immunity. When operating
together, innate and adaptive immunity can clear the organism.
Figure (right): If an individual lacks adaptive immunity, the innate

immunity has some limited capacity to fight pathogens. However, without
innate immunity, an individual would succumb to infection, because
innate immunity is necessary for first line of defense and for recruitment
of adaptive response.
Organs of the Immune System
There are two sets of organs that play a role in the development of immune cells and the activation of
these cells.
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1. Primary lymphoid organs are sites where white blood cells develop and mature. Examples include:
a. Bone-marrow (where B cells develop and hematopoiesis takes place)
b. Thymus (where T cells finish development)
An antigen is NOT required for the development of mature antigen-specific T and B

lymphocytes.
2. Secondary (peripheral) lymphoid organs:
Secondary lymphoid organs function

to bring together leukocytes and
antigens. They line the mucous
membranes of the respiratory,
digestive, and urogenital systems
where contact with pathogens is
highest. They are also found deeper
within the body.
Mature naïve lymphocytes get

activated in secondary lymphoid
organs. These sites include:
a. Lymph nodes which are

distributed all over the body,
collect Ag from tissue
b. Spleen which is an organ where blood-borne antigens, especially bacteria, encounter the
immune system)
c. MALT is a Mucous Associated Lymphoid Tissue and is comprised of tonsils (lymphoid tissue in
the respiratory tract), appendix and peyers patches (lymphoid tissue of the gut).
The trafficking of immune cells
The fluid, called lymph (which is similar in composition to plasma), drains tissue, collects in the
lymphatic vessels and passes through the lymph nodes on its way back to the blood circulation.
Lymph circulates in only one direction - from tissues, to lymph nodes, from lymph nodes to the
thoracic duct, back to the bloodstream, back to the tissues.
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Below are the steps that take place if the tissues are infected.
1. 1a. Antigen is carried to the nearby draining lymph nodes via afferent lymphatic vessels. It is
carried either by a cell called a dendritic cell, or
travels alone.
1b. Naïve B cells and T cells leave the primary

lymphoid organs to populate the blood pool. 1b
1c. High endothelial venules are specialized

1c
anatomical structures in the vasculature which
allow lymphocytes to leave blood and enter the
lymph nodes.
2. In the lymph node, antigen comes in contact

with lymphocytes to initiate an adaptive immune
response.
1c
3. Efferent lymphatic vessels transport lymph and 2
activated lymphocytes from the lymph nodes
back into lymphatic circulation, and then into a
blood circulation.
4. From circulation, activated lymphocytes go to

the tissues
5. At any given time many leukocytes recirculate

throughout the body.
Function of the adaptive immune system – response to an infection
Mature naïve T and B cells are those cells that developed in the primary lymphoid organ but
have not yet bound foreign antigen. These cells leave the primary lymphoid organs and recirculate via
blood through the secondary lymphoid organs.
• If a lymphocyte binds antigen, it
proliferates into a clone of lymphocytes that
differentiate into antigen-eliminating effector
cells.
• In a primary, first exposure, response
to antigen, the time it takes to select and
activate a specific T cell and B cell is 1 - 4
weeks long. At this time, not only the effector
cells that fight the current antigen but also
memory lymphocytes specific for the same
antigen are produced.
• On repeat exposure to the same
antigen, secondary response, the adaptive
response is faster, stronger and longer-
lasting. This is due to the expansion of
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antigen-specific memory lymphocytes formed during the previous primary response. Different
types of antibodies dominate primary and secondary responses.
It is important to realize that there are many instances when the activation of the adaptive immune
system in response to an antigen does not result in disease because the organism levels are kept
low. Only when multiplication of the organism extends beyond threshold level will the disease and
clinical presentation become apparent. We secrete antibodies in response to many pathogens that
we encounter however, only a small number of these organisms have actually made us feel sick.
Adaptive immunity can be acquired in two different ways:
1. Actively
• Actively acquired adaptive immunity requires 1-4 weeks to become established and may be
very long-lasting, from years to a lifetime. Active immunity is acquired by:
o Natural infection with the organism
o Vaccination with killed, live but weakened (attenuated) pathogen or inactivated toxin
(toxoid).
2. Passively
• Passive immunity protects as soon as the antibodies are transferred. Although it is specific
for the antigen, it lasts only weeks as the transferred antibodies are removed from the
circulation in a natural process called "turnover" and there are no B cells producing these
antibodies in the recipient’s body. Passive immunity is acquired from:
o Immune person by the transfer of antibodies such as mother to fetus
Practice questions:
1. The main advantage of passive immunization over active immunization is that:

a. it can be administered orally.
b. it provides antibody more rapidly.
c. antibody persists for a longer period.
d. it contains primarily IgM.
e. it provides more specific antibody.
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2. A lymphoid progenitor cell differentiates into ______ .
a. T cells
b. Macrophages
c. Erythrocytes
d. B cells
e. both a and d
3. The innate immune system is mainly mediated by _____.
a. T cells and B cells

b. Macrophages and neutrophils
c. Erythrocytes
d. Megakaryocytes
4. Cytokine release _____ vascular permeability.
a. increases
b. decreases
5. T cells finish development in the _____.
a. Thyroid
b. Bone marrow
c. Spleen
d. Thymus
e. MALT
6. T cell receptors can bind to _____.
a. Peptides only
b. Non-peptides only
c. Both peptides and non-peptides
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LECTURES 2 AND 3: GENERATION OF B CELL DIVERSITY AND B CELL DEVELOPMENT
Learning objectives:
• Understand the structure of antibody genes and proteins
• Understand the process of recombination

• Describe the steps in B cell differentiation
• A brief overview of B cell's fate after it finished differentiation in the bone marrow
To reach the lymph nodes, B cells and T cells travel

from bone marrow to the blood stream and then enter the
lymph nodes via the high endothelial venule. From the
lymph nodes, the lymphocytes exit via efferent lymphatic
vessels and enter tissues through signals received from
inflammation. The vasculature in the inflamed tissues has
a higher blood flow and endothelial cells that have
contracted and are therefore more permeable.
B cells’ job is predominantly to secrete antibodies

that fight against extracellular antigens. During B cell
development in the bone marrow, many different B cells
are generated from identical stem cells, yet all the B cells
differ from each other in their specificity. This
differentiation into antigen-specific B cells is antigen
independent. However, when a particular B
cell encounters an antigen in the periphery,
it gets stimulated by that antigen and by
additional signals in secondary lymphoid
organs. Activation then causes proliferation
of that particular B cell, production of
plasma cells that secrete the antibody
encoded by that B cell, and generation of
long-term memory cells that help fight the
antigen in the future after soluble antibodies
have been degraded.
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Immunoglobulin structure
Antibodies are tetramers of two identical "light" chains (pink) and "heavy" chains (blue).
• The variable region (idiotype) includes the N-terminal

end of the heavy and light chains, and is responsible
for antigen recognition. Specifically, an antibody binds
to an epitope of an antigen, also known as antigenic
determinant which usually constitutes a part of a larger
antigen (see Figure on the right).
• The constant region of an antibody, referred to as
isotype, is usually derived from one of the five major classes of the heavy chain found within
the constant portion of the heavy chain gene (the regions on the heavy chain are called mu,
delta, gamma, epsilon and alpha). When a heavy chain combines with a light chain an
antibody is produced. The antibodies are named after their isotypes. They are: IgM, IgD, IgG,
IgE, or IgA.
Genomic organization of immunoglobulin gene families

An antibody is encoded by two genes: heavy chain and light chain. Each person inherits:
a) 2 alleles of the Heavy chain gene (one allele from mom, one from dad). Within each gene
locus, there is a multiple of variable (V) regions, diversity (D) and joining (J) regions as well
as several constant region segments (isotypes).
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b) 2 alleles of the Kappa light chain gene (one allele from mom, one from dad) each having
multiple V and J regions as well as one C region.
c) 2 alleles of the Lambda light chain gene (one allele from mom, one from dad) each
having multiple V and J regions as well as a C region.
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B cell development and the generation of antibody diversity
Important Definitions:
Allotypes –the sequences of inherited immunoglobulin genes, which vary by individuals. They vary
because the genes that code for the L and H chains are polymorphic (there are multiple alleles of
these genes in a population), and individuals inherit two different alleles (or allotypes) of each gene.
The allotypic differences in heavy and light chain genes are clustered in the constant regions of the
genes.
Isotype refers to the constant region of an antibody. Even though there is more to an antibody than
the constant region, an antibody as a whole carries the name of its isotype. For example, IgM and
IgG are different isotypes; the constant region of their heavy chains (mu and gamma respectively) are
different antigenically. This nomenclature does not say anything about the antibody’s specificity. The
five immunoglobulin isotypes (or classes) are the following - IgG, IgM, IgA, IgD, and IgE have heavy
chains that are different. The IgG isotype is further subdivided into four subtypes (sub-isotypes),
IgG1, IgG2, IgG3, and IgG4, similarly IgA1 and IgA2 are different sub-isotypes. Kappa and lambda
chain genes are not isotypes of each other; they are distinct genes that have been duplicated in time.
They are considered different isoforms of the light chain.
Idiotypes are the antigenic

determinants formed by the specific
amino acids in the variable region. Each
idiotype is unique for the
immunoglobulin produced by a specific
clone of antibody-producing cells.
There are several factors that

contribute to diversity of idiotypes among B cells:
1. Somatic recombination of immunoglobulin genes leads to:

Combinatorial diversity which results from mixing and matching of various VDJ segments (due
to RAG)
Junctional diversity which causes imprecise joining of VDJ segments (due to TdT, discussed
later)
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2. Pairing of different light and heavy chains to form a functional antibody

3. Somatic hypermutation (post antigen encounter).
Factors 1 - 2 occur in the bone marrow.
The importance of somatic recombination is noted in one example of Severe Combined

Immunodeficiency Syndrome (SCID), a group of rare genetic disorders characterized by the disturbed
development of functional T cells and B cells, where recombination does not occur. In some people, a
deficiency in the enzyme that mediates somatic recombination, called RAG, arrests development of B
and T cells and therefore prevents mature naïve B cell and T cells from forming. These individuals
lack adaptive immune response and are highly susceptible to infections.
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Immunoglobulin gene rearrangement
In the figure to the right,

5’ 3’
the recombination of the a.
heavy chain is shown. It
involves the enzymes RAG
and TdT. The genomic DNA in
(Figure a) is not rearranged
and contains all of the
segments. The dotted lines 5’ 3’
represent the V, D, and J b.
regions in between the

adjacent segments.
5’ 3’
Specialized sequences called c.
recombination signal
sequences (RSS) mark the 3’ end of all the V regions, the 5’ end of all the J regions and both the 5’
and the 3’ ends of the D segments.
Figures “b” and “c” above show the process of recombination:

1. RAG first recombines a 3’ RSS of a particular D segment with a 5’ RSS of a J segment
creating a DJ joint.
2. After a D and J are joined, RAG recombines a 3’ RSS of a particular V region with the 5’
RSS of the D segment previously used by RAG for recombination. In figure above we can see that D2
joins with J4 to form D2J4 and V23 joins with D2J4. Note that in panel “b” of the figure above the 5’
Vs (V1 through V23) and the 3’ Js (J4 through J6) remain on the genomic DNA (b). Panel “b” of the
figure represents recombined DNA.
3. Figure c shows the final mRNA transcript. The transcription of the heavy chain gene begins
with the promoter of the V region that participated in recombination, in this case V23, and proceeds
all the way through the constant region mu portion, where transcription ends. This generates a pre-
mRNA (not shown) that contains V23-D2-J4-J5-J6-mu sequence. Note that while the pre-mRNA
contains only the V and the D segments that were used in recombination, it still contains the J
segments downstream of recombination. RNA processing then splices out the downstream Js (in this
case J5 and J6), so that the final transcript contains only the recombined portions of the variable
region and the mu portion of the heavy chain. In the figure “c” above, the final mRNA transcript would
read V23D2J4mu.
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Note that the

isotypes (gene
regions
encoded by the
constant portion
of the heavy
chain) are not
altered during B
cell
differentiation in
the bone
marrow (figure above). Heavy chain gene is, however, transcribed only through the mu region. The
figure above is also omitting the upstream V regions (V1 –V23) and the downstream J regions (J5
and J6) which should still be on the recombined DNA.
Overview of B-cell development:

The recombination of the heavy and light chain occurs during specific stages of B cell development.
• Stem cell (gives rise to common lymphoid progenitor CLP)

• Pro B cell
• Pre B cell
• Immature B cell
• Mature B cell
1. In the Pro-B
cell VDJ
gene
segments 1 2 1. Division
3 4
alternative RNA
2. Rearranging
of the light chain splicing
(k or lambda)
heavy chain
1. VDJ IgM and IgD expressed
1. VDJ rearranged
are Heavy chain VDJ rearrangement rearranged 2. VJ rearranged
2. Expression of 3. IgM expressed
rearranging µ protein on cell 4. Allelic
surface exclusion of
3. Allelic
via RAG 1 exclusion of µ
light chain
completed
gene completed
and RAG 2 Steps completed
Steps completed
enzymes
which utilize the recombination sequences (RSS). RSSs are composed of seven conserved
nucleotides (a heptamer) that reside next to the gene encoding sequence followed by a spacer
(containing either 12 or 23 unconserved nucleotides) followed by a conserved nonamer (9
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base pairs). Only a pair of dissimilar spacer RSSs are efficiently recombined (i.e. one with a
spacer of 12 nucleotides will be recombined with one that has a spacer containing 23
nucleotides). After the RAG enzyme cuts two RSS sequences, the TdT fills in the ends of
broken DNA.
2. In the pre-B cell the heavy chain called the mu chain is expressed as a transmembrane protein
on the surface of the cell. This is the product of either mom’s or dad’s heavy chain allele
recombination. The mu chain expression signals the other heavy chain allele to be shut down,
in a process called allelic exclusion.
The pre-B cell undergoes division and subsequent light chain rearrangement in the daughter cells.
• This division step creates more diversity of B cell specificities because all daughter cells of
a particular pre-B cell will have identical heavy chains but not the same light chains. Due to
the randomness inherent to the process of recombination the light chains recombination will
yield different VJ joints in the different daughter cells.
• Light chain recombination occurs in an orderly fashion such that k (kappa) light chain gene
rearrangement occurs first (on either mom’s or dad’s allele), and only when both alleles are
exhausted (i.e. kappa rearrangement was unsuccessful), then lambda light chain gene is
rearranged if necessary.
• However, if a successful recombination event occurred, the recombination of the light chain
seizes, causing the expression of recombined gene only. This ensures the allelic exclusion
of the light chain.
3. Immature B cell- Once the light chain is rearranged, it combines with the heavy chain in the
ER, and a functional IgM is expressed on the surface.
As a result of the above processes, only one antibody specificity is produced per B cell.
Monospecificity of B cells is ensured by allelic exclusion: as soon as the heavy chain gene
segments are rearranged, heavy chain protein is expressed on the cell surface.
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Immature B cell samples then the

environment in the bone marrow
(see Figure on the right). Only those
B cells that do not recognize antigen
in the bone marrow (and are
therefore not self-reactive) will
proceed to the next step.
4. Mature B cell - In the mature

stage, the B cell begins to
express both IgM and IgD
isotypes (bottom). This is due to
the mRNA processing that allows
a longer mRNA transcript to be
produced at this stage
(containing both the mu and delta region). This transcript is alternatively spliced to yield
variable (VDJ) region mRNA adjacent to the mu OR the delta constant region. Despite
alternative splicing, which is done at the mRNA level, the constant region of the heavy chain
DNA is unaltered, but the chromatin structure changes so that the delta region of the gene is
available for transcription. The remaining isotype regions are still there on the gene, but are not
accessible for transcription until after the antigen in encountered. The mature B cell leaves the
bone marrow and populates the blood stream.
Alternative RNA splicing

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A look ahead: after B cell encounters the specific antigen

After it encounters antigen, and provided it receives additional stimulatory signals, the B cell
gets activated. This causes the B cell to divide and for some of the daughter cells to release IgM as a
pentamer in a secreted form. Other daughter cells undergo two additional processes that result in a
better affinity of the antibody for its antigen through a process of somatic hypermutation and a
change in the heavy chain’s constant region through a process called isotype switching.
Somatic hypermutation (figure right)
The somatic hypermutation mechanism involves specific mutations in the

variable region of the antibody gene in dividing B cells. For some daughter B
cells, these mutations result in an antibody that is more specific to the initial
antigen than was the original antibody molecule; these B cells are selected to
differentiate into plasma cells in a process known as affinity maturation.
Some of the daughter cells with high affinity antibody do not secrete their
antibody and are kept as memory cells with membrane bound immunoglobulin.
Plasma cells and memory cells that are generated as a result of somatic
hypermutation accumulate random mutations in their DNA. Selection by antibody
binding ensures that the antibody with the highest affinity for its antigen will be selected.
Isotype switching (Figure below right)
Unlike simultaneous expression of IgM and IgD seen in the mature B cell, isotype switching
causes a DNA loss in the
constant region of the heavy
chain gene. This involves
recombination between the
switch region contained before
the mu portion and the switch
region located before one of the
other isotypes (other than delta).
All intervening DNA between the
two switch regions (including the
mu, the delta, and any other
isotypic region up to the second
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switch region) is removed. The B cell now expresses a different isotype encoded by a nucleotide
sequence downstream of the mu and delta regions.
B cell activation Isotype switching results in a functionally different Ab
The summary of various processes that effect antibody variable and constant regions
Practice questions:
1. Two distinct IgM-producing, naïve B cells are likely to have:
a. Same idiotypes, different isotypes

b. Different idiotypes, same isotypes
c. Different idiotypes, different isotypes
d. Same idiotypes, same isotypes
e. Antibodies that cannot recognize any antigen
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2. The lack of which of the following enzymes would result in SCID?
a. Ligase
b. DNA dependent DNA polymerase
c. RAG
d. DNA dependent RNA polymerase
e. None of the above
3. Diversity is an important feature of the immune system. Which one of the following statements
about it is INCORRECT?
a. Humans can make antibodies with many different VH X VL combinations
b. A single cell can synthesize IgM antibody, then switch to IgA antibody
c. The hematopoietic stem cell carries the genetic potential to create more than 104 different B cells
d. A single B lymphocyte can produce antibodies of many different specificities, but a plasma cell is
monospecific
e. Heavy chain has more diversity than light chain
4. Your patient became ill 10 days ago with a viral disease. Laboratory examination reveals that the
patient's antibodies against this virus have a high ratio of IgM to IgG. What is your conclusion?
a. It is unlikely that the patient has encountered this organism previously

b. The patient is predisposed to IgG-mediated hypersensitivity reactions
c. The information given is irrelevant to previous antigen exposure
d. It is likely that the patient has an autoimmune disease
e. The patient has recovered from his/her infection
5. You find a patient that has a rare disease that arrests their B-cell development in the immature
stage. You analyze B cells in this patient. What do you expect to see? (How would the answer
change if I asked about what you would find in the serum of the patient?)
a. IgM only
b. IgG only
c. IgA only
d. IgM and IgD
e. None of the above
6. You see a patient during rounds that has been exposed to heavy amounts of radiation in an
industrial accident. The patient’s bone marrow has been particularly damaged, and is functionally
useless at this time. What is the only source of idiotypic diversity in this patient’s remaining B cell
population?
a. Combinatorial variability
b. Junctional variability
c. Heavy/Light chain pairing variability
d. Somatic hypermutation
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7. Which of the following is the immunoglobulin that is initially seen on the primary immune response?
It is present as a monomer on B cell surfaces but as a pentamer in serum.
a. IgG
b. IgM
c. IgE
d. IgA
e. IgD
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LECTURE 4: ANTIGEN PROCESSING, MHC PRESENTATION, T CELL DEVELOPMENT
Antigen processing and MHC presentation
• Overview of MHC
• Describe the structure of MHC I
• List the genes that encode MHC I
• List the cells that express MHC I
• Draw out the interaction of T cells and MHC I
• Describe the structure of MHC II
• List the genes that encode MHC II
• List the cells that express MHC II
• Draw out the interaction of T cells and MHC II
• Understand the terms polymorphic and polygenic
Why do we need MHC presentation?
B cells can recognize

any unprocessed antigen found
in the extracellular space. B
cells then secrete antibodies
that can block these antigens.
T cells do not recognize lipid or
sugar antigens, they only
recognize proteins that have been processed into peptides and are presented on a host encoded
protein called major histocompatibility
complex (MHC) (right bottom figure).
Besides the functional differences,
there are also some structural differences
between T and B cell receptors. An
immunoglobulin consists of two copies of the
heavy and light chain proteins, the T cell
receptor is composed of a single beta chain
and a single alpha chain. TCRs are only found as transmembrane proteins and are never secreted.
There are also similarities between an immunoglobulin and a TCR. Both of these molecules
are heteroproteins (consisting of two distinct proteins each) and both TCR and immunoglobulin
contain variable and constant regions, such that the variable region differs between the receptors on
different cells.
26
The biological role of MHC proteins is to bind small peptides and to "present" these foreign or
non-foreign antigens at the cell surface for the inspection by T cell antigen receptors. Below are
examples of types of pathogens that are presented on MHC protein.
1. Endogenous pathogens are those that reproduce in host cell cytosol. They include
viruses, intracellular bacteria and protozoan parasites. Since antibody cannot enter infected cells,
cytotoxic T cells (CD8 T cells) must be activated to recognize and kill the infected cells. To be
activated by endogenous antigen, CD8 T cells use their TCR to bind endogenous antigen peptides
presented on membrane MHC I proteins of target cells.
2. Exogenous pathogens (Figure right) include bacteria, their soluble toxins, extracellular
parasites and fungi. They can often be phagocytosed and destroyed
by lysosomal enzymes or oxidative burst in neutrophils and
macrophages, especially with the opsonizing activities of
complement and antibody.
However, some pathogens persist unharmed and
protected in phagocytic vesicles of phagocytes and therefore
need to be dealt with. Regardless of whether the pathogen is truly
extracellular (i.e replicates outside a cell but is phagocytosed by
macrophage), or is intravesicular (replicates inside a vesicle in a
cell), a peptide form such pathogens is presented on MHC II proteins.
To recognize and respond to exogenous antigens, CD4 T cells (called helper T cells) must
use their TCR to bind antigen peptides presented on MHC II proteins. These helper CD4 T cells can
then differentiate into Th1 cells that help phagocytes destroy the bacteria within them (Figure bottom)
or into Th2 and Tfh cells that activate B cells.
27
Below is a schematic of various scenarios where T cells need to recognize antigen and respond to it.
In each case, MHC is necessary for the T cells to do their job.
MHC Structure
MHC I
• Class I MHC is a heterodimer of membrane-bound
alpha chain and non-covalently associated beta 2-
microglobulin.
• The genes for Class I alpha are highly polymorphic
(there are multiple alternative versions of each gene
in a population).
• The alpha chain has three domains named alpha1,
alpha 2, and alpha 3 and a region adjoining alpha3
that anchors it in the plasma membrane.
• Beta 2-microglobulin is encoded by a gene on
another chromosome. Beta 2-microglobulin
molecules are non-covalently associated with Class I
alpha chain and are not polymorphic.
28
• Class I MHC proteins are expressed on all nucleated cells. Leukocytes express the highest level
of MHC I and neural cells the least.
Antigen binds between MHC I alpha 1 and alpha 2 domains. The amino acid sequence of
these antigen-binding domains varies from allele to allele. Variability is maximized in the amino acids
which contact antigen.
MHC I can hold an 8-10 amino acid peptide. The cleft is closed at the ends, limiting the size of
the peptide which can bind in the peptide binding groove. The alpha 1 and alpha 2 domains also bind
TCR. CD8 protein binds MHC I alpha 3 domain, which has a species-specific constant amino acid
sequence.
There are three MHC I genes that encode MHC I proteins: these genes are called HLA-A,
HLA-B, HLA-C. Remember that an individual inherits two alleles of each of these genes.
For a particular MHC I to hold a peptide, this peptide needs to have certain anchor residues
that fit into the pockets on MHC I. Anchor residues are amino acids in the peptide that interact with
residues in the MHC binding cleft. For a given MHC allele, the residues within a certain position must
be related - for example, all hydrophobic or all acidic amino acids. The MHC I peptide is anchored by
its carboxy and amino terminals and two other anchor residues in the middle of the peptide. The
amino acid residues lining the peptide binding sites of MHCs vary among different allelic variants and
determine the identity of the anchor residues that can bind to a particular MHC. For example, dad’s
and mom’s allelic variants of HLA-A in an individual will bind peptides with different anchor residues.
Keep in mind that while T cells and B cells each bind a unique epitope of an antigen and are mono-
specific, individual MHC I proteins can present many different peptides (one at a time) provided that
these peptides have the anchor residues with affinity for the peptide binding groove. MHC II proteins
also bind peptides via anchor residues.
Examples of anchor residues Schematic of TCR and MHC binding
29
MHC II
• MHC Class II is a non-covalently bound heterodimer of

alpha and beta chains.
• Peptides bind MHC II alpha 1 and beta 1 domains.
These domains vary from one MHC II allele to the next.
In addition to binding antigen, alpha 1 and beta 1
domains also contact the variable region of TCR.
• CD4 binds to the non-polymprophic region of MHC II.
• MHC II peptide-binding site binds longer (13-50
residues) peptides than MHC I.
• Peptides which bind to Class II are not anchored by their
amino and carboxyl termini.
• Class II-binding peptides contain 2 anchor residues that must be of certain types to bind a
particular allele.
• MHC II proteins are found on: B cells, dendritic cells, macrophages and thymic epithelial
cells.
• Three gene loci encode MHC II : HLA-DP, HLA-DQ, HLA-DR. Since MHC II protein is a
heterodimer composed of two proteins (alpha and beta chains) each of the MHC II loci
codes for alpha and beta chain. The MHC II proteins are coded for by HLA-DP alpha and HLA-
DP beta genes, HLA-DQ alpha and HLA-DQ beta genes and HLA-DR alpha and HLA-DR beta
genes. Remember that each individual inherits two allels of each gene.
MHC Function
MHC I processing (Figure right)
1. Newly synthesized, partly folded 5
MHC I alpha chain (light blue) binds

the chaperone calnexin (dark
4
blue) in the lumen of the ER.
Chaperone binding prevents mis-
3b
folding that otherwise would occur
1
in the absence of antigen peptide.
2
3a
30
2. When beta 2-microglobulin binds to the MHC I alpha chain, calnexin dissociates.
3. MHCI/beta 2-microglobulin form a complex with other chaperones such as calreticulin (3a) and
tapasin (3b), and the TAP transporter.
4. When MHC I binds peptide, it is released from theTAP transporter and from other chaperones.
Unbound peptides from the ER are thought to be transported back into the cytoplasm for
reprocessing and retransport.
5. The MHC I -peptide complex is transported through the Golgi (5) to the plasma membrane (6).
MHC I lacking a peptide returns to the cell to be reloaded, it does not remain empty on the
membrane. Althoug MHC pathway has evolved to help us fight infection, in uninfected cells,
membrane MHC I presents self peptides.
Viruses have evolved mechanisms which interfere with TAP function or with transport of MHC I to the
plasma membrane which allows them to evade destruction by cytotoxic T cells.
MHC II processing (Figure right)

Exogenous
antigens are processed in
the endosomal
processing pathway. 5
Bacteria, soluble protein
antigens, phagocytosed 4
antibody-coated viruses
and viral pieces released 3
from infected cells are 2
examples of exogenous
antigens. The endosomes
become increasingly
acidic, turning into
1
lysosomes, as they move
away from the plasma membrane farther into the cytoplasm. Increased acidity activates proteases
that cut the antigen into peptides. However, some bacteria and parasites have actually adapted to
survive and multiple in the endosomal vesicles; these are also considered exogenous (intravesicular)
antigens.
31
Steps in MHC II processing:

1. During translation MHC II alpha and beta chains are transported into the ER lumen, where they
assemble with another polypeptide, called the Invariant Chain (Ii). CLIP (Class II-associated
invariant chain peptide) is a short portion of the invariant chain which occupies the peptide binding
site. Invariant chain blocks association of MHC II with endogenous peptides present in the ER
lumen.
2. The invariant chain also directs the transfer of Class II MHC through the trans-Golgi to a
specialized vesicular compartment called phagolysosome (also referred to as MHC class II
compartment or MIIC), where the invariant chain is degraded by the low pH.
3. A chaperone molecule (DM) is involved in loading the MHC class II molecule with a peptide.
4. In the presence of antigen, MHC II’s groove (which opens up in the lysosomal compartment due to
the low pH and the help of DM) will bind pieces of bacterial proteins that also got processed in the
lysosome. The low pH displaces CLIP in favor of higher affinity peptides.
5. The complex is moved to the cell surface to be presented to a T cell.
In the absence of infection, APC present MHC II containing self-peptides.
MHC and diversity of antigens being presented

There are two main reasons for why collectively MHC proteins offer diversity for antigen
binding.
1) MHC genes are highly polygenic (multiple gene families on chromosome 6 encode MHC I
and MHC II). MHC I family contains: 3 genes called HLA-A, B, and C. MHC II family contains 3 gene
loci called DP, DQ, and DR which are arranged in pairs encoding alpha and beta chains of MHC II
proteins.
Organization of
HLA locus
2) MHC genes are highly polymorphic (each gene has alternative alleles in a population). HLA-
A contains almost 1000 alleles, HLA- B contains ~1500 alleles, HLA-C contains ~500 alleles. There is
also great variation in the number of alpha and beta alleles encoding HLA-DP, HLA-DQ, and HLA-
DR. The existence of three versions of MHC I and MHC II, and the fact that it is likely that an
32
individual inherits different alleles of each HLA gene from their parents allows for presentation of a
vast number of peptides.
Clinical correlation:
Bare lymphocyte syndrome is a partial or complete deficiency in MHC I or MHC II proteins.
People with bare lymphocyte syndrome have an increased susceptibility to viral and opportunistic
infections. Symptoms range from none to severe combined immune deficiency, depending on the
number of MHC loci that are expressed.
Summary of T cell
and MHC
interactions
33
T cell development
• Describe the basic structure of the thymus (cortex, cortico‐medullary junction, medulla, TEC, DC,
thymocytes, macrophages)
• Describe the steps in the differentiation of T cells
• Structure of TCR
• RAG, TdT, double negative cells, double positive cells
• Know the principle behind positive and negative selection
Cellular organization of the thymus

The thymus is an essential organ for the
development of T cells. In mice and humans with
DiGeorge's syndrome, the thymus fails to form fully
which results in a lack of T cells and a severe
immunodeficiency.
The figures on the bottom demonstrate structural organization of the thymus (higher) and the
stages of T cell development (lower).
Top figure: bone marrow precursors
enter at the outer, sub-capsular region
and migrate through the cortex toward
the center of the lobe called medulla.
The cortex is composed of a dense
reticular network of a highly specialized
thymic cortical epithelial cells (TECs),
which appear to be the only cell type
capable of mediating positive selection
of thymocytes. In addition to the cortical
epithelial cells, there are a few other
cells found in the thymic medulla: a distinct
type of medullary epithelial cells, bone
marrow-derived dendritic cells and
macrophages. Dendritic cells and
macrophages appear to be the most efficient
at triggering negative selection.
34
Overview of T cell development

Bottom right figure: T cells normally undergo development in the thymus. Cells arrive from the bone
marrow via the blood with no obvious T cell characteristics. They enter into the lobes and
subcapsular area. They begin to divide and migrate down toward medulla. The most immature
progenitors are called double negative (DN) because they do not express CD4 or CD8. CD4-CD8- DN
T cells also lack the signaling proteins collectively called CD3. These cells expand in number mainly
under the influence of IL-7 produced in the thymus. They then undergo rearrangement of beta-chain,
followed by rearrangement of the alpha-chain, and upregulation of CD3, CD4, CD8. Cells that
express CD3, CD4, CD8, and a TCR are called double positive (DP) thymocytes and these are the
cells that will undergo positive and negative selection. After positive selection the DP cells begin to
differentiate into either CD4 or CD8 single positive (SP) T cells. High affinity cells die in the thymus.
As a result of positive and negative selection >95% of thymocytes die within the thymus.
Specifics of T-Cell Development: Rearrangement of T Cell Receptor Genes
a. b. c. d. e.
Stem cell to immature double-positive T cell differentiation (stages a – d above)
TCR gene segments rearrange during intrathymic T-cell development. Similar to B cell
development, this is a somatic recombination process that involves the RSS recognition elements
(this follows the 12-23 rule to ensure proper VDJ recombination between heptamer/nonamer regions).
The enzymes that are used in T cell recombination are the same as the ones used in B cell gene
rearrangement (e.g. RAG-1 and RAG-2, TdT). However, the process of T lymphocyte maturation
also has some unique features, primarily related to the specificity of different subsets of T cells for
peptides displayed by different classes of MHC molecules.
35
Steps in Figure above:

a. Double negative lymphocyte precursor
b. Pro-T cell. TCR β gene recombination occurs in a pro-T cell. Vβ, Dβ and Jβ gene segments
rearrange with the help of RAG and TdT. Transcription and splicing of VβDβJβ exons to the exon
coding for the constant region of the beta chain (Cβ) generates the mRNA for a T cell β-chain protein.
If VDJ recombination is successful in one of the two inherited loci, a TCR β-chain protein is
synthesized. It is expressed on the surface in association with an invariant protein called pre-Tα (not
shown), to form the pre-TCR complex of pre-T cells.
c. Pre-T cell. The surface expression of the beta chain marks the pre-T cell stage. If a complete TCR
β chain is not produced in a pro-T cell, that cell dies. The pre-TCR complex delivers intracellular
signals to promote survival, proliferation, and TCR α gene recombination, and to inhibit VDJ
recombination at the second TCR β-chain locus (allelic exclusion).The alpha chain begins to
rearrange in the dividing pre-T cell (between stages “c” and “d” in the figure above). The Vα gene
segment rearranges to a Jα segment to create a functional exon; then transcription and splicing of
this VJα exon to Cα generates the mRNA for a functional T cell α -chain protein.
d. Immature DP T cell. When the alpha chain is rearranging, the dividing pre-T cells also upregulates
CD4, CD8 and CD3 proteins. After the alpha chain is done rearranging, combining with the beta
chain to form a TCR, and CD3, CD4, and CD8 proteins are expressed, the resulting cell is called the
double positive T cell. After rearrangement, each T cell bares about 30,000 T cell receptors on its
surface.
Because the T cell receptor cannot by itself
transmit a signal (TCR has no intracellular domain)

the signaling is performed by a complex of
transmembrane proteins called the CD3 complex
(see Figure above). The CD3 complex is a complex of
3 proteins that are stably associated with the TCR on
36
the cell surface. All of the CD3 molecules associate with protein tyrosine kinases following receptor
activation and thus signal the cell interior that the cell has encountered its antigen (see below).
Immature double-positive to mature single-positive cell differentiation (stages d – e above)
Positive selection: The CD4/CD8 decision (lineage commitment) is determined when T cells
are positively selected by self-MHC. T cells that have "low affinity" for self MHC presenting self-
peptide are allowed to survive. T cells need to be able to recognize MHC I or II because in the
periphery this will be the main molecule that the TCR will interact with on the target cell. If a T cell
does not recognize any of the host’s MHCs it will not be able to function in the future. So T cells that
have rearranged their receptors in such a way that they do not recognize either MHC I nor MHC II die
by apoptosis, a process also referred to as "death by neglect". This preservation of self MHC-
restricted (i.e., useful) T cells is the process of positive selection. Next, the cells stop expressing
either CD4 or CD8 depending on whether their TCR bound to MHC I or MHC II. During positive
selection, the T cells also become functionally segregated: the CD8+ T cells are capable of becoming
CTLs on activation, and the CD4+ cells are helper cells.
37
Negative selection: T cells that

survived during positive selection will then Summary of Positive and Negative
selection
encounter a wide range of self-peptides
presented by self MHC molecules expressed
on dendritic cells and macrophages further in
the medulla. Those T cells with high affinity
receptors for MHC I or II and self-peptide
undergo clonal deletion (also called negative
selection) through induction of apoptosis. Any
disturbance in this process can lead to the
escape of auto-reactive T-cells that can trigger
autoimmune disease. These selection
pressures ensure that the mature T-cell
population which leaves the thymus can react
with foreign antigen (presented by self MHC I
and self MHC II), but does not react with self-
antigen presented in host MHC I or MHC II.
The summary of positive and negative
selection is illustrated in the figure on the right.
TCR diversity
Overall, the amount of diversity in the TCR is comparable with that of the immunoglobulin
genes. The TCR has a different requirement for diversity than the immunoglobulin. Immunoglobulins
have to bind to a wide variety of ligands. The ligand for a T-cell receptor is always inside an MHC
molecule. TCRs have a relatively invariant shape and most of the diversity is focused on the antigenic
peptide occupying the center of the surface in contact with the receptor.
Since the TCR does not undergo somatic hypermutation, most of the diversity in TCR is
generated during rearrangement. The limited number of V region gene segments may generate the
diversity needed for MHC recognition, whereas the enormous diversity generated at the junctional
regions by TdT facilitates recognition of antigen. One reason that T cells do not undergo somatic
hypermutation is that it could be disastrous if a T cell receptor was to arise in the periphery that
reacted with a self-protein. Remember that T cells spend a long time undergoing thymic education
38
just to avoid these self-reactive problems. Additionally, somatic hypermutation might prevent the T
cell from recognizing self-MHC.
Practice questions:
1. Processing and presentation of antigen for presentation on class I MHC differs from the
corresponding process involving class II MHC in that only in processing for class II:
a. Is the MHC peptide synthesized by the ER.

b. Does the MHC acquire antigen after it has passed through the ER.
c. Does β microglobulin associate with the MHC in the ER.
d. Are cytoplasmic proteins passed into phagolysosomes for hydrolysis.
e. Is tapasin necessary for proper loading of antigen.
2. What is the earliest recombination event in B cell development?
a. Rearrangement of the kappa chain

b. Allelic exclusion of the lambda chain
c. Rearrangement of the heavy chain
d. Allelic exclusion of the heavy chain
e. Expression of IgM on the surface of the cell
3. Cytotoxic T cells induced by infection with virus A will kill target cells
a. from the same host infected with any virus.

b. infected by virus A and identical at class I MHC loci of the cytotoxic T cells.
c. infected by virus A and identical at class II MHC loci of the cytotoxic T cells.
d. infected with a different virus and identical at class I MHC loci of the cytotoxic cells.
e. infected with a different virus and identical at class II MHC loci of the cytotoxic cells.
4. Each of the following statements concerning class II MHC proteins is correct EXCEPT:
a. They are found on the surface of both B and T cells.

b. They have a high degree of polymorphism.
c. They are involved in the presentation of antigen by macrophages.
d. They have a binding site for CD4 proteins.
5. Which of the following is not expressed by double positive immature T cell in the thymus?
a. CD3
b. CD4
c. CD8
d. HLA-DP
e. Beta chain
39
6. Which of the following molecules is expressed first on a surface of a developing T cell?

a. CD3
b. CD40L
c. CD4
d. CD8
e. Beta chain
7. Which of the following molecules is expressed by a pre-T cell and by an immature double positive
T cell.
a. T cell receptor
b. CD4
c. CD8
d. Beta chain
e. Alpha chain
40
LECTURES 5 AND 6: ACTIVATION OF IMMUNE RESPONSE
• Overview of Immune response

• Diapedesis and entrance into lymph node
• Understand the differences between the three types of antigen presenting cells
• Delineate the activation and the effector phase of CD8 response
• Delineate the activation and the effector phase of Th1 response
• Delineate the activation and the effector phase of Th2 and Tfh response
• Briefly describe the induction of peripheral anergy
• Understand the T cell independent B cell response
• Describe the role of haptens in immune response
T cell activation
T cell activation is MHC dependent as illustrated below:
MHC I MHC II
Expressed by All nucleated cells APC’s
Present Intracellular peptides (self and Extracellular peptides (engulfed

viral peptides) pathogens)
Acquires peptide in Vesicles, after fusion with acidic

Rough ER
endosomes
Associated with Beta 2 Microglobulin
Encoded by α chain: HLA-A, -B, -C genes

HLA-DR, -DP, and –DQ genes
β macroglobulin chain:
chromosome 15 (Β2)
Binds to TCR, and CD8 receptor TCR, and CD4 receptor
41
Summary of CD4 T cell differentiation and activation
Immune response activation occurs in secondary lymphoid organs. T cells and B cells enter
secondary lymphoid organs
from the blood stream,
specifically from a region
called the high endothelial
venule (HEV) in a process
called diapedesis (see Figure
right). Diapedesis is mediated
by chemo-
attraction, rolling adhesion,
tight adhesion and transmigration. The HEVs are located in the T cell zones of lymphoid tissues and
are lined by specialized endothelial cells, which express carbohydrate ligands such as ICAM-1 and
Gly-CAM that bind to naïve T and B cell-expressed LFA-1 and L-selectin. HEVs also display
chemokines that are made only in the lymphoid tissues (such as CCR7) that bind CCR7R. These
interactions slow lymphocytes down and arrest them in the bloodstream.
Once in the lymph node, naïve T cells are not activated until they receive stimulation from a
professional antigen presenting cell (APC). There are several cells that can function as a professional
APC and activate naïve T cells. These cells include; dendritic cells, macrophages and B cells. The
table (below) compares these three populations of cells.
42
Antigen is usually encountered on a professional APC in a secondary lymphoid organ. If the

TCR recognizes antigen displayed on MHC molecules and also receives a second costimulatory
signal, the T cell
becomes activated. The
activated cells then
proliferate and undergo
clonal expansion and
differentiation into
effector cells, most of
which are short-lived.
The effector T cells can
move into peripheral
tissues and other organs
to handle pathogen
infection directly, or they
can migrate to germinal
centers to help activate B
cells with specificity for
the same antigen to
secrete antibody. The
CD8+ and CD4+ T-cell
subsets can develop with
different effector
functions. By virtue of the different specificities of CD4+ and CD8+ effector T cells, the immune
response can monitor extracellular pathogens (e.g., bacteria and parasites) and intracellular
pathogens respectively. Dendritic cells are the most versatile of all APCs because their sole function
is to activate T cells.
43
Simplified diagram of the types of cell-mediated immune reactions designed to eliminate different
types of microbes. CD4+ helper T cells secrete cytokines that recruit and activate other leukocytes to
phagocytose (ingest) and destroy microbes. CD4 T cells can also recruit B cells to secrete
antibodies. CD8+ cytotoxic T lymphocytes (CTLs) kill any infected cell containing microbial proteins
in the cytosol or nucleus, eliminating cellular reservoirs of infection.
Microbial infections may occur anywhere in the body, and some infectious pathogens are able to infect and
live within host cells. Pathogenic microbes that infect and survive inside host cells include all viruses that
work by infecting phagocytic and nonphagocytic cells and living and replicating in the cytoplasm of these
cells. In addition, many bacteria, fungi, and some protozoa that are ingested by phagocytes resist the killing
mechanisms of these phagocytes and thus survive in the vesicles or in the cytoplasm.
The different classes of T cells differ in the cellular locations of microbes they recognize and in the nature of
the reactions they elicit. In general, CD4+ T cells recognize antigens of microbes in phagocytic vesicles and
secrete cytokines that recruit and activate other leukocytes that kill the microbes, whereas CD8+ cells
recognize antigens of microbes that are present in the cytosol and directly destroy the infected cells.
44
CD8 T cell activation

Phases of activation:
T cell responses follow several phases
of activation. As shown in the diagram on
the right, CD8 T cells with particular
antigen specificity are very rare in the
periphery. However when the antigen
specific activation of such T cells occurs,
they expand (II) and their levels increase
to a much higher level than originally
found.
However, not all effector cells remain
alive and many activated T cells undergo a contraction phase (III), when they die by apoptosis. Only
a limited number of memory T cells remain in the body to fight this antigen in the future. Note that
the number of antigen specific memory T cells is still higher than the original number of such T cells
(IV).
CD8 T cell activation:
Dendritic cells can be exposed to cytoplasmic endogenous antigen in the tissue either by direct
infection with these antigens, or by phagocytosis and cross-presentation of the antigen on MHC I. A
naïve T cell specific for that antigen recognizes a peptide from the antigen presented on the MHC I
molecule of the dendritic cell. The interaction of a TCR with MHC peptide binding groove and a co-
receptor with non-polymorphic region of MHC provide a primary signal for T cell activation. For CD8 T
cells, this primary signal is the TCR-MHC binding and the binding of CD8 co-receptor to the non-
polymorphic region of MHC I.
A secondary signal (costimulation) is also required for T-cell activation: the binding of the B7
costimulatory molecule on the APC to the CD28 on the T cell provides costimulation. The name
costimulator derives from the fact that these molecules provide stimuli to T cells that function
together with stimulation of TCR by MHC/antigen complex. The best-defined costimulators for T cells
are two related proteins called B7-1 (CD80) and B7-2 (CD86), that will collectively be referred as B7
in this book. These proteins are expressed on APCs and their expression is increased when the
APCs encounter microbes. CD28-mediated signaling is essential for the responses of naive T cells; in
the absence of CD28-B7 interactions, antigen recognition by the TCR is insufficient for T cell
activation, and in fact has an opposite effect – deactivating the T cell.
45
After receiving the two signals, the naïve CD8 T cells then become activated cytotoxic T cells
(CTLs). During activation the activated T cells begin to secrete IL-2 which they bind in an autocrine
manner and which causes them to proliferate. CTLs begin to synthesize perforin and granzyme. They
then travel from the lymph nodes to the blood via the thoracic duct and exit the blood stream in the
infected tissue. In the tissue they find the infected cells by recognizing MHC I and the specific peptide
presented on these cells. The secretion of perforin and granzyme directly into the target cells induces
apoptosis in these cells. However, since non-infected cells do not present pieces of the pathogen,
they are not affected by the CTLs.
CD8 response
A.
B.
D.
C.
Induction of CD8 T cell response: (A) Dendritic cells (DC) pick up antigen and present a peptide on MHC
I. These DC travel to the lymph node. (B) Naive CD8+ T cells recognize peptides presented by DC in
peripheral lymphoid organs. The T lymphocytes are stimulated to proliferate and differentiate into effector
cells by 1) TCR/CD8 signaling and 2) CD28 signaling. The T cells respond by producing cytokines, such as
interleukin-2 (IL-2), and expressing receptors for these cytokines, leading to an autocrine pathway of cell
proliferation. Some of the progeny differentiate into effector cells, which serve various functions in (cell
mediated immunity) CMI, and memory cells, which survive for long periods. (C) Effector T cells and other
leukocytes migrate from efferent lymphatic vessels to blood vessels. (D) The T cells exit in peripheral tissues
by binding to endothelial cells that have been activated by cytokines produced in response to infection in
these tissues. (E) CD8+ cytotoxic T lymphocytes (CTLs) kill infected cells.
46
T cell inactivation (for both CD8 and CD4 effector cells):
Inactivation involves a molecule CTLA-4. Like CD28, CTLA-4 binds B7. In contrast to CD28,
CTLA-4 is transiently expressed only following T cell activation. Also, unlike CD28 signaling, the
signal delivered via CTLA-4 down-regulates T cell function and inhibits excessive expansion of
activated T cells.
CTLA-4 knockout mice exhibit massive lymphoproliferative disorder called ALPS (acute
lymphoproliferative syndrome) and spontaneous autoimmune disease that is uniformly fatal by 4-5
weeks. Therefore, CTLA-4 is a negative regulator of T cell function. In numerous animal transplant
models, treatment with CTLA-4 Ig (soluble CTLA-4 which competes with host’s CD28 for B7 binding),
prolonged graft survival, reduced the lethality of graft-vs.-host disease, and in some cases resulted in
donor-specific tolerance.
In addition to CTLA-4, activated T cells express FasR (CD95). The binding of the inducible
CD95 ligand (CD95L) to CD95 on activated T lymphocytes results in apoptotic cell death. There are
many cells that express FasL. This activation-induced cell death is implicated in the control of
immune cell homeostasis and immune response termination.
CD4 cell activation
CD4+ T-cells
have been
subdivided
into many
different
subsets on
the basis of
their cytokine
production
and their
functions (Figure right).
• Th1
o Th1 cells participate in both cell-mediated immunity and antibody-mediated immunity.

They are essential for controlling intravesicular pathogens such as certain bacteria,
e.i., Listeria and Mycobacterium tuberculosis (the bacillus that causes TB). They also
provide cytokine-mediated "help" to cytotoxic T lymphocytes — perhaps the body's most
potent weapon against intracellular pathogens. They are the most potent IL-2
producers. Their role in antibody-mediated immunity will not be discussed here.
47
• Th2
o These provide help for B cells and are essential for the production of IgE antibodies and
assist in the production of some subclasses of IgG as well. Antibodies are needed to
control extracellular pathogens which, unlike intracellular parasites, are exposed to
antibodies in blood and other body fluids.
• Tfh
o These also provide help to B cells enabling them to develop into antibody-
secreting plasma cells. This occurs in nests of lymphoid cells - called follicles - in
the lymph nodes. The most abundant helper T cells there are B-cell helpers
called follicular helper T (Tfh) cells.
• Th17
o These protect surfaces (i.e., skin and lining of the intestine) against extracellular
bacteria. These are characterized by production of IL-17 and recruitment of neutrophils.
Th17 cells may play a role in a range of inflammatory conditions including uveitis,
multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, and
rheumatoid arthritis.
In addition, there is another related subset of T cells that dampens rather than promotes
immune responses. These cells are designated 'regulatory T cells or (Treg) and their function is to
inhibit autoimmunity and protect against tissue injury.
Upon activation, CD4 T cells start as a THo cell that can develop into Th1, Th2, Th17, Tfh and
other T cells with different functions, these functions are determined by the initial DC and cytokine
interactions. Dendritic cells themselves respond to the antigenic environment around them and then
become the regulators of the T cell response. The different types of Th cells are also defined by the
cytokines they secrete and the responses that these T cells induce are determined primarily by
cytokines. In general, T helper (Th) cells are CD4+ cells that “help” other immune cells perform their
functions. Similar to CD8 T cells, CD4 T cells also need peptide presentation on MHC II and B7 co-
timulation in order to be activated.
Generation of Th1 cells (see diagram below)
• A. When a dendritic cell encounters an intravesicular antigen it expresses a peptide from that
antigen on MHC II.
• B. The DC travels to the lymph node and secrete interleukin-12 (IL-12). Naïve CD4 T cells
present in the lymph node need two signals to get activated 1. Triggering of TCR and CD4 by
48
MHC II, and 2. Triggering of CD28 by B7. DC provide the MHC II/peptide and the B7 needed to
activate naïve CD4 T cells in the lymph node.
• C. Activated CD4 T cells differentiate into Th1 cells in the presence of IL-12.
Th1 cells return to the tissue and are responsible for the activation of macrophages that express
the antigen recognized by the Th1 cell.
• Macrophages also need two signals to get activated in order to destroy the intravesicular bacteria
that parasitized them. These intravesicular bacteria often have evolved mechanisms to disarm the
vesicles formed by the macrophages.
• D. In the tissue, Th1 lymphocytes express CD40L, which engages CD40 on the macrophages,
and the T cells secrete interferon-γ (IFN-γ), which binds to IFN-γ receptors on the macrophages.
This combination of signals activates the macrophages to produce microbicidal substances that
kill the ingested microbes.
o Activated macrophages produce toxic substances such as nitric oxide (NO) and oxygen
radicals which either destroy or help control the bacteria. Th1 cells do not kill the
macrophage.
49
o Activated macrophages also secrete cytokines that induce inflammation. Tumor

necrosis factor (TNF) is a potent vasodilator, interleukin-1 (IL-1) increases the
expression of adhesion factors on endothelial cells to enable transmigration and
induces fever, IL-6 raises temperature, CXCL8 recruits neutrophils, and IL-12 which in
addition to directing Th1 cell differentiation also recruits and activates NK cells that in
turn strengthen macrophage.
Th1 response (in

the presence of
A.
IL-12)
B.
D.
C.
o Macrophage activation also leads to the induction of their professional APC properties.
• In addition to macrophage activation, cytotoxic T cells also get stimulated by Th1 cells, via IL-2
and IFN-γ that Th1 cells secrete.
• In addition to helping macrophages fight intra-vesicular cacteraTh1 cells can also modulate some
isotype switching in B cells to isotypes that will help bacteria opsonization (discussed later).
Th2 and Tfh cells

T helper type 2 (Th2) and T follicular helper (Tfh) cells are a distinct lineage of CD4+ effector T cell
that secretes IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-21 as well as other cytokines. These cells are
required for humoral immunity and play an important role in coordinating the immune response to
50
extracellular pathogens as well as bacterial toxins. Th2 differentiation occurs in the presence of IL-4,
whereas Tfh cells differentiate in the presence of IL-21. Unlike Th1 cells which travel to the tissue to
find infected macrophages, Tfh and Th2 cells remain in the lymph node and travel to the germinal
centers.
Th2 (and Tfh) cell activation and B cell stimulation:

In order to get optimally stimulated, each B cell needs to bind antigen via its antibody, receive
cytokine signals from the Th cells and interact with the CD40L on the helper T cell (via the CD40R on
the B cell).
Optimal B cell activation results in three population of cells:
1) IgM-producing short-lived plasma cells
2) Somatically hypermutated and isotype switched memory cells and
3) Somatically hypermutated and isotype switched long-lived plasma cells.
Steps 2 and 3 require germinal center formation. The antigen which stimulates Th2 (and Tfh)
response is usually soluble and extracellular, so that it can travel to the lymph node and binds to a B
cell that expresses antigen specific antibody.
Th2 response (in the presence of IL-4)

Steps in B cell activation
• A. DC can phagocytose
extracellular antigen in the A.
tissue, it will then carry the B.
antigen from the tissue to
the lymph node,
presenting the peptide on
E.
MHC II.
• B. Antigen specific B cell
also phagocytose the C.
extracellular antigen when
it travels to the lymph
node, process it and
present a peptide on MHC
II. D.-Th2
51
• C. The DC can then activate a CD4 naïve T cells using the two signals needed for activation
(MHC II/antigen and B7).
• D. In the presence of IL-4 (or IL-6, IL-21 and TGF-beta for Tfh) in the lymph node, the CD4 T cell
will differentiate into a Th2 (or Tfh) cell.
• E. Activated T helper CD4 (Th2 or Tfh) cells can then bind to the antigen-specific B cell presenting
a piece of antigen on MHC II. Th cells will then activate the B cell which they recognized.
• In addition to activating B cells, Th2 cells also stimulate and recruit specialized subsets of
immune cells, such as eosinophils and basophils, to the site of infection or in response to
allergens or toxin leading to tissue eosinophilia and mast cell hyperplasia. This induces
mucus production, goblet cell metaplasia, and airway hyper-responsiveness. Because of
their influence on the production of IgE antibodies and allergic responses, over activation of
Th2 cells appears to be responsible for the exacerbation of allergies.
B cell differentiation and activation summary
Isotype switching (Figure right) – review of Lecture 2

Remember that all isotype
information is carried on the
constant portion of the heavy
chain gene. Isotype switching
requires CD40L/CD40R
signaling to induce an enzyme
AID to open up the
heterochromatin of the heavy
chain. Subsequently, the
different cytokines secreted by
the Th2 and Tfh cells will
dictate which isotype needs to
undergo switching.
52
Th2 cells secrete IL-4 to induce IgE production by B cells. Tfh cells secrete IL-6, IL10, IL-21 to
induce switching to isotypes other than IgE. Remember that isotype switching causes recombination
and a permanent DNA loss between the switch region of the mu region and a switch region of
another isotype.
Somatic hypermutation (Figure bottom right) - review of Lecture 2

During isotype switching, the B cells also undergo another process called somatic hypermutation.
In this process deaminating mutations are introduced into the
variable portions of heavy and light chain genes. As a result,
new amino acids are synthesized in the variable regions.
The daughter B cells with the highest affinity for antigen will
survive and become plasma cells or long-lived memory cells.
B cells are selected for highest affinity via a process of
affinity maturation during which the selection and survival
of B cells with the highest antigen-binding affinity takes place
as antigen levels drop due to antigen clearance. Somatic
hypermutation also requires AID enzyme.
Since isotype switching occurs at the same time as
somatic hypermutation, resulting B cells will not only
have a high affinity antibody expressed on their
membrane but also the antibody will have an isotype
other than IgM. When secreted, these antibodies will have
characteristics such as long life-span, ability to activate
macrophages and also to activate complement cascade as well as cross various tissue barriers.
Daughter cells that secrete high affinity, isotype switched antibody serve as long-lived plasma cells
(as opposed to IgM secreting plasma cells which are short-lived). Remember, that after activation,
each B cell will only have antibody with one specificity and one isotype expressed.
Note that while isotype switching and somatic hypermutation require CD40R engagement on the
B cell, antigen binding and cytokine signaling, without CD40 signaling, may be enough for IgM
secretion. Clinically, a CD40R deficiency syndrome, called hyper-IgM syndrome, is characterized
by a lack of isotype switched antibodies but abundance of IgM.
Most of the activated T cells (whether CD8 or CD4) die by apoptosis after they have fought
against the infection. This restores homeostasis to the T-cell pool. A few effector cells mature into
53
memory T cells, which have the ability to respond faster and more effectively on re-encountering
antigen.
Dendritic cells as regulators of immune responses
How do dendritic cells know whether to secrete IL-4, IL-12 or other cytokines necessary for the
differentiation of Th2, Th1, and Tfh responses, respectively? DC and macrophages express Toll-like
receptors (TLRs) which
recognize structurally
conserved molecules derived
from microbes. These
microbe-derived molecules
are called pathogen-
associated molecular
patterns (PAMPs). PAMPs
are broadly shared by
pathogens but are
distinguishable from host
molecules. Depending on the
TLR triggered during
infection, APCs will secrete a
certain profile of cytokines.
These cytokines then
influence T cell development during the induction of an immune response.
Peripheral tolerance
Although central tolerance in the thymus ensures that self-reactive T cells die and do not leave the
thymus, it may happen that a self-reactive T cell makes its way out of the thymus and into the
periphery. Therefore and additional mechanism of tolerance induction must occur in the periphery.
Induction of tolerance outside the thymus is called peripheral tolerance.
There are several ways that peripheral tolerance can be induced:
1) Clonal deletion: a T cell that binds repeatedly (because of high concentration of self-antigen) will
undergo programmed cell
death Anergy induction
2) Anergy: T cells recognize
antigen in MHC but do not
receive costimulatory
signals remain inactive but
do not die. These cells are
called anergic cells.
3) Active suppression: self-
reactive T cells are kept
nonfunctional when self
antigen is presented at low
levels. The cells reacting at
low levels differentiate into
54
regulatory cells, that secrete regulatory cytokines in order to prevent other cells from reacting to
that antigen.
T cell independent B cell responses
B cells need at least two signals to initiate activation. For maximal antibody production most B
cells requires T cell help as described above. Such responses are thus called T-cell-dependent
responses, and the antigens that can activate B cells is this manner are called T- cell-dependent
antigens. With a T-dependent antigen, the first signal comes from antigen cross linking BCR and the
second from the Th2 or Tfh cell. T-dependent-antigens usually contain protein so that peptides can
be presented on B cell’s MHC II to the Th2 or the Tfh cells. Th2 and Tfh cells then help trigger B cell
proliferation and differentiation into plasma cells and memory cells.
T-dependent responses require that a B cell’s antibody and theTh2 or the Tfh cell that this B
cell interacts with recognize epitopes on the same antigen. The T and B cell epitopes don’t
necessarily need to be identical; for example, a T cell can respond to an internal viral protein
presented on an MHC II of a B cell, while the B cell can have an antibody specific to the viral coat
protein.
Despite the need for T cells to activate B cells, it has been found that a lack of a thymus does
not prevent all B cell activation. T cell dependent T cell independent
Therefore, sometimes B cells

can be activated in a T cell-
independent manner. Such
responses are called T-cell
independent- B cell
responses. These responses
require the stimulant antigen to
be a T-cell-independent
antigen, meaning the antigen
alone has the properties
required to deliver all
necessary signals to activate B cells. Examples of such antigens include repeating carbohydrate
epitopes found on the capsule of several bacteria. Binding of these antigens simultaneously to
several antibodies on the same B cell causes this B cell to divide and to respond with IgM synthesis.
Most pathogens, however, do not have many repetitive epitopes and therefore are not able to cross-
link several antibodies on the membrane of a single B cell.
55
Unlike T-cell-dependent-B cell activation, T-cell-independent-B-cell activation pathway does

not result in production of memory cells nor appreciable class-switching. The ability of B cells to be
activated without T cell help depends mainly on the nature of the pathogen.
56
Haptens:
Haptens are substance that are non-immunogenic but are foreign (or self, in autoimmune
reactions) and can react with an antibody on a B cell. Haptens are small molecules that normally do
not induce an immune response when administered by themselves (they are usually carbohydrates
that can’t be
Figure:
presented in
Top. Injection of hapten alone yields MHC), but can
no antibody response in the rabbit.
Collection of rabbit serum and induce an
performing ELISA results in no immune
fluorescence. response when
Bottom. Conjugating the hapten to coupled to a
a carrier protein, prior to injection,
carrier protein.
stimulates an immune response in
the rabbit. This is evidenced by
serum fluorescence seen in ELISA.
Concept of hapten - Right bottom figure: Here an antigen is made up of a hapten (orange, blue,
green) which is a carbohydrate lacking repetitive epitopes and a protein (pink). If the hapten alone is
administered it would not elicit an immune response. Attaching the protein (pink) to the hapten can
convert an otherwise non-immunogenic carbohydrate hapten into a T-dependent antigen.
• 1. The carbohydrate-specific B cell

binds the antigen via the blue-green
hapten carbohydrate. 2. Peptide (not
hapten)
• 2. The MHC of the B cell, however, presented to
presents a pink peptide (not the 1. Binding the Th cell
hapten) to Tfh cells. hapten
• 3 and 4. Tfh cell then activates the
B cell through CD40 and cytokines
• 5 and 6. B cell then produces and
secretes antibodies specific for the
blue-green hapten carbohydrate.
Cooperation between B cells and T
cells allows them to recognize the
same antigen through various epitopes
of that antigen. So while an epitope
recognized by a Tfh or Th2 cell is
constrained to be a peptide, the epitope recognized by the B cell can be protein, carbohydrate or lipid.
These epitopes are derived from the same antigen. This linked recognition of antigen by T and B
cells is important in manipulating haptens in order to make them immunogenic.
57
Examples of hapten-protein conjugates include: 1) the Hib (Haemophilus influenza b) vaccine

and 2) urushiol, a toxin found in poison ivy. When absorbed through the skin from a poison ivy plant,
urushiol reacts with skin proteins to form hapten conjugates which then causes a skin reaction.
LECTURE 7:
FUNCTIONS OF ANTIBODIES, NATURAL KILLER CELLS, BLOOD TYPES, ANTIBODY PROFILE

IN INFANTS
• Overview
• Describe the differences between neutralizing and opsonizing antibodies.
• Describe the role of complement in immune response.
o Classical vs Alternative cascade
o C3 convertase
o C5 convertase
o Deficiency in MAC
o Deficiency in C3
• Draw out the mechanism behind Natural Killer induced cell death.
• o How a lack of MHC I causes NK to kill targets
• o Role of antibody in NK mediated killing (ADCC)
Antibody functions: So far we have discussed in detail what antigen binding is like, however it is
also important to understand what an antibody can do to the antigen it bound.
1. Neutralization:
2. Antibodies can neutralize viruses in Neutralization
a number of ways. They may
interfere with virion binding to
receptors, block uptake into cells
and/or prevent uncoating of the
genomes in endosomes.
58
3. Opsonization: Opsonization
This is a process
by which a Opsonization
pathogen is
marked for
ingestion prior to
being destroyed
by a phagocyte.
Opsonization
involves the
binding of an
opsonin, (i.e.,
antibody), to a
molecule on the pathogen's cell membrane. After opsonin binds to the membrane, phagocytes
are attracted to the pathogen.
4. Activation of complement:
The following are several important consequences of complement activation which lead to the
destruction of pathogen.
a. Inflammation
b. Opsonization
c. Lysis of bacteria
Complement
There are two major pathways that can initiate the complement cascade. These pathways are
called the Classical pathway and the
Alternative pathway. Although the Alternative
pathway most likely began earlier during
evolution, it was discovered after the
Classical pathway and therefore received the
name “Alternative”. Both pathways lead to
inflammation, opsonization of the pathogen
and direct lysis of the pathogen.
59
Classical pathway
The classical pathway of complement is triggered by binding of IgM or IgG isotypes to a

pathogen (right figure below). Usually two Fc regions are needed to recruit the first complement
component. So a single IgM pentamer or two IgG monomers suffice. Early events involve a series of
proteolytic steps. A large precursor protein is cleaved to yield one large active fragment which usually
binds to the surface of the pathogen and a small peptide fragment that does not bind the membrane
and often mediates inflammatory responses.
Steps in classical pathway

activation (Figure right)
• The first component is 7

C1 (made up of C1q,
1 6 +
C1r, C1s). It is activated
+
when antibodies (IgM or
3
IgG) are bound to a 8
2 + 4 + 5
pathogen. Binding
activates an enzymatic
activity in C1r, which
causes C1s to act as an active serine protease. (1)
• The activated C1 now acts on the next 2 components of the pathway: C4 and C2. First, C1
cleaves C4 into C4b and C4a (2). C4b binds covalently to the surface of the pathogen (not
shown).
• This bound molecule of C4b now binds one molecule of C2, making it susceptible to cleavage by
C1. (3)
• C2 is cleaved into C2b and to C2a. (4)
• C4b and C2b remain on the surface of the pathogen and function as C3 convertase (aka C4b2b).
• C3 convertase (C4b2b) cleaves C3 into C3b and C3a. (5)
C3 cleavage results in the deposition of many molecules of C3b on the pathogen surface, which
can be recognized by complement receptors on the phagocytes and cause engulfment.
• Binding of C3b to C4b2b generates C5 convertase (aka C4b2b3b). (6)
• C5 is now cleaved to generate C5b, which forms part of the membrane attack complex and C5a
which is a mediator of inflammation. (7)
60
• C5b, C6, C7, C8 and several C9s together form the membrane attack complex (MAC) which
punctures a hole in the pathogen. (8) This occurs because C8 can change conformation to expose
hydrophobic domains that insert in the lipid bilayer and C9s can polymerize to form a pore in the
membrane.
• Peptides C4a, C3a and C5a are local mediators of inflammation.
Steps in the alternative pathway

activation (Figure right)
• The alternative pathway (right

5
figure) can be activated in the
absence of antibody and is
+
considered to be part of the +
innate response. 3
• The first fragment bound on + 4 6
1 +
the surface of the pathogen is 2
C3b (the first one bound in the

classical pathway is C4b).
• In the blood, C3 can be spontaneously cleaved by blood proteases to generate C3a and C3b. (1)
Normally, this complex is rapidly inactivated. However, if there are pathogens around, some C3b
can become covalently attached to the surface of the pathogen and be stabilized.
• Next, factor B binds to C3b. (2)
• This binding renders factor B susceptible to cleavage by Factor D, (3) yielding Ba and Bb. Bb now
forms C3bBb on the surface of the pathogen and Ba is an inflammatory mediator.
• The complex C3bBb is the C3 convertase of the alternative pathway and can cleave many
molecules of C3. (4) C3 convertase generates more C3a and C3b.
• C3b can also bind to pre-formed C3bBb to generate C3bBb3b. This is the C5 convertase and it
cleaves C5 into C5a and C5b. (5)
• MAC is formed exactly the same as for the classical pathway. (6)
• Note that the cleavage of complement component C3 is central to both pathways of complement
activation. When C3b is generated by C3 convertase of either cascade, it binds to the surface of a
pathogen and can initiate the alternative pathway or function as an opsonin.
• From the point of C3 cleavage into C3a and C3b, the pathways converge and C3b is one of the
major opsonins, binding to complement receptors on phagocytes.
61
Natural killer cells

Natural killer cells provide early defenses against intracellular infection. NK cells kill virally
infected and tumor cells while sparing normal cells. There are two distinct ways in which NK cells can
kill their target.
1) NK cells can destroy virally
Antibody-dependent cytotoxicity
infected cells with the aid of
antibodies in a process called
antibody dependent cellular
cytotoxicity (ADCC) (Figure
right).
• In ADCC the target cell

expressing an immunogenic
molecule is bound by
antibodies of the IgG isotype.
• NK cells have Fc receptors for IgG, so they can recognize the Fc portion of the same antibody that
coated target.
• Once the NK cell binds to the IgG’s Fc portion with its Fc receptor, the NK cell is triggered to
secrete perforin and granzyme to kill the target cell.
• NK cells can also cause apoptosis in the target cell by engaging the cell’s FasR with NK-
expressed FasL.
• Macrophages also have Fc gamma receptors and so they too can kill IgG coated targets.
However, unlike NK cells, macrophages kill the target by phagocytosis and degradation of the
target in the vesicles (not shown).
2) NK cells can cause death in target cells that express a lack of (or have a limited amount of) MHC I
(see Figure below, right).
• This ability is conferred upon the NK cell by its expression of both activation receptors that
interact with certain ligands on target cells and killer cell inhibitory receptors (KIRs) that interact
with class I MHC molecules on normal cells.
• KIRs have inhibitory cytoplasmic domains, so when KIR is engaged by MHC I, the cell received a
signal that suppresses its activation.
• So when MHC I is present on the target cell, the NK cell is inactivated and doesn’t kill the target
(top panel).
62
• A lack of MHC I on a cell shuts down the negative feedback via KIR, allowing the activation
receptor signaling to dominate, resulting in a target cell death.
• One of the most important roles for these cells is in the early phases of infection with intracellular
pathogens, such as some viruses and bacteria.
• Many viruses have the ability to downregulate
MHC I in the host cell. A benefit of
downregulating MHC I is that the infected cell
can no longer be recognized by CTL, and viral
replication can proceed unchecked.
• However in this scenario an NK cell can destroy
the target because the target cell lacks MHC I
expression and the NK cell’s function is to
detect and get rid of such cells.
• In addition to recognizing the lack of MHC I, NK
cells may also be able to recognize changes in
cell surface glycoproteins induced by viral or
bacterial infection.
• NK cells also secrete type I IFNs which inhibit viral replication during the early phases of infection.
NK cell activity can be increased by IL-12, which is also produced by phagocytes upon contact
NK and Macrophage role in ADCC is exploited by certain anti-

cancer drugs. In the Figure to the right, Rituximab-opsonized B cells
are subject to attack and killing by at least three pathways.
(A) Binding of rituximab causes activation of the complement

cascade, which generates the membrane attack complex that can
directly lyse B cells by complement-mediated cytotoxicity.
(B) Complement activation deposits C3b fragments on the B cell and

on the antibody itself. The Fc portion of rituximab and the deposited
C3b fragments allow for recognition by both Fc receptors and
complement receptors 1 and 3 on macrophages, which leads to
phagocytosis and antibody-dependent cell-mediated cytotoxicity.
(C) Binding of rituximab allows interaction with natural killer cells via
FcRIII and complement receptor 3, which leads to antibody-
dependent cell-mediated cytotoxicity.
Abbreviations: ADCC, antibody-dependent cell-mediated
cytotoxicity; FcR, Fc receptor; MAC, membrane attack complex; NK,
natural killer. Figure from Nature Immunology.
63
Blood types
The ABO blood group system is the most important blood type system (or blood group system) in
blood transfusion. There are two antigens and two
Phenotype Phenotype antibodies that are mostly responsible for rejection in the
ABO system. The two different types of antigens (also
Genotype
Genotype
called agglutinogens and isoantigens) are called type "A"

and type "B". The gene for this system has three alleles.
The A and B alleles code for enzymes that produce the
type A and B sugar antigens respectively. A third version
of this gene, the O allele, codes for a protein that is not
functional and does not produce antigenic surface
molecules. Each biological parent donates one of their two
ABO alleles to their child, and so a child inherits two
alleles of the ABO antigens, one from each parent (see
figure, left). The genes coding for the enzymes that
produce ABO antigens are expressed co-dominantly.
A blood test can be used to determine a person’s blood type. Although a blood test can tell you
which antigens are expressed on your RBCs, and thus determine your blood type phenotype, it is
not possible to determine the exact genotype from a blood test unless the person is AB or O.
What is the mechanism behind rejection of blood in a blood transfusion? It is know that people
with type A blood will develop antibodies to type B (called anti-B Ab isoantobodies) early in childhood.
People with type B blood develop antibodies to A (anti-A Ab isoantobodies). People with type O blood
develop both anti-A and anti-B Abs and people with type AB blood lack both types of Abs. These
antibodies are preformed without a known sensitization reaction and are of IgM isotype. These IgM
antibodies would react with the red blood cells
that express the antigen they recognize. People
do not develop Abs to their own blood group due
to negative selection of self-reactive B cells.
There are various theories for the unusual nature
of these preformed Abs but no one knows for
certain when or how they are formed. The figure
to the right illustrates the antigen and antibodies
that define the ABO system. To determine a
64
blood type, the patient’s red blood cells are mixed with various antibodies and agglutination is
measured. However a blood test will not determine your genotype. For example if a blood test
determines that an individual is Type A, it will not tell you whether his/her genotype is AA or AO (both
of which yield Type A blood).
Blood transfusion
When a transfusion of blood is required, it is necessary to make

sure that the recipient does not have antibodies that will attack the
donor’s red blood. Much of the routine work of a blood bank involves
testing blood from both donors and recipients to ensure that every
individual recipient is given blood that is compatible and is as safe as possible. This is done through
testing the recipient’s serum and the donor’s RBC. If the blood is compatible, no agglutination
occurs (see “a”, figure above). If the blood is incompatible, a visible agglutination forms (“b”, figure
above).
Incompatible blood between donor and recipient may lead to acute hemolytic reaction, RBC
destruction, lack of blood supply to the kidney, renal failure and shock. Antibodies that attack RBCs of
the donor are of IgM isotype and bind components of the complement system possibly leading to
massive hemolysis. Therefore a Type A individual can only donate to Type A and AB, Type B can
donate to B and AB, AB can only donate to AB, and Type O can donate to any type blood individual.
A postive aggutination reaction (which would lead to a physiological rejection) occurs when the donor
and the recipient are incompantible (specifically when the recipient has antibodies against donor
RBCs), as for example Type A donor and Type B receipent (below left). Based on these reactions a
compatibilty table of donor/recipient pairs can be made (below right).
Recipient Recipient
Group A Group B Group AB Group O Group A Group B Group AB Group O
65
Rh incompatibility – Similar to the ABO group system, another inherited factor, called the Rhesus
factor (Rh) can give rise to blood group incompatibility between individuals. Unlike A and B antigens
of ABO group, Rh factor is a protein, not a sugar, expressed by red blood cells. Therefore Rh factor is
directly encoded by a gene. Also, unlike antibodies to the ABO groups, Rh antibodies are not
preformed, but are only produced after an exposure to the Rh factor.
Figure right: The inheritance of Rh trait can be predicted by a simple Mating combinations between an Rh+ father and Rh- mother
conceptual model in which there are two alleles for this trait, D and d.
Rh + Rh +
Individuals who are homozygous dominant (DD) or heterozygous (Dd)
are both phenotypically Rh+. Those who are homozygous recessive
Rh -
Rh -
(dd) are phenotypically Rh-. Since an Rh+ father can have either a DD
or Dd genotype, there are 2 mating combinations possible with
differing risks as shown below. Although there is a chance that an Rh+
father and Rh- mother conceive an Rh- child (mating combination 2), if
the father is Rh+ and the mother is Rh-, doctors assume that mating
combination 2 is possible and that there may be an incompatibility
problem and act accordingly.
Mating combination 1 Mating combination 2
Figure below:
• Rh incompatibility is a condition that develops when an Rh-negative woman conceives an Rh

positive baby (A). This can only occur when the woman is Rh negative but the father is Rh
positive. Incompatibility will occur in this situation only if the Rh negative mom conceives an Rh
positive fetus. An Rh negative fetus will not pose a problem.
• During pregnancy, and more commonly during delivery (or miscarriage), red blood cells from the
unborn baby can cross into the mother's bloodstream through the placenta (B).
• If the mother is Rh-negative, her immune system treats Rh positive fetal cells as if they were a
foreign substance and makes antibodies against the fetal blood cells (C).
• These anti-Rh antibodies are of IgG isotype and have the potential to cross the placenta. This is a
problem for a second Rh positive fetus, rather than the first because by the time the IgG against
the Rh is made, the first baby is delivered. In the second or subsequent pregnancies, the IgG from
dad mom
Anti Rh Ab
A B C D
66
the mother that crossed the placenta to the Rh positive fetus can cause red blood cells to be
broken down (D).
During hemolytic disease of the newborn (which can happen with Rh incompatibility),
infant’s RBCs are destroyed and they release bilirubin, which causes an infant to become yellow
(jaundiced). The level of bilirubin in the infant's bloodstream may range from mild to dangerously high.
After birth, the infant may have yellowing of the skin and whites of the eyes, anemia and low muscle
tone (hypotonia) as well as lethargy. Occasionally Rh incompatibility can result in hydrops fetalis
(also called erythroblostis fetalis), an abnormal collection of fluid in at least two different fetal
compartments. This can lead to swelling, hemolytic abnormalities and miscarriage.
Currently the treatment that prevents the development of Rh antibodies in the mother is
administration of immune globulins called RhoGam. These immunoglobulins are against the Rh
factor and are IgG isotypes, but they are modified so that they themselves can’t cross the placenta
back to the fetus. RhoGam is ready to neutralize the Rh positive RBCs of the baby if/when they cross
to maternal circulation and thus these fetal Rh+ RBCs can’t sensitize maternal B cells specific for the
Rh factor.
Antibody profile in infants
A baby's immune
system is not fully
developed until he/she is
about six months-old. In the
meantime, the anibodies
passed by pregnant
mothers through the
placenta to the fetal
circlculation protect the
baby.
IgG is the only
antibody that crosses the placenta to the fetus during pregnancy. IgG antibodies are the smallest but
most abundant making up 75-80% of all the antibodies in the body. These antibodies help protect the
fetus from developing an infection inside the womb. Immediately after birth, the newborn has high
levels of the mother's IgGs in the bloodstream. This is called passive immunity because the mother is
"passing" her antibodies to her child. This helps prevent the baby from developing diseases and
infections.
67
During the next several months, maternal IgGs are steadily degraded in the child’s
bloodstream. When healthy babies are about two to three months old, the baby will start to gain the
ability to produce his/her own IgGs. Between 3 and 6 months, the baby will experience the body's
natural low point of antibodies in the bloodstream. This is because the maternal antibodies have
decreased, and young children, who are making antibodies for the first time, produce them at a much
slower rate than adults.
The IgMs, however, can be produced by the baby even in utero. By the time the baby is born
his/her ability to secrete IgM is about 50% that of an adult. By 6 months the baby has the same
potential as an adult to produce IgMs. However, since these antibodes have low affinity, they do not
offer as much protection as IgGs.
Practice questions:
1. Which immune cell utilizes a pore-forming molecules to damage the target cell membrane?
a. NK cell
b. Th2 cell
c. Th1 cell
d. B cell
e. Red blood cell
2. Serum drawn from a three-day-old infant would contain which of the following?
a. Infant IgG2
b. Infant IgA
c. Maternal IgM
d. Maternal IgG1
e. Maternal IgE
3. Which pair of molecules are both opsonins?
a. IgG1 and C5a

b. IgA1 and IgG2
c. IgE and IgG4
d. IgG3 and IgM
e. IgG4 and C3b
4. A CD8+ T cell binds to an antigen-MHC1 complex on a dendritic cell. The CD28-B7 interaction
happens concomitantly, but no IL-2 is present yet. What is currently happening?
a. The CD8 T cell is activated only

b. Clonal expansion of the T cell only
c. Clonal expansion and activation of the T cell
d. Perforins and Granzymes are released into the lymph node
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5. A patient’s immune response to a pathogen shifts from primarily IL-12 mediated to IL-4 mediated.
What can be said of the subsequent change in the immune response?
a. The TH1 response increases

b. Macrophage stimulation increases
c. CD8 effects are enhanced
d. B cell stimulation increases
6. What step of the classical complement pathway will fail if the function of C3 convertase is blocked?
a. C1 will not be activated

b. C2 will not be split into C2a and C2b
c. C4 will not be split into C4a and C4b
d. Phagocytosis will be diminished
7. Which cell will be spared by a Natural Killer cell?
a. cell expressing MHCII only

b. cell expressing MHCI only
c. cell coated with IgG
d. cell expressing Fc gamma receptor and no MHC I
8. A worried mother of a 3 month-old baby comes to your clinic with concerns that her baby has had a
runny nose. You draw an antibody titer and find high levels of IgM and low levels of IgG. What can we
say about this child’s immune system.
a. He has a congenital IgG deficiency

b. His immune system is normal
c. He has an over-abundance of IgM
d. He is unable to isotype switch from IgM to IgG
9. You are on NICU service in your intern year when you are presented with a severely jaundiced
neonate. Upon looking into the history, you find that the mother declined RhoGam therapy due to her
religious beliefs. This is her second baby. What can be said about the maternal and fetal blood types?
Mother Fetus
a. B+ A+
b. O+ B-
c. AB- A+
d. A- AB-
10. The deletion of which of the following molecules would cause over-proliferation of T cells?
a. B7
b. TCR
c. MHC
d. Fas
e. CD40
11. Which of the following collectively describes the antibodies found in a neonate that is nursed?
69
a. Same idiotype, different isotypes

b. Same isotype, different idiotypes
c. Same idiotype, same isotype
d. Different idiotypes, different isotypes
e. Different allotypes, same idiotype
12. Which of the following processes changes a B cells’ idiotype and occurs in the lymph node?
a. Allelic exclusion
b. Isotype switching
c. RNA splicing
d. Somatic hypermutation
e. Somatic recombination
13. Which of the following DNA sequences would most likely to occur in the heavy chain gene in an
immature B cell?
a. V1, V2…Vn; D1, D2,…Dn; J1, J2…….J6; mu, delta, gamma3

b. V1, V2, D2, J5, J6; mu, delta, gamma3….
c. V1, D4, J2; gamma3, gamma1…
d. V1, V2…Vn; J1, J2….J6; kappa
e. V1, J3, lambda
14. You have a patient with a rare genetic disease that prevents their lysosomes from reaching their
normal, low pH. What will you find bound to their MHC II proteins?
a. tapasin
b. calcineurin
c. bacterial antigen
d. CLIP protein
15. You see a patient in the clinic with advanced AIDS. His T cell count is 40 (extremely low), but you
notice that the surviving population is almost all Th1-type cells. What immune function will be the
most preserved?
a. Infected cell apoptosis

b. Macrophage activation to kill pathogens
c. B cell antibody production
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LECTURE 8: HYPERSENSITIVITY REACTIONS

• Overview of hypersensitivity
• Describe the mechanism of Type I hypersensitivity
• Provide clinical examples of Type I hypersensitivity
• Understand the principle of chronic desensitization
• Describe the mechanism of Type II hypersensitivity
• Explain the role of ADCC in Type II hypersensitive
• Provide examples of foreign-antigen versus self‐antigen-induced diseases in Type II hypersensitivity
• Describe the mechanism of Type III hypersensitivity
• Provide examples of foreign-antigen versus self‐antigen-induced diseases in Type III hypersensitivity
• Describe the mechanism of Type IV hypersensitivity
• Provide examples of foreign-antigen versus self‐antigen-induced diseases in Type IV hypersensitivity
Hypersensitivity results from the damage done to the body by an immune response. Immune
responses can damage the body during antigen removal, causing swelling and pain from
inflammation, or during lysis of virally-infected cells by cytotoxic T cells. Usually these are mild.
Occasionally, when the damage is too great, hypersensitivity can become life-threatening.
Hypersensitivities are classified into four types based on the mechanism of tissue damage
(Figure above). Most of what we call "allergy" is Type I hypersensitivity, in which IgE is produced in
71
response to an antigen called an allergen. However IgG can also mediate tissue damage, as seen in
Type II and Type III hypersensitivities. Additionally, T cells are implicated in Type IV hypersensitivity.
TYPE I HYPERSENSITIVITY
Type I (immediate) hypersensitivity is

mediated by IgE and mast cells. Mast cells are
commonly found at sites in the body that are
exposed to the external environment, such as the
skin. In these locations, they are found in close
proximity to blood vessels, where they can
regulate vascular permeability and effector-cell
recruitment. Although they do not have direct
cell–cell contact with local populations of antigen-
presenting cells (such as the Langerhans cells in
the skin), mast cells can modulate the behavior of
these and other neighboring effector cells
through the release of mediators such as
histamines, prostoglandins and leukotrienes.
72
IgE-mediated allergic
reactions/syndromes include: hay fever,
skin inflammation (urticaria), food
allergies, asthma, and systemic
anaphylaxis (Table, right). One in five
people in developed countries are
thought to have some form of Type I
hypersensitivity.
The risk of developing a Type I
hypersensitivity (also called atopy) is
linked to family history and IgE levels. A
child's risk of becoming atopic is
greater if s/he has parents who are
atopic. People with higher IgE levels
tend to be atopic more often than
people with lower IgE levels, although
the linkage is not absolute.
It is not clear what makes certain
molecules allergens for atopic people.
However, allergens fall into groups of
molecules including: grasses, pollens,
animal and food products that usually reach mucosal surfaces at very low doses. They are
usually proteins, since only protein molecules can be presented to T cells and elicit T cell help which
is necessary to ensure IgE
production by B cells. Several of
these allergens are proteases. In
addition to proteins, haptens have
also been associated with Type I
hypersensitivity; this occurs when
the haptens conjugate a soluble
protein which makes the haptens
become immunogenic. The
Figure on the right illustrates
the effects that mast cells in
Type I hypersensitivity have on
various organs.
The main symptoms
associated with Type I
hypersensitivity are due to the
release of toxic mediators
synthesized by mast cells. These
mediators cause inflammation and
tissue damage the final effect depends on the tissue. Examples of the effects that the release of toxic
mediators have on various target organs is shown in the figure above.
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The mechanism of Type I Hypersensitivity
In this response, exposure to allergens generally occurs at mucosal membranes in the

respiratory and digestive tracts. Antigen dose is generally low, favoring activation of Th2 responses.
The allergen is usually low molecular weight and very soluble, so it can diffuse through the mucus
membrane. There are two phases in Type I hypersensitivity: sensitization phase and activation
phase.
Sensitization:
Figure on the right:
• Similar to the initiation of a Th2 response, the allergen in Type I hypersensitivity is first introduced
through mucosal or skin barrier into
A. Sensitization
the tissue (A).
• The DC uptake and present a peptide
portion of the allergen on MHC II.
This DC can then activate an
allergen-specific naïve CD4 T cell
(B). The cytokine balance during C.
activation favors Th2 cell
B.
differentiation (D). E.
F.
• The soluble, extracellular allergen
D.
also travels unprocessed to the
lymph nodes, where antigen-specific
B cells can bind to it via their antibody. The B cells then process and present epitopes from the
antigen on the MHC II (C).
• The allergen-specific Th2 cell can then activate the allergen-specific B cell which is presenting the
peptide from the allergen to the Th2 cell (D). The same signals are necessary for the B cells to get
activated as discussed before. The prevalence of IL-4 favors not only Th2 differentiation but also
induces B cells to isotype switch to IgE. Note that the specific epitope recognized by the B cell
does not have to be the same as the epitope presented on MHC II of the B cell. For example, the
B cell could recognize a surface carbohydrate molecule on the antigen but present a peptide from
within the antigen.
• The terminally differentiated plasma cells secrete IgE (E).
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• IgE then enters the bloodstream and binds to the Fc epsilon receptors (FcεRI) expressed by mast
cells lining mucosal surfaces (F). The IgE bound to mast cells remains after the antigen has been
cleared from the body.
• Sensitization results in IgE binding to mast cells in smooth muscle, blood vessels, mucosal linings
and connective tissues around the respiratory and digestive tracts. Sensitization usually does not
result in symptoms.
Activation:
After sensitization, a subsequent exposure to the same allergen cross-links the mast cell-bound IgE
molecules, activates the FcεRIs and triggers the release of various pharmacologically active
substances. Mast cell degranulation is preceded by increased Ca++ influx.
Activation
Activation phase has two sub-phases: Immediate/Early and Late
Immediate/Early-phase (occurs within seconds)

• Preformed mast cell mediators (such as histamine, heparin, and proteolytic enzymes) stored in
cytoplasmic granules are rapidly released.
• Proteolytic enzymes break down tissue matrix proteins.
• Histamine causes respiratory smooth muscle contraction, increased mucus release,
vasodilatation, and increased capillary permeability.
75
• Increased blood flow and fluid release at mucus membranes and into tissues washes away the
antigen.
• In the respiratory tract, mucus production, vasodilation and smooth muscle contraction
leads to runny nose, watery eyes, sneezing, coughing, sinus congestion and constricted
airways.
• In the gastrointestinal tract, smooth muscle contraction and fluid release cause
cramping, diarrhea, and vomiting.
• In the skin, localized swelling causes hives (urticaria).
Late-phase response (follows hours after the initial reaction)
• Leukocytes, such as eosinophils and neutrophils are attracted to the inflammation.
• More cytokines are released
• White blood cell production is stimulated in the marrow
Testing for Type I hypersensitivity
Skin testing for allergies is

used to identify the substances
that trigger allergy symptoms. It is
performed by applying a very
small amount of an allergen to
your skin dermis, and then
evaluating the skin's reaction. If an
individual has previously been
exposed to an allergen and has
mounted a Type I reaction, he will
have a built-up of mast cell-bound
IgEs in his skin. Upon exposure to
antigen, these IgE’s will cause the
mast cell to degranulate locally
and produce a swelling as a result
(see figures below).
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Treatment/Management of Type I hypersensitivity:
Drugs: Antihistamines, Corticosteroids (anti-inflammatory), Cromolyn sodium (stabilizes mast cells),

Epinephrine (blood vessel constriction)
Type I desensitization
People with Type I allergy may receive chronic desensitization through injections of allergen
beginning with very low doses
that are increased over many
months. Desensitization is not
effective for every allergen or
for every individual, and its
mechanism of action is unclear.
It is thought that the
effectiveness of this
procedure results from the
induction of high levels of
IgG antibodies, which can
prevent allergic reactions by
competing for the allergen
and preventing it from reaching mast-cell bound IgE, or that the treatment induces suppressor T
cells (probably Th1 cells) which block B cell activation and IgE synthesis in response to the allergen.
77
TYPE II HYPERSENSITIVITY
Type II reactions occur as a result of immune mediators responding to an antibody bound to
cell surface antigen. The cells with such antigens can be extrinsic (foreign) or intrinsic (host-
derived). The antibody produced is usually IgM and/or IgG. The results of binding of these classes of
antibodies to the cell surface are shown in the figure below:
1. Complement activation
Classical complement activation by IgG 2.
causes the release of inflammation-
promoting anaphylatoxins and may 1.
lead to formation of the membrane
attack complex (MAC) and lysis of the
antibody-coated cell.
2. Antibody-dependent cell-
mediated cytotoxicity (ADCC)
ADCC is a mechanism of immune
defense whereby an effector cell of The effects of Type II hypersensitivity
the immune system actively lyses a target cell, whose membrane-surface antigens have been bound
by specific antibodies. The most common cells participating in ADCC are natural killer (NK) cells and
macrophages. NK cells and macrophages express CD16, which is an Fcgamma receptor (called
FcgammaR1). This receptor recognizes, and binds to the Fc portion of an antibody, such as IgG. The
IgGs, in turn, are bound via their idiotypes to the surface of a pathogen-infected target cell. When NK
cell’s FcgammaR1 is triggered, the NK cell releases perforin and granzymes to kill the target cell.
Macrophages also express FcgammaR1 and can also destroy the antibody coated target cell.
However they mediate target cell death through phagocytosis. Type II reactions may lead to cell
death or inflammation.
Clinical examples of Type II hypersensitivities:
There are two categories of antigens (foreign and self) that can induce Type II
hypersensitivity:
Examples of foreign antigen-induced Type II hypersensitivity:
1. Blood transfusion can also result in Type II Hypersensitivity to blood group antigens such as
A, B and Rh. Remember that in blood transfusion, if the blood types are not matched, the
recipients’ IgM may bind to donor red blood cells. This causes complement activation and
destruction of donor RBCs in the recipient.
78
2. Drugs like aspirin and penicillin, which often complex with erythrocyte membrane
proteins, may induce the host to synthesize IgG antibodies which then bind the drug on the
drug-coated erythrocytes and damage them.
3. Hyperacute graft rejection, a rejection in which preformed antibodies to blood group antigens
(ABO/Rh system) or transplantation antigens (MHC system) can cause immediate, severe, and
non-reversible damage to the graft. Since the destruction is mediated by antibodies bound to
the donor tissue, this reaction is considered Type II.
Host Ab
kidney kidney
Examples of autoantigen-induced Type II hypersensitivity:

Antibodies are produced to self-membrane proteins.
1. Myasthenia gravis (MG) – This disease is caused by the
production of antibodies to the nicotinic acetylcholine
receptors on muscle cells. It affects mainly somatic nervous
system which controls skeletal muscles. For example,
oculomotor nerve supplies both the muscles that move the
eyelid (e.g. levator palpebrae superioris) and also carries
parasympathetic innervation of pupil. If the entire
oculomotor nerve is damaged, the pupil dilates and the
eyelid droops. However, in MG, levator palpebrae
superioris can’t respond to the stimulation by the alpha-
motor neuron, whereas the parasympathetic nerve’s function is unaffected. The result is: the
eyelid droops but the pupil constricts normally. Other symptoms of MG include difficulty talking,
difficulty climbing stairs and lifting objects, difficulty swallowing, and difficulty maintaining
steady gaze. Symptoms typically progress from top to bottom. Weakness in arms and legs
represent flaccid paralysis.
79
2. Graves disease – This disease results from the production of antibodies to thyroid stimulating
hormone (TSH) Receptor.
• TSH is secreted by the pituitary gland in
response to hypothalamus-released thyrotropin
releasing hormone (TRH).
• TSH receptors are located on the thyroid
follicular cells that produce the thyroid hormones
T3 and T4. Normally, TSH acts on the TSH
receptor to stimulate T3 and T4 production by the
thyroid gland. T3/T4 cause glycogenolysis,
gluconeogenesis, muscle wasting, increased
activity of the heart and skeletal muscle and
overall increased metabolism.
• After T3 and T4 are made, there is a negative
feedback loop, where the T3/T4 hormones act on
pituitary gland to block TSH production.
• In Graves’ disease receptor antibodies act as
agonists, mimicking TSH hormone and
resulting in overproduction of thyroid
hormone T3 and T4. Excessive binding of TSH antibody to the receptor causes continual
T3/T4 production, resulting in hyperthyroidism. One symptom of Graves’ is the enlargement
of the thyroid gland (goiter). Other symptoms include anxiety, irritability, fatigue, rapid or
irregular heartbeat, increase in perspiration, sensitivity to heat, weight loss, and bulging
eyes (Graves' ophthalmopathy).
3. Autoimmune hemolytic anemia – Hemolytic anemia (HA) occurs when the bone marrow is
unable to increase production of red blood cells (RBCs) to make up for the premature
destruction of RBCs. In the autoimmune type of HA, antibody against the erythrocyte
membrane protein is produced. Some of the symptoms include fatigue, shortness of breath,
and rapid heartbeat. Anemia also causes jaundice because unconjugated bilirubin is released
from the dying RBCs. Bilirubin, a breakdown product of hemoglobin, accumulates in the
circulation and in the tissues causing yellowing. Unconjugated bilirubin in the plasma is
conjugated by the liver and is returned back to the blood. Excess conjugated bilirubin present
in the urine can give urine an unusually dark color.
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TYPE III HYPERSENSITIVITY
Type III reaction occurs when excess of immune complexes deposit in the tissues. Immune
complexes are usually formed when antibodies bind to soluble antigens. After immune complexes
are formed they are usually cleared by phagocytes but if the clearance is delayed the complement
cascade is activated and this may result in tissue damage. (Figure on the bottom)
Although phagocytes play an important role in immune complex removal, red blood cells are also
very important in carrying immune complexes to the spleen and liver for destruction. Often, numerous
immune complexes, too small to bind FcR on phagocytes, are removed from the circulation by
erythrocytes which bare complement receptor CR1 (it’s a receptor for C3b). CR1 expressed by the
red blood cell binds to C3b deposited on to the Fc portion of the antibody attached to an antigen.
These complexes are brought to the spleen by the red blood cells and are degraded before they can
cause any damage.
Under certain conditions, such as when the antigen persists in the body for long periods or when
high levels of antigen are encountered at one time, immune complexes reach such high levels that
they are no longer soluble or easily removable. These immune complexes can then deposit and stay
in tissues. Common sites of complex deposition and tissue damage are: blood vessel walls,
kidneys, and joints. Immune completes often deposit in organs where blood is filtered at high
pressure to form other fluids, such as urine and synovial fluid.
Immune complex deposition can result in either localized reactions or in generalized (more
systemic) reactions. The antigen causing the antibodies to deposit can either be foreign or self (in
which case the antibodies are auto-antibodies).
Example of localized, foreign antigen-induced reaction:
1. Arthus reaction - In this condition, a preformed IgG (formed during the sensitization phase)
forms local immune complexes with the antigen. The antigen is usually something that a
person has been primed to before. For example, arthus reaction may result from a tetanus
booster shot that is re-administered after an accident. In this situation, an individual would
81
already have IgGs formed during prior immunizations. The tetanus toxoid booster shot
administered after accidents and these preformed IgGs will form complexes, locally, in the
tissue where the shot was administered. These complexes activate complement, which in turn
releases mediators such as C5a, which creates a local inflammatory response. The lesion
contains primarily neutrophils, deposits of immune complexes and complement. Macrophages
infiltrating in later stages may be involved in the healing process.
Example of generalized, foreign antigen-induced reaction:
1. Serum sickness - generalized form of Type III hypersensitivity.

In the pre-antibiotic era, immune horse serum (containing antibodies specific for a
particular antigen) was often given to treat many infections. Antibodies from the horse serum
helped clear the infection in the human. However individuals receiving horse’s serum
sometimes developed their own immune response against the Fc region of the horse antibody.
A similar situation can occur in a person receiving a therapeutic passive immunization
with horse neutralizing antibodies to rattlesnake venom. In both cases, a person receiving
horse antibodies for the first time makes a primary response to the foreign IgG protein 7-10
days after administration. After 7-10 days, enough human anti-horse IgG antibody has been
produced to form immune complexes in the circulation and deposit in small vessels. The
immune complex consists of human antibody binding to the Fc portion of the horse antibody.
Since the passive immunization is given as an IV it activates complement, neutrophils and
macrophages systemically,
Symptoms of serum sickness are systemic and include: fever, chills, rash, arthritis, and
sometimes kidney damage. Note that on second and subsequent exposure to horse
antibodies, the patient would begin experiencing serum sickness within a couple of days since
isotype switching to IgG had already occurred.
Examples of generalized, autoantigen-induced reactions:

1. Rheumatoid arthritis (RA)
• Rheumatoid arthritis is a chronic inflammatory disorder
that typically affects the small joints in your hands and
feet. Unlike the wear-and-tear damage of osteoarthritis,
rheumatoid arthritis affects the cartilage lining of the
joints, causing a painful swelling.
• In autoimmune RA, patients develop an auto-IgM
antibody against their own IgG which forms an immune
complex that contributes to arthritic joint inflammation.
RA usually affects joints on both sides of the body
equally. Wrists, fingers, knees, feet, and ankles are the
most commonly affected.
• In RA, the synovial fluid fills with polymorphonuclear
leukocytes that have encountered and engulfed immune
complexes.
• Joint symptoms may include: morning stiffness, which
lasts more than 1 hour, warm joints, and joint pain often
felt on the same joint on both sides of the body. Once the
inflammatory reaction is established, the synovium
thickens, the cartilage and the underlying bone begins to disintegrate and evidence of joint
destruction accumulates. Over time, joints may lose their range of motion and may become
82
deformed. In addition to causing joint problems, rheumatoid arthritis sometimes can affect
other organs of the body — such as the skin, eyes, lungs and blood vessels.
2. Systemic Lupus Erythematosis (SLE)

• Lupus is another chronic inflammatory disease that occurs when your body's immune system
attacks healthy tissue in your body. Lupus results from a combination of your genetics and
your environment.
• Many different body systems are affected by SLE including: joints, skin, kidneys, liver, blood
vessels, brain, nervous system, heart and lungs.
• Mechanistically, in SLE, autoantibodies are produced against the patient’s own DNA and
histones. Additionally autoantibodies against platelets can be detected. The course of the
disease is unpredictable, with periods of illness alternating with remissions. Symptoms of lupus
include severe fatigue, painful or swollen joints, headaches, rash on cheeks and nose called
“butterfly” rash, hair loss, anemia, blood-clotting problems and Raynaud's syndrome (fingers
turning white/blue and tingle when cold).
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A schematic of Type III reactions: Events required triggering lupus
RA
Arthus rxn Farmer’s Serum SLE
lung sickness
TYPE IV HYPERSENSITIVITY
Type IV reaction is called delayed-type hypersensitivity (DTH) because its effects are felt 48-
72 hours after antigen contact (rather than within minutes to hours). In contrast to Type I, II and II
reactions which are mediated by antibodies, Type IV response is mediated by antigen-specific Th1
cells, macrophages, or by CD8 T cells.
As in other hypersensitivities, the initial response is called sensitization, and may not result in
symptoms. The initial sensitization phase, i.e. the priming of immune cells, takes 1 - 2 weeks to
develop, the same amount of time as in Type I, II and III reactions. Upon second contact with antigen
activation phase occurs.
Clinical examples of Type IV reactions include self and foreign antigens:
Foreign-antigen induced reactions:

1. Local skin reaction to poison ivy (right).
Sensitization phase: In the case of poison ivy,

during sensitization phase an allergen chemical (called
urushiol) is absorbed into the skin which then elicits a
CD8 immune response that is remembered on
subsequent allergen exposures. Urushiol is low-
molecular-weight and can easily penetrate the
keratinocytes of the superficial epidermal layers. Urushiol
is also lipid-soluble and can therefore cross the cell
84
membrane and conjugate intracellular self-proteins. This makes self-proteins look foreign. The
modified proteins are then processed by the proteosome within the cytosol, and are translocated into
the endoplasmic reticulum. The modified peptides are then presented on MHC class I molecules of
the keratinocytes and local DCs. After reaching the lymph nodes, DC present MHC I-bound peptides
to naïve CD8 cells. CD8 T cells are activated and CD8 memory cells form. Urushiol is such a strong
sensitizer that it can produce intense inflammation in weak concentrations and can cause
sensitization in 10 to 14 days after only one exposure.
Activation phase: Within 24-72 hours after re-exposure to urushiol, the allergen
penetrates the epidermis, modifies intracellular proteins and recruits CD8 T memory cells to the skin.
This causes an overt inflammatory reaction to the allergen. This is called contact dermatitis. This
dermatitis can persist for 3 to 4 weeks even after the antigen is removed and is characterized by
erythema, edema, and vesiculation resulting from destruction of epidermal cells and activation of the
dermal vasculature. Itchy rash is a common feature. Other examples of delayed type hypersensitivity
(DTH) where CD8 cells are involved include reactions to proteins in insect venom and contact
sensitivities to metals such as nickel.
The reason that in Type IV reaction, the symptoms take a 2-3 days to manifest upon re-
exposure (rather than a few minutes or hours, as in Type I, II and III reactions) is because Type IV
reaction is T cell rather than antibody mediated. Memory T cells are not present in hight numbers in
the tissues and have to be recruited from the vasculature to the skin. This recruitment and
subsequent action of memory cells on their targets takes several days.
In some cases contact hypersensitivity can cause a Th1 (rather than a CD8) response. Here a
Th1 cell may recognize macrophages presenting allergin within MHC II rather than MHC I. After
activation, these Th1 cells then secrete chemokines and cytokines such as IFNgamma which activate
macrophages. Activated macrophage secrete TNFalpha and TNFbeta which in turn upregulate
adhesion molecules on local blood vessels and causes vasodilation. Macrophages also secrete IL-3
and GM-CSF which leads to an increase of the bone marrow’s monocyte output.
2. PPD test (Tuberculosis skin test) – a test for a local skin reaction to the Mycobacterial
proteins (See figure to the right.
Sensitization to TB can occur during

prior exposure to Mycobacterium
tuberculosis. The exposure could occur
be via prior vaccination or via a natural
infection. The presentation of the
intravesicular TB antigens on MHC II
results in the production of memory Th1
cells to Mycobacterial proteins.
In a PPD test, when purified
tuberculin protein is injected into the
dermis, resident macrophages
phagocytose the particles and present
them on MHC II. These macrophages
recruit memory Th1 cells from the
nearby vasculature. The memory Th1 cells bind to tuberculin proteins presented by the macrophages
and activate macrophages to secrete more cytokines. This attracts more macrophages and
granulocytes from the blood. If a patient is positive for the TB screen test they will develop
inflammation in response to the injected antigen. The reaction can be visualized as induration and
erythema.
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3. Chronic Allograft rejection
Most graft rejection reactions are T cell-mediated responses. Transplanted organs express
donor MHC molecules, resulting in 2 pathways of antigen recognition (allorecognition) by T cells:
direct and indirect.
Direct pathway (Figure below): Allorecognition is the term used to define immunological
recognition of HLA antigens between genetically disparate individuals within the same species.
Specifically, allorecognition
refers to direct recipient T Direct pathway-After transplantation, dendritic cells (DCs) from the donor migrate
out of the graft into the recipient’s lymph nodes and present donor peptides on their
cell recognition of MHC own major histocompatibility complex (MHC) to the T-cell receptor (TCR) of the
molecules on donor cells. recipient’s T cells. This elicits an anti-donor T-cell allorecognition response that
In graft rejection, donor destroys the graft.
APC may travel out of the graft
and into the host’s regional
lymph node (See panel “a” in
the figure to the right). When
these donor’s MHCs are
recognized by the host T cells
the T cells are activated (“b”).
a. b. c. d.
These host T cells can then
migrate back to the graft,
recognize the MHC expressed
by the donor tissue and cause inflammation and rejection of the graft (“c –d”). Both CD8 and CD4 T
cells are involved, but the destruction of the graft is mediated mainly by CTLs (CD8 cells).
The mechanism behind the direct pathway is thought to involve the fact that there is no
mechanism for eliminating host T cells whose TCRs have a high affinity for allogeneic MHC
molecules. Since alloMHC is not present in a recipient’s thymus, these allo-reactive T cells are not
Direct pathway: Recognition of allogeneic major histocompatibility complex (MHC) molecules by T lymphocytes. Recognition
of allogeneic MHC molecules may be thought of as a cross-reaction in which a T cell specific for a self MHC molecule-foreign
peptide complex (A) also recognizes an allogeneic MHC molecule whose structure resembles that of a self MHC molecule-
foreign peptide complex (B and C). Peptides derived from the graft (labeled self peptide) may not contribute to allorecognition
(B), or they may form part of the complex that the T cell recognizes (C). The type of T cell recognition depicted in B and C is
direct allorecognition.
negatively selected and exit into the periphery.

The process of negative selection eliminates host T cells that strongly react to self MHC but
leaves a small percentage of T cells to recognize foreign MHC. Approximately 5% of T cells in an
individual’s peripheral pool are alloreactive. These T cells have passed the requirements of positive
and negative selections in the host, but have a TCR with specificity for allogeneic MHC.
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Indirect pathway: After transplantation, recipient DCs migrate into the graft, and take up and
process proteins from the donor cells. These donor peptides are then presented on recipient MHC to
recipient T cells in the lymph nodes, again eliciting an immune response. It is thought that the direct
pathway is responsible for acute rejection and that the indirect pathway is responsible for chronic
rejection.
Examples of autoantigen-induced reactions:
1. Type I diabetes (right)

Type I diabetes is a chronic condition in which the pancreas
produces little or no insulin, a hormone needed to allow glucose
to enter cells to produce energy. The far more common type 2
diabetes occurs when the body becomes resistant to the effects
of insulin or doesn't make enough insulin.
In Type I diabetes the destruction of beta cells in the
pancreas plays the most important mechanism. MHC class I-
dependent CD8 T cell responses are an essential component of
both the initiation and progression of pancreatic cell destruction,
ultimately leading to type 1 diabetes. Initially CD8 cells play the
most important role in pathogenesis. Later in the disease CD4 T
cells and insulin-specific autoantibody-producing B cells also
play a role.
2. Multiple sclerosis (MS) (right)

Multiple sclerosis has been defined as "multiple
white matter lesions separated in space and time". In
this disease the immune system eats away the
protective myelin sheath that covers the nerves in the
central nervous system. The activation of CD4+
autoreactive T cells and their differentiation into a Th1
phenotype are crucial events in the initial steps, and
these cells are probably also important players in the
long-term evolution of the disease. Although Th1 cells
induce the initiation of MS, damage to the myelin of the
central nervous system is most likely mediated by other
components of the immune system such as: antibodies,
complement, macrophage-secreted factors and other
factors produced by innate immune cells.
Damage to myelin in the white matter of the CNS
interferes with the communication between the brain
and the rest the body. Many patients have experience demyelination of the optic nerve which
controls vision. Inflammation of this nerve causes optic neuritis and visual defects. Damage to
spinal cord pathways (myelopathy) is yet another common presentation. This can include damage
to descending motor pathways, producing spastic paralysis or damage to sensory tracts (usually
the dorsal columns). In the latter case, paresthesias (sensation of tingling, tickling, pricking) may
be very distressing.
3. Celiac disease (bottom)

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Celiac disease is defined as a disorder in which there is an abnormality of the small intestinal
mucosa manifested by contact with the gluten of wheat and certain other cereal grains. Nutrients from
food are normally absorbed
by the villi in small intestine.
If the villi are damaged, the
person cannot absorb
nutrients properly and ends
up malnourished, no matter
how much he or she eats.
Glutens have a high
proline and glutamine
content. The high proline
content renders these
proteins resistant to
complete proteolytic
digestion by gastric enzymes
in the human intestine. This
can result in the
accumulation of relatively
large peptide fragments (as
many as 50 amino acids in
length) in the small intestine.
These peptide can penetrate
into the gut mucosa. In the
mucosa gluten peptides are
modified by tissue
transglutaminase. Certain
MHC II alleles, such as HLA-DQ2 and HLA-DQ8 expressed by APCs, can then bind and present
“gluten” peptides to populations of CD4+ T cells in the lamina propria of the small intestine. Intestinal
CD4 T cells are then activated which in turn cause inflammation of the intestine. In celiac disease, in
addition to activated T cells, antibodies to transglutaminase can also be found.
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Practice questions:
1. Fran walks outside on a beautiful day and takes a deep breath of ragweed pollen, to which she
has a strong Type I hypersensitivity. Which event below will NOT occur within 30 minutes due to
this hypersensitivity?
a. A local inflammatory response in the nose is induced, resulting in a runny or stuffy nose.
b. IgE specific for ragweed pollen is synthesized by B cells in the local lymph nodes.
c. Mast cells respond to the antigen-IgE signal by releasing preformed histamine
d. Ragweed pollen antigen binds to IgE present on mast cell FceRI in the respiratory tract.
e. Systemic effects of hypersensitivity such as anaphylactic shock may occur.
2. The principal difference between cytotoxic (type II) and immune complex (type III) hypersensitivity
is:
a. The class (isotype) of antibody

b. The site where antigen-antibody complexes are formed
c. The participation of complement
d. The participation of T cells
e. The time frame of sensitization phase
3. A patient with rheumatic fever develops a sore throat from which beta-hemolytic streptococci are
cultured. The patient is started on treatment with penicillin (first time treatment), which he
continues for the prescribed course, and the sore throat resolves within first several days.
However, 14 days after initiation of penicillin therapy the patient develops a fever of 103°F, a
generalized rash, and proteinuria. This MOST probably resulted from:
a. recurrence of the rheumatic fever.

b. a different infectious disease.
c. an IgE response to penicillin.
d. an IgG-IgM response to penicillin.
e. a delayed hypersensitivity reaction to penicillin.
4. You see a 26 year-old female in the clinic complaining of cold intolerance, weight loss, palpitations
and bulging eyes (exopthalmos). You suspect Grave’s disease. What is the mechanism behind
her symptoms?
a. The pituitary overproduces TSH due to stimulation by IgG.

b. The thyroid overproduces T3 and T4 due to stimulation by IgG
c. The pituitary overproduces TSH due to a lack of feedback inhibition by the thyroid
d. T4 is peripherally converted to more bioactive T3 by autoimmune stimulation
5. You see a patient in the ER who has been bitten by a rattlesnake. You administer murine anti-
venom antibodies intravenously. The patient is saved, but you notice a diffuse petechial rash on all
four limbs at the one-week follow-up appointment. What is the cause of this rash?
a. Micro-thromboembolus from direct Ab-mediated platelet activation

b. Cytotoxic T-cell mediated vascular damage
c. Localized swelling from mast cell degranulation
d. Accumulation of antigen-antibody complexes in the vasculature
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MERP Medical Virology
VIROLOGY MODULE
VACCINES
Learning Objectives
• List the composition of some common, routinely administered vaccine

• Understand the components of toxoid vaccines
• Understand the components of recombinant vaccines
• Understand the components of conjugate vaccines
• List advantages and disadvantages of live versus killed vaccines
A historical blurb
The first vaccine was developed in the 18th century. It was a vaccine against smallpox disease
which was caused by a virus from the Poxviridae family. In this vaccine, rather than smallpox, cowpox
virus was used as an immunizing agent (cowpox is structurally similar to smallpox,). At first lesions
from cowpox infected milk-maids or lesions from udders of cattle that had cowpox were used for
inoculation of susceptible people. Eventually the live virus was purified and used directly instead of
lesion fluid. Smallpox vaccine is an example of a live vaccine. Prior to the discovery that cowpox
protects against human smallpox, a more dangerous procedure was used. Lesions from smallpox-
infected people were scratched and directly inoculated into non-infected people. This procedure
resulted in some recipients getting very sick. Today vaccines exist in many other forms, in addition to
live vaccines.
Common types of vaccines:

Most bacterial and viral vaccines are administered by injection.
Viral vaccines
Killed (also called inactivated) vaccine is composed of viral particles that have been treated
with chemicals, heat, or irradiation such that they are no longer alive (ex. Influenza virus
vaccine, rabies virus vaccine).
Live (also called attenuated) vaccines contain live virus with a reduced ability to grow in
humans. These are more potent than killed vaccines because they more closely mimic a real
infection. Most viral vaccine are live (ex. vaccine against measles, mumps, rubella).
Subunit (also called recombinant) vaccines are vaccines that use only a particular
component of the virus rather than an entire virus. Usually these components are given as
proteins, which were translated in bacteria or yeast (ex. Hepatitis B, HPV vaccine).
Bacterial vaccines
Live-attenuated vaccines- There is a small number of whole cell live-attenuated bacterial
vaccines: ex. Salmonella, Mycobacterium tuberculosis.
Subunit vaccines-
Toxoid vaccines - Since many bacterial diseases result from toxic proteins secreted by
bacteria, rather than the bacterium itself, a purified inactivated version of a particular
toxin, called toxoid, can be used as a vaccine against such bacteria. These vaccines
are made by purifying a toxin and inactivating it with a chemical called formalin
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(example of toxoid vaccines are diphtheria toxoid against Corynebacterium diptheriae

and tetanus toxoid against Clostridium tetani).
Capsular conjugated and unconjugated vaccines - Many bacteria have very pathogenic
capsules (a polysaccharide structure covering the surface of bacteria). This structure is
not immunogenic in that it cannot fix complement very well, nor stimulate other
components of the innate system, but it can elicit an adaptive immune response.
Therefore subunit vaccines have been developed where injected purified capsule
stimulates and antibody response. Often these vaccine are administered as conjugate
vaccines where the capsule is conjugated to a carrier protein to make the capsule more
immunogenic (more on this later).
Adjuvants
To be effective a vaccine must create a state of inflammation. Therefore helper substances that
induce inflammation are used in conjunction with a vaccine. These substances are called adjuvants.
An example of an adjuvant is alum-a form of aluminum hydroxide.
Advantages and disadvantages of live and killed vaccines

Live- attenuated vaccines can, in some instances, revert to becoming pathogenic. This
happens rarely, and if the vaccine is thought to provide much more benefit than harm, it is still
recommended.
In general, live-attenuated vaccines have some advantages over inactivated vaccines: they
are more effective, provide life-long immunity and do not need repeated boosters.
On the other hand the advantages of inactivated vaccines include their stability and ease of
transportation, as well the lack of a risk associated with vaccine-related illness. However these
vaccines are not as potent as live vaccines. When a person is vaccinated by either of the above
mentioned vaccines, he develops active immunity.
Passive immunization
Passive immunization is used when exposure to pathogen has already occurred and there is
not enough time to induce active immunity, or prophylactically in children with immune deficiencies.
Passive immunization involves administration of specific antibody which has been produced in a
human or animal in response to vaccination or environmental exposure to the pathogen. Examples
include antivenin for snake bite, Rhogam (human anti-Rh) to block formation of IgG anti-Rh
antibodies in an Rh-negative pregnant mother, and human gamma globulin given to children who
have immune deficiencies. Passive immunization can confer fast but short-term protection. When the
antibodies come from another species, serum sickness can develop.
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Administration of vaccines
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LECTURES 9 and 10: INTRODUCTION TO VIROLOGY
Learning Objectives
o Understand the basics of viral life cycle

o Describe the main differences between non‐enveloped and enveloped viruses
o Describe the differences between RNA and DNA viruses
o Describe in detail the steps in the replication of the RNA viruses
o Describe in detail the steps in the replication of the DNA viruses
Although historically viruses were grouped according to the disease symptoms they produced and the
range of host species they infected, today classification is based largely on architecture of the virus
particle (the virion) and the nature of the genetic material. Further distinctions can then be made on
the basis of biological properties and antigenic relationships.
Below you will find some general features of all viruses:
• Filterable agents
• Obligate intracellular parasites
• Outside the cell, they consist of particles called virions
• Viruses use the energy and the protein synthesis machinery of the host
• The protein outer shell of a vision is called a capsid, a capsid is always encoded by the viral
genome. Sometimes on the outside of the capsid the virion may contain other ingredients (e.g.,
lipids, carbohydrates), but these are derived from the host cells.
• A virion’s genomic material may be DNA or RNA genomes, but not both.
• Viruses have genes that that encode proteins needed for viral genome replication which host
cells can’t provide. Viruses also encode proteins that make up their capsid.
• Viruses may contain lipids and carbohydrates but these are derived from the host cell.
• Viruses assemble their components (genomes and capsids) before they exit the cell.
• The overall processes of the viral replication cycle is referred to as “assembly”.
• Viral infection can be lytic (cause cell death), persistent (no cell death) or transforming
(immortalization of a cell).
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Viral life cycle (see figure right)

1. Adsorption (Attachment)
Viruses can only enter cells that express viral receptors
and are able to replicate in cells with appropriate
biosynthetic machinery. Below are some examples of
viral attachment proteins and their respective receptors
on host cells.
2. Penetration
Viral entry depends on the nature of the virus (enveloped versus naked). Following attachment, viruses may enter the
host cell through receptor mediated endocytosis (naked and enveloped viruses are capable of this) or by receptor
mediated fusion (only some enveloped viruses are capable of doing this).
3. Uncoating
Uncoating is a process where viral capsid is degraded by viral enzymes or host enzymes. This can occur in the vesicle
after receptor mediated endocytosis or in the cytoplasm after receptor mediated fusion.
4-5. Early transcription/translation
In this step, host (or rarely viral) DNA dependent RNA polymerase transcribes viral genes that are responsible for viral
replication and genes that encode proteins that serve as transcription factors for late genes. These are than translated by
host’s translation machinery.
6. Replication
In the case of DNA viruses, viral genome is replicated either by host or viral DNA dependent DNA polymerase. In the case
of RNA viruses the viral genome is always replicated by viral RNA dependent RNA polymerase.
7-8. Late transcription/translation
Late transcription and translation usually applies to DNA viruses. In this step, host DNA dependent RNA polymerase
transcribes late viral genes that code for structural proteins and then the host translation machinery translates this late
mRNA.
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9. Assembly
Viral genomes are packaged with viral capsids and if the virus is enveloped, the nucleocapsid is delivered to the plasma
membrane.
10. Release
Viruses may escape from the host cell by causing cell rupture (lysis). Most naked viruses cause cell lysis as they exit.
Enveloped viruses typically "bud" from the host cell. During the budding process, a virus acquires the phospholipid
envelope containing the embedded viral glycoproteins. This enveloped can be derived from one of several membrane
bound compartment within a cell. Depending on the efficiency of viral replication, budding may or may not result in cell
lysis. Some enveloped viruses can move between cells via syncytia formation, during which the infected cell fuses with a
non-infected cell to form a multinucleated giant cell. For those viruses capable of forming syncytia, CD8 response is
critical to control viral infection
Enveloped versus non-enveloped viruses

Viral properties (such as stability) in the external environment, as well as its ability to enter and exit
the cell, depend on its external structure.
Non-enveloped/Naked viruses
There are several characteristics of naked viruses that are important for their pathology:
Basic structure of naked

• In naked viruses the outer
most layer is made of a
protein capsid
• The capsid consists of 12
vertices, 20 triangular faces, and 30 edges. The capsid is usually made of many different
proteins assembled symmetrically into an icosahedral shape.
• Capsids are usually environmentally stable and resist changes in
temperature, resist acidic conditions, detergents and drying.
• Naked viruses often survive in poorly treated water.
• Naked viruses survive the adverse acidic conditions in the gut.
• Naked viruses are usually released by cell lysis.
• Naked viruses are easily spread on fomites, hand-to-hand touch,
dust and small droplets.
• VAPs are part of the capsid
• Antibody may be sufficient for immunoprotection against naked
viruses.
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Enveloped viruses
There are several characteristics of enveloped viruses that are important for their pathology:
Basic structure of enveloped viruses
• The outer most layer of these viruses is made up of a lipid bilayer membrane embedded with
glycoproteins.
• Enveloped viruses are environmentally labile/unstable and are disrupted by heat, acid,
detergents and drying.
• Enveloped viruses must stay moist to retain infectivity.
• Enveloped viruses are spread in large droplets such as bodily secretions, organ
transplantations and blood.
• Enveloped viruses can be released by budding, exocytosis or cell lysis.
• Enveloped viruses modify host cell membrane during replication.
• Enveloped viruses do not need to kill the cell to spread; can spread from cell- to-cell via
syncytia.
Syncytia formation by enveloped viruses
• Enveloped viruses may need Ab and cell mediated response for control and protection.
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Viral replication steps 2 and 3 depend on whether the virus is enveloped or naked (Figure below)
2. Penetration
Viral entry depends on the nature of the virus (enveloped
versus naked). Following attachment, viruses may enter
the host cell through receptor mediated endocytosis
(naked and enveloped viruses are capable of this) or by
receptor mediated fusion (only some enveloped viruses
are capable of doing this).
Receptor mediated endocytosis
Receptor mediated fusion
3. Uncoating
Uncoating is a process where viral capsid is degraded by viral enzymes or host enzymes. This can happen inside
the endocytic vesicles or inside the cytoplasm depending on the virus.
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Viral replication steps 4 through 8 depend on whether the virus is DNA or RNA (Figure below)
4-5. Early transcription/translation
In this step, host (or sometimes viral) DNA dependent RNA
polymerase transcribes viral genes that code for proteins
responsible for viral replication and genes that encode
proteins that serve as transcription factors for late genes. The
mRNAs for these genes are translated by host’s translation
machinery.
6. Replication
In the case of DNA viruses, viral genome is replicated either
by host or viral DNA dependent DNA polymerase. In the
case of RNA viruses the viral genome is always replicated by
viral RNA dependent RNA polymerase.
7-8. Late transcription/translation

Late transcription and translation usually applies to DNA
viruses. For most DNA viruses, host DNA dependent RNA polymerase transcribes late viral genes that code for
structural proteins and then the host translation machinery translates this late mRNA. Note that RNA viruses
rarely have early and late transcription but rather carry out transcription and/or translation in one step.
DNA versus RNA viruses
Properties of DNA viruses

DNA virus
• DNA molecule is stable
• Because it is stable, DNA genome can
remain in the infected cell for a long time
• Many DNA viruses establish a persistent
infection.
• DNA genome resides in nucleus (except
for POX which resides in the cytoplasm).
• Viral genes interact with host proteins to
get help with transcription and replication
(except POX which carries out its own
transcription).
• For DNA viruses, viral gene transcription is temporally regulated (early genes code for
replication machinery, late genes code for structural proteins).
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• Larger DNA viruses can promote their own replication via a DNA dependent DNA polymerase
that these viruses encode.
• In order to best utilize host machinery, all DNA viruses must have a way to make the target cell
divide, or they must infect a previously dividing cell.
Properties of RNA viruses

Positive RNA virus
• Unlike DNA, RNA molecule is
transient and labile.
• Most RNA viruses replicate in
cytoplasm (except Orthomyxo,
which replicated in the nucleus)
• Cells cannot replicate RNA. RNA
viruses must encode RNA d.
RNA pol which has low fidelity so
RNA viruses are prone to
mutation.
• The genome structure and
polarity determines how viral
mRNA is generated and proteins
are processed.
• RNA viruses can be negative, positive or double stranded depending on the structure of their
RNA genome.
• All (-) RNA viruses are enveloped.
• All (-) RNA viruses and dsRNA viruses must carry RNA dep. RNA polymerase in the virion.
• Retroviruses are unique. These viruses have a positive sense RNA genome but they must be
converted into a dsDNA by the viral encoded and carried enzyme Reverse Transcriptase
(RNA dependent DNA polymerase) prior to integration into the host cell.
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Negative RNA virus replication
Viral replication steps 9 (Assembly) depends on both the nature of the viral coat structure (i.e., naked
or enveloped) and on whether it’s DNA or RNA whereas step 10 (Release) depend on whether the
virus is naked or enveloped (figure right)
9. Assembly
Viral genomes are packaged with the viral capsids and

if the virus is enveloped, the nucleocapsid is then
delivered to the plasma membrane or ER to acquire
the envelope.
10. Release
Viruses may escape from the host cell by causing cell
rupture (lysis). Most naked viruses cause cell lysis as
they exit. Enveloped viruses typically "bud" from the
host cell. During the budding process, a virus acquires
the phospholipid envelope containing the embedded
viral glycoproteins. Depending on efficiency of viral
replication, budding may or may not result in cell lysis.
Note that some viruses exit via exocytosis instead of
budding, which means that they acquire their
membrane in an internal organelle, such as ER, and
then are released out of the cell via a vesicular pathway. Some enveloped viruses can move between cells via
syncytia formation, during which the infected cell fuses with a non-infected cell to form a multinucleated giant cell.
For those viruses capable of forming syncytia, CD8 response is critical to control viral infection. Note that a
particular virus may exit one cell by lysis and another cell through budding or another mechanism.
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Figure: Budding of an enveloped

virus
General clinical progression of viral infections
Many viruses initiate infection in the oral mucosa or upper respiratory tract. Disease signs may
accompany viral replication at the primary site. The virus may replicate and remain at the primary site,
or it may disseminate to other tissues via the bloodstream or via the mononuclear phagocytes and
lymphatic system. Alternatively, some viruses spread via neurons. The transport of virus in the blood
is termed viremia. This primary viremia may not result in a large number of virions. However, in the
blood, viruses taken up by the phagocytic macrophages may be delivered to other tissues.
Replication of a virus in these other tissues, such as the endothelial lining of blood vessels, or the
liver can cause the infection to be amplified and initiate the development of a secondary viremia
which can then cause sufficient virions to be released to infect more target tissues such as skin, brain
etc.
The outcomes of viral infections are shown

in the figure to the right. Viral infection of a target
organ may cause lysis of the target cells within the
organ. Alternatively, some viruses establish
persistent infections in which the infected cell is
not killed and the viral genome remains within the
cell. These persistent infections can be divided into
chronic and latent types. For a chronic infection
to happen, the virus must be released from the cell Outcomes of viral infections
gently through exocytosis or through budding from
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the plasma membrane. If the infected cell is killed by the virus, CTL or ADCC chronic infection of that
cell will end.
Certain DNA viruses can cause latent infections of a cell. This occurs if the infected cell
lacks the machinery for transcribing all the viral genes. The factors required by such a virus to make
progeny may be expressed only at certain times such as during cell activation. For example, HSV
establishes a latent infection in neurons that lack the nuclear factors required to transcribe the early
viral genes, but stress and other stimuli can activate the cells to allow viral replication. This then
allows HSV to come out of its latent stage and produce progeny.
Below is a map of viral families and their characteristics.
Practice questions:
1. A patient is sick with severe diarrhea. The examination of a stool sample reveals an icosahedral-shaped virus with
long fibers protruding from the virus. What else is a likely characteristic of this virus?
a. It is naked
b. It is enveloped
c. It is an RNA virus
d. It is a DNA virus
e. It is a retrovirus
2. All of the following describe killed vaccines except:
a. Very stable under different environmental conditions

b. May be composed of dead virus or viral particles
c. May be used to protect against either viral or bacterial infections
d. Less commonly used as viral vaccines of choice
e. More potent than live vaccine because they mimic infection
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LECTURE 11: POXVIRIDAE and PARVOVIRIDAE
Learning Objectives
• Describe the family of Poxviridae
• Characterize the diseases caused by these viruses
• Describe Parvovirus B19
• Characterize the diseases caused by these viruses
POXVIRIDAE
Unique properties of Poxviruses:
• The largest, most complex viruses
• Brick-shaped
• The receptor for pox virus is
a glycosaminoglycan
expressed in the host
membrane
• The only DNA virus that
replicates in cytoplasm
• Because it replicates in the
cytoplasm it encodes and carries all proteins needed for mRNA synthesis
• Assembles in cytoplasmic Guarnieri bodies (inclusion bodies) where it acquires membranes
• Encodes soluble interferon, IL-1beta, and TNF-alpha receptor homologues that block the host
cytokines
• Virus stimulates cell growth by encoding a protein similar in sequence to a growth factor called
EGF. This protein is secreted and binds to growth factor receptors on infected cells.
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Replication of Poxviruses (Figure left):
In humans, the most deadly Pox virus was smallpox

which caused a severe disease but has now been
eliminated by intensive international vaccination.
Current interest in poxviruses centers on their
possible use as vaccine vectors.
Human diseases
1. Smallpox
This is the only disease that has been globally
eradicated.
Transmission of smallpox is through close contact
with infected persons, usually via infection with a
droplet from the pharyngeal secretions. As little as
100 particles can cause infection. The ulcers in pharynx shed the virus. Incubation period for
smallpox is 10 - 12 days. The virus multiples in the respiratory tract, then in lymphatics, which brings
the virus to the bloodstream. Circulation carries the virus to the skin and respiratory tract.
Major illness is the most common form of small-

pox. It is characterized by abrupt onset of fever
and prostration followed by a macular rash
(head, limbs, hands and feet rather than trunk,
including palms and soles) on the third day of
fever.
Rash progresses from papules to vesicles
which become pustular, ulcerated,
scabbed, and eventually healed but with
scarring ("pock marked face"). Often skin
pustules become contaminated with
bacteria that normally reside in the skin.
Vesicles are also found inside the mouth.
An infected person needs cell-mediated
and humoral response for resolution. Viral
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replication results in cell lysis. Smallpox has 15 to 40% mortality rate (Figure on the left illustrates
smallpox pathogenesis).
As result of an unprecedented international WHO campaign of 'seek and contain' by
vaccination, the natural case of smallpox was diagnosed in 1977. Vaccinia is a viral strain which has
been used for immunization against smallpox. It has evolved from cowpox. In man, it caused a
localized pustule with scar formation. In immunocompromised persons or eczematous persons it
sometimes caused a severe generalized vaccinia infection. Routine immunization of all children in
USA stopped in about mid 1980's.
2. Cowpox is acquired by humans, usually through milking cows; it then manifests as ulcerative
lesions (sometimes called "milker’s nodules") on the hands of dairy workers. It is a local infection, not
nearly as morbid as smallpox. Because of antigenic similarity between cowpox and smallpox, people
who recover from a cowpox infection are protected against smallpox.
3. Molluscum contagiosum (image on the right) is a minor infection that

presents with infectious warty papules on the skin. These papules have a
central umbilication and are found in a cluster of 5 - 20. They become softer
with time and sometimes contain white waxy material. Disease is
transferred by direct contact with infected human and as a venereal
disease. Contact sports are associated with disease transmission. The lesions are on the epidermal
layer of the skin and are not found on palms and soles. Diagnosis is usually based on clinical
findings.
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PARVOVIRIDAE B19
Unique properties of Parvoviruses
• Parvoviruses are among the smallest, simplest eukaryotic viruses

• Particles are naked, icosahedral, 18-26 nm in diameter and consist only of protein (50%) + DNA
(50%).
• They have a ssDNA genome that encodes two genes (a gene for capsid and a gene for a
transcription factor)
• The virus uses host’s DNA d. DNA pol for replicating its genome
• The capsid resists inactivation
• It can cross the placenta
• It infects mitotically active erythroid precursors in the bone marrow and establishes a lytic infection
• Replication occurs in the nucleus
• All parvoviruses are highly dependent on cellular functions for genome replication
Replication of Parvoviruses
Human diseases:
• All are caused by one serotype (B19) but infection

can result in different disease manifestation.
• B19 is spread by respiratory route and from
mother to fetus.
• It replicates in mitotically RBC precursors.
• Major sites for replication include: bone

marrow and fetal liver.
Individuals at risk:
1. Children:
Children with B19 present with erythema
infectiosum (also called slap cheek and fifth
Postulated replication of parvovirus (B19) based on
information from related viruses. The internalized
disease) which is a mild systemic rash illness.
parvovirus delivers its genome to the nucleus, where the
sinagle-stranded (plus or minus) DNA is converted to
2. Parents of kids with B19 who have not been
double-stranded DNA by host factors and DNA
exposed in childhood can present with a rash and
polymerases present only in growing cells. Transcription,
replication, and assembly occur in the nucleus. Virus is arthritis.
released by cell lysis.
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For both children and adults, the most common disease caused by B19 is erythema
infectiosum, sometimes called "fifth disease" from a historical enumeration of the rashes or slap
cheek. B19 is spread by the respiratory route, and the rash appears after about 17 days.
B19 causes a biphasic disease (see figure below, left):

Phase 1: Infectious phase is due to influenza-like symptoms which are caused by viremia
Phase 2. Noninfectious phase is due to circulating immune complexes of Ab bound to virions
which results in erythematous rash, and arthritis.
• The distinct feature of the rash is the red cheeks, with circumoral pallor.
• On the rest of the body it is a lacy, pink macular rash that fades quickly, but may reappear after a
warm bath.
• Adults with fifth disease may also have a rash which is often accompanied by painful joint pain
usually in the hands, feet, or knees. Some adults will have only joint pain but no other symptoms.
The joint pain usually lasts 1 to 3 weeks, but it can last for months or longer.
Slap cheek
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B19 serology, clinical features and hematological changes
Mechanism of spread of parvovirus

within the body.
3. People with chronic anemia will develop a severe reduction of RBCs, a drop in hemoglobin (Hb),
and will often require a blood transfusion. A severe reduction or blockage in RBC production in the
bone marrow is called aplastic crisis. The bone marrow in these patients is hypocellular. The shut-
down in red cell production is temporarily until the virus is eliminated by the immune system, usually
within 10 days. However in people with anemia symptoms may be more severe and include malaise,
high fever and joint pain.
4. Pregnant women - transmission of B19 virus from mom to fetus, may cause
hydrops fetalis (congestive heart failure and edema, right figure) due to rapid
death of fetal RBC precursors.
• A maternal infection prior to pregnancy means maternal acquired IgGs will

cross the placenta and protect the fetus from congenital defects.
• In the case of seronegative mothers, 10% of primary maternal infections
will result in fetal loss.
• Hydrops fetalis stems from fetal anemia. Less oxygen-carrying RBCs
means less oxygen arrives to the tissues so the heart needs to pump a much greater volume
of blood to deliver the same amount of oxygen. Hydrops causes an abnormal accumulation of
fluid in 2 or more fetal compartments, including liver and/or heart. This can cause heart failure.
Attempt to increase RBC production in fetal liver and spleen increases these organs.
Antibody to B19 is important for resolution and prophylaxis. There is no vaccine for this
infection. Laboratory diagnosis of acute B19 is also based on the presence of IgM antibodies.
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LECTURE 12: PAPILLOMAVIRIDAE, POLYOMAVIRIDAE and ADENOVIRIDAE
Learning Objective
• List the features of Papilloma and Polyomaviridae
• Describe the clinical presentation of various HPV serotypes
• Describe prevention methods for HPV 6, 11, 16, 18
• Describe the clinical presentation of Polyomaviridae
• Describe the clinical presentation of Adenoviridae
PAPILLOMAVIRIDAE AND POLYOMAVIRIDAE
• Both of these families used to belong to the same

family, called Papovaviridae.
• Now they are split into individual families:
PAPILLOMAVIRIDAE and POLYOMAVIRIDAE.
• Since these viruses are very similar, we will
characterize the properties unique to them
collectively and then describe the disease caused by
each family separately.
Human papovaviridae and their disease
Virus Disease
Papillomavirus Warts
Polyomavirus
BK virus Renal disease*
JC virus Progressive
multifocal
leukoencephalopathy *
*Disease in immunosuppressed patients
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Unique properties of Papilloma and Polyoma:
• The virus has a naked icosahedral capsid

• Human papilloma viruses (HPV) have more
than 100 serotypes
• Polyoma viruses have two main serotypes:
JC and BK
• These viruses go through early and late
transcription process.
• They can cause lytic or latent infections in
humans depending on what cell is infected. Papilloma can also immortalize certain human cells.
• These viruses promote cell division by binding and inactivating host cell factors that control cell
growth and cell death, specifically through inactivation of cellular pRB and E2F proteins.
o Normally, host pRB protein restricts the cell's ability to replicate DNA by preventing its
progression from the G1 to S phase of the cell division cycle. It does so by inhibiting
E2F family of transcription factors which induce S phase.
o Normally, host P53 plays a role in apoptosis and cell cycle arrest.
o Papilloma’s E6 binds and inactivates p53 while E7 binds and inactivates pRB.
o Polyoma T antigen inactivates both p53 and pRB.
• Less prevalent human polyoma viruses include the KI, WU, and the Merkel cell carcinoma
polyomaviruses. Only recently has a polyomavirus been
associated with human cancers.
Papilloma viruses:
HPV infects basal layer of epidermis via micro-
abrasion. HPV may be acquired by close contact, sexual
contact, and passage through infected birth canal. Viral
specific tissue tropism depends on viral serotype.
The various viral replication stages depend on
epithelial cell growth and differentiation stage. Papilloma Development of papilloma (wart). Human
papillomavirus infection promotes the outgrowth of the
viruses are persistent in basal layer keratinocytes that are basal layer, increasing the number of prickle cells of the
stratum spinosum (acanthosis). These changes cause
initially infected but cause active, persistent infection in the skin to thicken and promote the production of keratin
(hyperkeratosis), thereby causing epithelial spikes to
differentiated keratinocytes. At least 40 identified HPV types form (papillomatosis). Virus is produced in the granular
cells close to the final keratin layer.
infect the genital tract. Studies found that if a college woman
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has at least one different partner per year for four years, the probability that she will leave college with
an HPV infection is greater than 85%
Mechanisms of
spread of
papillomaviruses
within the body.
Human Papillomaviridae diseases
1. Warts (Image, right)

Most types of HPV cause benign outgrowth called warts.
Warts are usually on hands and feet, may have a long incubation
period of 3-4 months. The shape of these warts is dome-shaped or
flat. They often regress spontaneously or may be removed.
2. Benign head and neck tumors
These tumors are epithelial cell tumors of the oral cavity and tumors of the larynx which may
extend into trachea and the bronchi. Although they are benign, they could be a problem in children
due to obstruction of the airways. They are causes by HPV6 and
HPV11. Each year, about 300 infants are born with the virus on their
vocal cords because of maternal transmission (there is a 1-3% chance
that an infected mom will pass HPV to baby). See Figure on the right.
3. Anogenital warts are genital warts called condyloma acuminatum.
They occur on the squamous epithelium of perianal
area and the genitals. These are usually caused by HPV6
and 11 which are not associated with malignancy in healthy
people. Sometimes HPV is hidden from the immune
response which results in a persistent infection.
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4. Cervical carcinoma
Cervical carcinoma begins as a genital tract infection (a common STD) and is usually due to
HPV-16 and HPV-18. In most cases these infections will resolve on their own without causing cancer.
In rare cases, infection does lead to cancer (usually takes 15 years from original infection). In the
cells that became cancerous, HPV DNA is found integrated into the host DNA. HPV virus is the most
common cause of cervical cancer and is the most common infection of the reproductive tract. It is
also the most common sexually transmitted infection (STI) in US.
Cervical cancer is thought to develop in stages from
neoplasia to severe dysplasia and cervical carcinoma.
Signs of abnormality include an increased number of
koilocytes which are squamous epithelial cells with
enlarged nucleus and perinuclear cytoplasmic
vacuolization. Subsequent stages of cervical cancers
are also identified by cytological changes. It is more
common in women than men, and is usually
asymptomatic in both. When symptoms are seen
women experience irregular, intermenstrual (between
periods) or abnormal vaginal bleeding, pelvic pain,
fatigue, weight loss, loss of appetite, as well as vaginal
discomfort or odorous discharge.
Progression of human papillomavirus
(HPV)-mediated cervical carcinoma. a.
HPV infects and replicates in the basal
epithelial cells of the cervix, maturing and
releasing virus as the epithelial cells
progress through terminal differentiation.
Growth stimulation of the basal cells may
or may not produce a wart. b. In some
cells, the circular genome integrates into
host chromosomes, inactivating the E2
gene. c. Expression of the other genes
without virus production stimulates growth
of the cells and possible progression to
neoplasia.
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Clinical Syndromes Associated with Papillomaviruses
Outcomes of genital infection with HPV. About 70% of cervical cancers are
caused by HPV infections with two HPV types, 16 and 18. Although most
cervical cancers are caused by HPV 16 and 18, infection with these
serotypes most often leads to resolution.
Prevention:
Vaccine: A recombinant vaccine (Gardasil) is available for serotypes 6, 11, 16, 18.
In 2015 Gardasil 9 was introduced. Covering nine HPV types, five more HPV types than Gardasil,
Gardasil 9 has the potential to prevent approximately 90 percent of cervical, vulvar, vaginal and anal
cancers. It is approved for the prevention of cervical, vulvar, vaginal and anal cancers caused by HPV
types 16, 18, 31, 33, 45, 52 and 58, and for the prevention of genital warts caused by HPV types 6 or
11. Gardasil 9 adds protection against five additional HPV types—31, 33, 45, 52 and 58— which
cause approximately 20 percent of cervical cancers and are not covered by previously FDA-approved
HPV vaccines.
Diagnosis:
Warts can be diagnosed microscopically based on histological appearance. Hyperplasia of the prickle
layer of the skin and excess keratin production is visible. For specificity, PCR is the method of choice.
Tissue specimen such as cervical swab (PAP smear) can be used for PCR.
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Polyoma viruses:
Two main types of Polyomaviruses exist:
BK and JC, both of which enter through
inhalation of infectious particles via the
Mechanisms
respiratory tract. They are then spread by of spread of
polyoma-
viremia to other organs, predominantly the viruses within
the body. CNS,
kidneys. While both can establish a latent infection Central
nervous
in the kidney they are normally blocked from system; PML,
progressive
replication by the immune system of multifocal
leukoencephal
immunocompetent hosts. The infections with BK opathy.
and JC viruses in immunocompetent individuals

are asymptomatic. However in
immunocompromised people, these viruses can
cause severe disease.
Human diseases
BK virus-
• This infection is usually asymptomatic, but with impairment of the cellular immune system the
virus can reactivate and lead to tissue damage.
• In recipients of bone marrow and solid organ transplants, the reactivation can be associated with
disease in urinary tract and kidneys. The disease include: hemorrhagic cystitis, nephropathy
and ureteral stenosis.
• BKV was first discovered in 1971 from the urine of a kidney transplant recipient who had
developed ureteral stenosis (narrowing of the ureteral lumen) 4 months after transplantation.
• Specific identification of the virus is now routinely done in organ transplant recipients using PCR
analysis or immunofluorescence of BK in the urine.
• Biopsy of the kidney can also indirectly reveal the presence of BK virus.
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JC virus
This virus causes progressive multifocal leukoencephalopathy (PML) which is a subacute

demyelinating disease in people with severe immunosuppression (often seen in AIDS patients). To do
so, JC crosses the blood-brain barrier and replicates in the endothelial layer of the capillaries. In the
brain lytic infection of oligodendrocytes causes demyelination. This causes multiple neurological
symptoms such impaired coordination and paralysis.
PML is diagnosed by histological examination of the brain tissue in biopsy.

• One can see areas of demyelination surrounded by oligodendrocytes and lesions in the white
matter (See Figure on the bottom left).
• To detect the virus in brain tissue, PCR can also be used.
astrocyte
ADENOVIRIDAE
Unique Properties
• More than 55 serotypes exist
• Structure consists of a naked icosahedral capsid with a
linear, non-segmented, dsDNA genome
• The capsomers are composed of hexons and penton fibers.
Penton fibers are found at each of the vertices of the
icosahedron
• Human adenoviruses are grouped A through G by DNA homologies and by serotype (more than
55 human types) according to various properties including the oncogenicity of viruses in newborn
rodents.
• Serotype is mainly a result of differences in the penton fibers which determine the nature of tissue
tropism and disease.
• Spread by aerosol, close contact, fecal-oral, finger-to-eye, inadequately chlorinated swimming
pools, fomites.
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• Infects mainly mucoepithelial cells in the respiratory tract, GI tract, conjunctiva, and cornea
• Usually causes direct cell damage by lysis.
• Can be persistent in some tissues such lymphoid tissues (tonsils, adenoids, Peyer’s patches).
• In order to induce cells division, E1A binds to and inactivates a cellular protein, pRB, the product
of the retinoblastoma gene. Remember that pRB prevents excessive cell growth by inhibiting cell
cycle progression until a cell is ready to divide.
• E1B binds to and inactivates p53. Remember that p53 is another tumor suppressor protein which
initiate apoptosis and inhibits the cell cycle.
• Antibody is important for prophylaxis and resolution.
Human diseases
Time course of adenovirus respiratory infection
Mechanism
of
adenovirus
spread
within the
body. Rare
Adenovirus infections are very common and most are asymptomatic. Most people have been
infected with at least 1 type by the age of 15. Virus can be isolated from the tonsils and adenoids that
have been surgically removed, indicating latent infections.
There are many different serotypes that cause various clinical diseases. Collectively these
viruses can infect and replicate in epithelial cells of the respiratory tract, GI (because they are
resistant to acid), eyes, liver. Some persist for years in adenoids and are shed after initial infection.
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Examples of diseases include:

1. Acute febrile pharyngitis is a disease characterized
by cough, nasal congestion, sore throat, fever and
headache. It is usually in infants and children and is
a common respiratory disease.
2. Pharyngoconjunctival fever is a disease similar to
acute febrile pharyngitis but also conjunctivitis is
present. It frequently presents as outbreaks in
swimming pools in summer camps.
3. Acute respiratory disease occurs in military recruits in epidemic form. People with this disease get
fever, fatigue, cough and pharyngitis. (Link to article: http://www.cnn.com/2007/HEALTH/conditions/12/19/killer.cold/index.html)
4. Pneumonia is often a complication of acute respiratory disease, in children 10% of pneumonias
are due to Adenovirus.
5. Eye infections- These could present as serious infections such as epidemic keratoconjunctivitis.
Both palpebral and bulbar conjunctivas (membranes that cover inner layer of eyelid and outer
layer of the eye) are inflamed. It is very contagious and is characterized by keratitis (inflammation
of cornea that leaves round subepithelial opacities in the cornea for up to 2 years).
6. Gastroenteritis and diarrhea- two serotypes account for 5-15% of cases of viral gastroenteritis in
kids. The diarrhea caused by Adenovirus often persists longer than diarrhea caused by other
viruses. It is usually watery, non-bloody diarrhea and lasts 3 -10 days. Diarrhea may be
accompanied by vomiting, abdominal pain and fever. Although many viral subtypes can replicate
in intestinal cells and are present in stools, most are not associated with disease.
Diagnosis:
These are usually diagnosed clinically but virus can be isolated from stool or throat swab and
grown on a variety of epithelial cells. One will see characteristic cytopathic effects: rounding and
clustering of swollen cells. Adenovirus also causes characteristic intranuclear inclusion bodies.
Clinical sample can also be used directly and assayed by immunofluorescence, and hemagglutination
tests. PCR and electron microscopy are esp. useful for diagnosing GI infections.
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LECTURE 13: HERPESVIRIDAE
Learning Objectives
Describe clinically and structurally the family of Herpesviridae. List the features that are common to all
Herpesviridae. Differentiate clinically among the different disease caused by Herpesviridae.
• Describe HSV disease/clinical presentation

• Describe where HSV causes latency/recurrence
• Describe the effect of HSV on the central nervous system
• Describe the role of HSV in neonatal disease
• Describe Tzanck smear and inclusion bodies
• Describe VZV ‐ disease/clinical presentation (specifics about the rash and how it differs
from other rashes)
• Know where VZV causes latency/recurrence (chicken pox versus shingles)
• Describe VZV diagnosis and prevention
• Describe the clinical features of diseases caused by EBV
• Know where EBV causes latency and recurrence
• List the target cells for EBV
• Describe the importance of T cells in control of all Herpes (including EBV)
• Describe the link between EBV and Burkett’s lymphoma
• Describe EBV diagnosis (detail the importance and time course of heterophile antibodies,
atypical lymphocytes, EBV specific antibodies)
• Describe CMV disease mechanism and list populations at risk
• Describe the mechanism of CMV spread
• Be able to describe the characteristic histology of CMV
• HHV6 (roseola)‐ describe the rash and differentiate between this rash and all the other
childhood rashes
Unique Properties of Herpes viruses:

HSV, VZV, EBV, CMV, HHV-6
• Herpesviruses are large enveloped
viruses with icosahedral capsids and
double-stranded DNA genomes.
• Herpesviruses encode many enzymes,
including their own DNA d. DNA
polymerase, that promote viral DNA
replication and are good targets for
antiviral drugs.
• DNA replication and capsid assembly occurs in the nucleus.
• Virus is released by exocytosis which may lead to cell lysis. The virus may also spread from cell
to cell via syncytia.
• While Herpes viruses cause lysis of certain cells, Herpes family will cause persistent infections in
which certain cells harbor the virus long term.
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• Herpesviruses can also cause immortalizing infections (ex. Epstein-Barr virus).

• Herpesviruses are ubiquitous.
• Cell-mediated immunity is required for control.
Herpes replication cycle:
Receptor mediate fusion of Herpes virus

and cell membrane
• Attachment of the viral

attachment proteins to host
receptors mediates fusion virus
into the host cell (steps 1- 4 in
Figure above and Figure to the
right). Fusion with the plasma
membrane releases the core and
the tegument proteins into the
host cytoplasm (steps 4, 5,
right).
• The capsid is transported to the
nuclear pore where viral DNA is
released into the nucleus (steps
6, 7).
• Host RNA polymerase II
mediates transcription of the
immediate early and early viral
mRNA which is translated in the
cytoplasm (9-12). These encode
proteins involved in replication
of the viral DNA.
• Genome replication occurs by
bidirectional replication (13).
• Next the host transcribes and
translates late genes, encoding
structural proteins (14-17).
• Virus assembles in nuclear viral
factories (19) and buds through
(19a, b) or is exocytosed through
(20a, b) the inner lamella of the
nuclear membrane.
• Golgi can be modified by the
insertion of herpes glycoproteins
(19d). Virus travels through 120
Golgi (21).
• Virus is exocytosed at the
Herpes viruses can trick immune system by:

• Direct infection of immune cells
• Inhibition of T cell antigen recognition:
 Inhibition of cell-surface class I MHC expression
 TAP inhibition (transporter associated with antigen presentation)
• Inhibition of natural killer cell attack
• Sequestration of chemokines
• Encoding cytokine and chemokine homologs
Types of infections caused by

Human Herpes viruses
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HSV 1 and 2
Both HSV-1 and HSV-2 cause painful lesions that
present as clear vesicle surrounded by a red area.
Infection HSV-1 and HSV-2 is initiated by breaks

in the skin or through mucosal membranes. Although
both HSV1 and HSV2 are common, and typically not
dangerous, they can cause serious disease if they
spread to eye or brain, or if they infect
immunosuppressed people or neonates. These viruses can be latent in trigeminal ganglia (HSV-1) or
sacral ganglia (HSV-2).
Symptoms of HSV-1 and HSV-2 overlap:
The virus is spread in bodily fluids such as saliva (HSV-1) and vaginal secretion (HSV-2).
• Preceded by a prodrome of localized burning
• Pruritus over the site
• Vesicles develop within 24 hours of initial symptoms
• Lasts about 1 week
• Common infection which can occasionally cause serious disease if they spread to eye or brain,
or if they infect immunosuppressed people or neonates.
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Exercise: Look at the figure on the right, which is another depiction of Herpes cell cycle and work
through each step on your own.
Replication of herpes simplex
Review of replication cycle (right figure):

• The virus binds to specific receptors
• After binding, the virus fuses directly with
the plasma membrane (no entry into low
pH endosomes/lysosomes is necessary).
• The nucleocapsid then delivers the DNA
genome to the nucleus.
• Transcription and translation occur in
three phases: immediate early, early, and
late. Immediate early proteins promote
the takeover of the cell; early proteins
consist of enzymes, including the DNA-
dependent DNA polymerase; and the late
proteins are structural and other proteins,
including the viral capsid and
glycoproteins.
• The genome is replicated before
transcription of the late genes.
• Capsid proteins migrate into the nucleus,
assemble into icosadeltahedral capsids, and are filled with the DNA genome. The capsids filled
with genomes bud through the nuclear and endoplasmic reticulum (ER) membranes into the
cytoplasm, acquire tegument proteins, and then acquire their envelope as they bud through the
viral glycoprotein-modified membranes of the trans-Golgi network.
• The virus is released by exocytosis or cell lysis.
• It also appears to be able to pass through intercellular junctions and thereby spread from cell to
cell.
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Clinical course of genital herpes infection. The time course and

Disease mechanism for HSV (right figure): symptoms of primary and recurrent genital infection with herpes simplex
virus type 2 are compared. Ann Intern Med 98:958-973, 1983
• Disease is initiated by direct contact
with the infected fluid and usually
begins in the mucoepithelium of the
oral or genital tract.
• Viral infection causes direct
cytopathologic effect (CPE) on the cell.
CPE is a direct change in the host cell
which may range from lysis of the cell
to the formation of multinucleated giant
cell (syncytia).
• HSV lesions usually begin with a clear
vesicle containing infectious virus with
a red (erythomatous) base. These are
usually painful.
• Patient may experience tender regional lymph nodes during lesion eruption.
• Pus-containing (pustular), encrusted lesions and ulcers may develop.
• Systemic symptoms are due to interferon secretion by the infected cells and recruited immune
cells.
• Although some released virions will be blocked by antibody, some virions can avoid antibody
by cell-to cell spread, which helps maintain the infection.
• Cell-mediated immunity is required for resolution
• In most infected individuals the virus establishes latency in sensory neurons.
• The virus can be reactivated by stress or immune suppression.
• Recurrent disease is usually shorter in duration and is more abrupt.
• In addition to the direct damage of the infected cells by the virus, cell-mediated
immunopathological effects contribute to symptoms
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Examples of human diseases
1. Herpetic gingivostomatitis-
lesions in the mouth, palate,
pharynx, gingivae (gums of
mouth) and tongue, often
seen in kids
2. Cold sores, fever blisters-
lesion in corner of mouth,
next to lips
3. Herpes pharyngitis- severe
sore throat, fever, chills, headache, and malaise, usually in adults
4. Herpetic whitlow- infection of the finger through a cut in the skin (often in nurses who attend HSV
patients or thumb sucking children)
5. Eczema herpeticum- severe disseminated HSV skin infection acquired by kids with underlying
eczema condition. It causes spread of infection from skin to adrenal glands, liver and other
organs.
6. Genital herpes- genital lesions accompanied by itching, pain and in females vaginal discharge
7. Herpes encephalitis- (usually HSV-1) - lesions on temporal lobes leading to destruction of the
lobe, increased red blood cells in CSF and seizures
8. Herpes meningitis (usually HSV-2) - a complication of genital HSV infection. It Neonatal HSV
usually resolves by itself. Meningitis is characterized by headache, neck

stiffness, fever and irritability accompanied by nausea.
9. Neonatal HSV (usually HSV-2)- can be acquired in utero, but more commonly
through the passage of infant through vaginal canal, or postnatally. Infection of
the neonate’s mucoepithelium will result in a disseminated infection in the liver,
lung, CNS which can result in severe disabilities.
10. Keratoconjunctivitis- From the skin, the virus reaches the cornea via the sensory ophthalmic
branch of the trigeminal nerve, it then causes infection of conjunctiva, corneal ulcers and
vesicles in the eyelids.
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HSV Latency
HSV-1 Latency
After a primary infection, Herpes stays dormant in peripheral nerve ganglia. Following one of a
variety of stimuli, vesicles erupt on the muco-cutaneous junctions of the nose or mouth. These
are more localized than the primary infection and heal more rapidly (7-10 days). The eruption is
often preceeded by paraesthesia of the involved area.
Location of HSV-1 latency- HSV-2 latency – sacral dorsal root ganglia
Reactivation of HSV often follows stressful conditions, immunosuppression or sun exposure.
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Diagnosis
• Direct analysis of clinical sample will show characteristic CPE in a Tzanck smear (Tzanck
smear is scraping of base of lesion) or in a PAP smear or biopsy.
• Tzanck smear will demonstrate:
o Syncytia (Figure below: A)
o Cowdry Type A intranuclear inclusions (Figure below: B) and ballooning cytoplasm
• Tissue sample can also be stained for viral antigen using IF
• Viral DNA can be amplified from the sample using PCR
A B
Syncytia
Cowdry Type A
Treatment:
To treat Herpes, Acyclovir can be administered. This is a modified nucleotide (with a guanine
base) that is phosphorylated inside a cell by HSV’s thymidine kinase. Although the drug is absorbed
by all cells, since the nucleotide can only be phosphorylated by a viral enzyme, acyclovir functions
specifically in the infected cells.
The last 2 phosphates are added to acyclovir by the by host’s enzymes. Once acyclovir is
converted to the triphosphate form it is incorporated by
viral DNA d. DNA polymerase. If, by chance host’s
polymerase gets access to Acyclovir triphosphate it will
have 200 fold lower affinity for it than the viral DdDpol.
This means that the host cell can still survive despite
being treated with acyclovir. Valtrex is a modified
acycolvir with an added valine group. Valtrex is better
absorbed by GI.
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Varicella-zoster (VZV)
Disease mechanism for VZV
• VZV causes a disease called chicken pox
(varicella) and upon recurrence it causes zoster
(shingles).
• Unlike HSV, VZV spreads predominantly through
the respiratory tract from where it enters
circulation and causes viremia and lesions all
over the body. The lesions are characteristic of
the chicken pox childhood rash.
Chicken pox
• VZV causes a lytic infection in the epithelial cells
of the lungs and keratinocytes, these cells are the Shingles if
reactivated
source of contagion. The other cells that host the Mechanism of spread of varicella-zoster virus (VZV) within the
body. VZV initially infects the respiratory tract and is spread to the
virus are not lysed by the virus. reticuloendothelial system and T cells and then by cell-associated
viremia to the skin.
• In addition to lysing cells, VZV can cause syncytia
• Interferon-α, natural killer cells and T cells limit the spread of the virus in the tissue, but antibody
is important for limiting the viremic spread of VZV. Passive immunization with varicella-zoster
immune globulin (VZIG) within 4 days of
exposure is protective. Cell-mediated
immunity is essential for resolving the
disease. The virus causes more disseminated
and more serious disease in the absence of
cell-mediated immunity. The more
pronounced immune response in adults is
responsible for the more severe symptoms
Time course of varicella (chickenpox). The course in young children, as
seen in adults as compared to children. presented in this figure, is generally shorter and less severe than that in
adults
• VZV is latent in the dorsal root and cranial
nerve ganglia.
• Upon reactivation, the virus is released along a particular neural pathway and infects the skin
causing a rash along the dermatome.
• Shingles results from immune-suppression and presents as lesions along a dermatome.
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Pathophysiology of human diseases
1. Varicella (chicken pox) - a childhood exanthema

Symptoms:
• 14 days incubation period
• Fever and rash appear at the same time.
• Presents as a maculopapular rash (vesicular rash):
o Macules are areas of skin discoloration that are usually
less than about half an inch (one cm) in diameter. They
are neither elevated nor depressed
o Papules are small, round elevations of the skin that
don't appear to contain any fluid.
o Rash becomes pustular and forms crusts
o Lesions last for about a week
• Rash is more severe on the trunk than extremities
• Rash present on the scalp
• In adults, a complication is interstitial pneumonia (due to
inflammation at the primary site of infection).
• Live chicken pox vaccine is now routinely given to kids.
• A strong cell-mediated response is needed for clearance of initial infections
• Latency in dorsal root ganglia
2. Herpes zoster (shingles)
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• Reactivation of the VZV causes it to spread along a nerve tract, causing burning sensation prior to
rash eruption.
• The typical rash appears in 2 to 3 days,
after the virus has reached the skin
• Rash consists of red patches of skin with
small blisters that look very similar to early
chickenpox.
• Rash often increases over the next 3 to 5
days.
• Blisters break forming small ulcers that
begin to dry and form crusts.
• The crusts fall off in 2 to 3 weeks, leaving
behind pink healing skin.
• Lesions appear along a single dermatome
and are only on one side of the body (unilateral).
• The trunk is most often affected, showing a rectangular belt of rash from the spine around one
side of the chest to the sternum.
• Older patients may experience postherpatic neuralgia which is a chronic pain syndrome
affecting the area innervated by infected nerves.
• Live vaccine available for shingles.
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Diagnosis:
Clinical presentation and
Histology: Skin lesion scraping
can be analyzed for Cowdry type
A intranuclear inclusion body, as
well as syncytia of the epithelial
cells.
Specific antigen tests: Skin
lesion scraping can be analyzed
by staining with a direct
fluorescent antibody against the
HSV antigens expressed on the
host cell.
Serology: Tests that detect
patient antibodies to VZV can be
used to screen people for
immunity to VZV. These tests are
usually ELISA or IF.
The figure on the right shows
various ways in which an
organism can infect the. The
orange layer shows epidermis, the yellow layer is the dermis. A pathogen can get to the skin from the
systemic infection in the blood (ex. VZV), they can get to the skin directly by being introduced into the
epithelium, and stay locally in the tissue (ex. HPV). Some pathogens are brought to the skin as part of
an immune complex, which then stimulates inflammation and a rash (ex. Hepatitis B).
Epstein-Barr Virus (EBV)
Disease mechanism of EBV

• EBV is shed from salivary glands
into saliva, so sharing of
toothbrushes and cups initiates
infection.
• EBV uses complement receptor
called CD21 as a means of
attachment to epithelial cells of the
oropharynx and nasopharynx as
well as attachment to B cells.
• EBV from infected saliva enters oral
epithelial cells and then spreads to
naïve resting B cells in the tonsils.
• EBV can be detected in saliva for 6 months after acute infection.
• EBV causes lytic infection of epithelial cells and in some B cells.
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• In epithelial cells lytic replication often follows

viral entry. In most B cells, however, lytic
replication only takes place after reactivation
of the virus from latency.
• EBV establishes life-long latency in
memory B cells.
• EBV may be reactivated when the memory B
cell is activated and turns into plasma cell
(especially in the tonsils or oropharynx) which
then may cause the shedding of virus into the
saliva.
• Although EBV may not directly kill naïve B
cells, T cells kill and limit B cell outgrowth. T cells are required for control of infection.
• Elevated T cell response (lymphocytosis) contributes to symptoms of infectious mononucleosis.
o Asymptomatic shedding is a characteristic of EBV.
o More than 90% of EBV-infected people intermittently shed the virus for life.
• EBV can rarely promote immortalization of B cells.
o Continual activation of B cells by EBV (and the absence of T cell response) causes
lymphoma
o EBV is associated with lymphoma in immunosuppressed people and African children
(Burkitt’s lymphoma) and nasopharyngeal carcinoma in China.
Diseases
While EBV is usually asymptomatic, EBV disease results from either an overactive immune response,
causing infectious mononucleosis or inadequate immune response which can lead to lymphoma.
Over-reactive immune response leads to:

1. Heterophile Ab-positive infectious mononucleosis:
This disease is usually milder in children than adults.
50% of adolescents experience symptoms. Classical
symptoms are: high fever, lymphadenopathy and
exudative pharyngitis. Splenomegaly and a rash may
be present. A great amount of energy is required to
power the T-cell response, leading to great fatigue
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(Figure, right). Rarely, EBV can cause complications like laryngeal obstruction, rupture of spleen and
neurological disorders.
Under-reactive immune response:

1. Lymphoproliferative disease
On infection with EBV, people lacking T cell immunity (such as people who are undergoing
immunesuppressive therapy, people with congenital deficiencies of T-cell function) or people with
chromosomal translocation of the c-MYC oncogene may get lymphoproliferative disease.
Specific B cell lymphoma of the jaw and
face called African Burkitt’s lymphoma is
endemic in children living in malarial regions
of Africa. It usually starts as tumors of the
jaw (mandibular or maxillary masses are
common) which causes facial bone
deterioration and loosening of the teeth. The
swelling of the lymph nodes in the neck and
below the jaw is common. The lymph nodes are non-tender and rapidly
growing. Other symptoms include night sweats, unexplained high
temperatures and weight loss.
Nasopharyngial carcinoma is endemic in Asia. It occurs in adults, and unlike Burkitt’s
lymphoma (which is of lymphocyte origin) has epithelial origin.
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Diagnosis of Mononucleosis
Mono is diagnosed several parameters:
1. Symptoms that include a triad of: fever, lymphadenopathy and exudative pharyngitis (and a fourth
possible symptom - splenomegaly).
2. Blood work:
• Atypical lymphocytes (called Downy cells,
see bottom figure) which are overly
activated T cells,
• Lymphocytosis - a sharp increase in

mononuclear cells
• Positive “Monospot test” – a test which
Clinical course of infectious mononucleosis and laboratory
detects IgM heterophile antibodies. These findings of those with the infection. Epstein-Barr virus infection
are transient short-lived antibodies may be asymptomatic or may produce the symptoms of
mononucleosis. The incubation period can last as long as 2 months.
characterized by having many different EA, Early antigen; EBNA, Epstein-Barr nuclear antigen; VCA, viral
capsid antigen.
specificities. Interestingly these antibodies
include specificities for antigens on horse and sheep red blood cells (see figure below).
Monospot test. A few drops of the patient's plasma are mixed
with RBCs from a goat or horse or sheep. The patient’s plasma
on the left (but not on the right) contains heterophile IgM
antibodies that agglutinate sheep or horse red blood cells.
• The findings of EBV-specific antibodies such as anti-EBNA antibodies.

3. PCR- EBV viral DNA can also be detected directly by PCR in patient samples such as saliva,
peripheral blood and biopsies.
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Cytomegalovirus (CMV)
• CMV can cross the placenta and is the

most common viral cause of congenital
defects.
• In US, about half of expectant mothers
have not been infected with CMV prior
to conceiving.
• About 1% to 4% of these previously
uninfected mothers have primary (or
first) CMV infection during their
pregnancy.
• About one third of women who become Outcomes of cytomegalovirus (CMV) infections. The outcome
of CMV infection depends very heavily on the immune status of
infected with CMV for the first time the patient.
during pregnancy pass the virus to their unborn babies.

• Women who had CMV before getting pregnant can also pass the virus to their unborn babies, but
this is less common.
• Although about 1 in 150 babies in US are born with CMV infection, only 1 in 800 children in US
are born with symptoms of CMV. This is due to the protective role of maternal IgG.
• CMV causes mild disease in children and adults, but very serious disease in immunocompromised
patients.
Disease mechanism of CMV
• Transmission of CMV occurs via blood, organ transplants, and other secretions: urine, saliva,
semen, cervical secretions, breast milk, and tears.
• CMV establishes a lytic infection in fibroblasts, epithelial cells and macrophages, and a latent
infection in lymphocytes, macrophages and bone marrow stromal cells.
• Secretory cells infected with CMV result in a persistent infection and viral shedding.
• Infection of the genitourinary system leads to clinically inconsequential viruria.
• Despite ongoing viral replication in the kidney, renal dysfunction is rare.
• Cell-mediated immunity is required for control and contributes to symptoms.
• CMV generally causes subclinical infection and may be shed asymptomatically.
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Diseases:
1. Congenital defects: 0.5-2.5% of infants in US are infected with CMV prior to birth but do not
necessarily have the disease. Less than
10% of the infected newborns show
birth defects including
hepatosplenomegaly with cirrhosis,
and microcephaly with mental or
motor retardation as well as hearing
loss. Other disease signs include:
thrombocytopenia, intracerebral
calcification, and rash. The congenital
disease is sometimes referred to as
cytomegalic inclusion disease.
• Perinatal infection: About 20% of women harbor CMV in the cervix. These women often
experience reactivation during pregnancy or labor. Infants may acquire CMV through birth canal
and infected cervix during delivery or through breast milk after delivery. This usually does not
result in a serious disease. Infants can also acquire CMV through transfused blood, which may
result in pneumonia and hepatitis.
• Infection of children and adults: CMV is a sexually transmitted disease, and because it is often
asymptomatic, many adults are infected with the virus. In some cases the infection results in
heterophile-negative mononucleosis, with similar symptoms to EBV, but less severe
pharyngitis and lymphadenopathy.
• Infection in blood and organ recipients: transmission via transfusion and transplantation can
result in CMV infection. Transmission in blood is usually asymptomatic, whereas transmission in
organ transplants may result in pneumonia and mild hepatitis. Blood is screened for CMV, and
CMV negative blood is reserved for transfusion to pregnant women and immunocompromised.
Organ donors are also screened for CMV.
• Infection of immunocompromised host. This is a serious problem. In this population, CMV
causes symptomatic, systemic infection which can result in recurrent disease. In
immunocompromised, CMV can cause pneumonia, hepatitis, retinitis, encephalitis, colitis.
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Diagnosis
• Histology and specific tests:

o Histological hallmark of CMV is an enlarged cell that
contains central “owl’s eye” basophilic intranuclear inclusion
body. Such a cell can be found in any tissue of the body, in
urine, and are thought to be of epithelial origin
o Blood, bronheoalveolar lavage* or urine can be tested for
viral antigen specifically using antigen-antibody based tests such as IF and ELISA or for
viral DNA via PCR.
• Serology: Seroconversion (test for patient’s IgM or increasing titer of IgG) is usually an excellent
marker for primary CMV infection.
• Viral isolation: CMV can also be grown in culture and then examined for CPE
*Bronheoalveolar lavage- diagnostic procedure conducted by placing a small fiberoptic scope into
the lung of a patient, and injecting sterile water into the lung and removing the fluid. The sterile
solution removed contains secretions, cells, and protein from the lower respiratory tract.
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Human Herpes Virus 6 (HHV6)
• HHV6 causes roseola and is another example of a childhood exanthema.

• Infection occurs early in life (before 3 years of age).
• Virus replicates in the salivary glands, is present in saliva of most adults and is spread to children
easily by oral secretions.
• HHV6 also infects lymphocytes, monocytes, epithelial and endothelial cells.
• Disease is characterized by rapid onset of high fever, followed by generalized rash which lasts 24-
48 hr. The rash usually spares the face.
• Disease resolves without complications, however the infection remains persistent for life. The
persistency is usually in T cells.
• In adults, it may cause very mild mononucleosis and lymphadenopathy.
Time course of symptoms of exanthem subitum (roseola) caused by human herpesvirus 6 (HHV-6).
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LECTURE 14: HEPADNAVIRIDAE
Learning Objectives
Be able to describe clinically and structurally the causative agent of Hepatitis B.
• Describe the features of Hepatitis B virus and its antigens: HBsAg, HBcAg, HBeAg
• Describe the vaccine composition and its administration
• Draw out HBV’s replication cycle
• Understand the serological charts for acute and chronic Hepatitis B infected individuals
• Describe Hepatitis B as a cause of cancer
• Relate serological markers to stages of disease
Hepatitis B
Unique properties:
• HBV is less sensitive to detergents than other enveloped viruses

• It is spread by blood, sexual intercourse, saliva, breast milk, transfusion and needle stick injury.
• HBV has a strict liver tropism.
• Virions (also known as "Dane particles") contain a circular, partially dsDNA genome.
• HBV encodes and carries reverse transcriptase.
• It replicates through circular full length RNA intermediate.
• HBV genome can integrate into host genome.
• 10% of infected people will have chronic infection with shedding.
• Persistent regeneration of the liver can result in cancer.
• HBsAg recombinant subunit vaccine available.
• As treatment for high risk newborns-passive immunization is given.
Important HBV Antigens
o HBsAg; surface (coat) protein. It is produced in

excess to what the virion need and is released as
small spheres and tubules into the blood.
o HBcAg; inner core protein, structural protein derived
from pre-core-mRNA
o HBeAg; secreted protein derived from pre-core-
mRNA and is made in a 1:1 ratio to core Ag; function
unknown
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Replication cycle of Hepatitis B (Figure: right)
1. In the first step, the virus attaches to the

liver cells via a receptor.
2. It enters through endocytosis.
3. Partially dsDNA synthesis is completed
either in the cytoplasm by viral reverse
transcriptase (RTase) or in the nucleus via
host’s DNA dependent DNA polymerase
(DdDpol).
4. Cellular RNA polymerase II transcribes a
longer-than-genome-length mRNA called
the pregenome (or replication
intermediate) and shorter, subgenomic
transcripts, all of which serve as mRNAs.
5. The shorter viral mRNAs are translated by
ribosomes attached to the cell's
endoplasmic reticulum. These proteins are
Replication of hepatitis B virus (HBV). After entry into the
destined to become HBV surface antigens hepatocyte and uncoating of the nucleocapsid core, the
partially double-stranded deoxyribonucleic acid (DNA) genome
in the viral envelope. is completed by enzymes in the core and then delivered to the
nucleus. Transcription of the genome produces four
6. The pregenome mRNA is translated to messenger RNAs (mRNAs), including an mRNA larger than
the genome (3500 bases). The mRNA then moves to the
produce a reverse transcriptase cytoplasm and is translated into protein. Core proteins
assemble around the 3500-base mRNA, and negative-sense
polymerase protein “P” which then binds to DNA is synthesized by a reverse transcriptase activity in the
core. The ribonucleic acid (RNA) is then degraded as a
a specific site at the 3' end of its own
positive-sense (+) DNA is synthesized. The core is enveloped
transcript (RNA-P complex). The before completion of the positive-sense DNA and then
released by exocytosis. HBsAg, hepatitis B surface antigen.
pregenome mRNA also codes for the core
and “e” antigens.
7. The RNA-P protein complex is packaged into the core and while packaging continues reverse
transcription takes place.
8. The viral DNA is synthesized by the viral reverse transcriptase using the encapsidated
pregenome as template. Remember that RTase has three functions: RNA to DNA synthesis,
DNA to DNA synthesis and RNAse H activity during which it destroys RNA in a RNA:DNA
hybrid.
9. The newly synthesized nucleocapsid acquires the viral envelope in the endoplasmic reticulum
(ER) and Golgi apparatus, with release of mature virions from infected cell by exocytosis.
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10. This mode of virus production does not result in cell death, thus contributing to chronic
infection of the liver.
Clinical Outcomes
• Incubation period of HBV is 2 - 5

months.
• HBV causes a more severe disease
than Hepatitis A.
• Asymptomatic infections occur
frequently.
• Infection is parenterally transmitted
(taken into the body in a manner other
than through the digestive tract).
Transmission occurs via blood (blood
transfusions, serum products, sharing
of needles, razors), tattooing, renal
dialysis, organ donation, sexual
intercourse, vertical transmission
(perinatal transmission from a carrier
mother to her baby during delivery). In vertical transmission for Hepatitis B the virus is passed
from mother to baby during delivery, not prior to
delivery.
Pathogenesis
The virus replicates in the liver and the progeny
virions, as well as excess viral surface protein are
shed in large amounts into the blood. Viremia is
prolonged and the blood of infected individuals is
highly infectious.
The diagram on the right illustrates the
transmission of Hepatitis B. After the introduction of the virus into the blood, it infects the liver. If an
individual has previously formed antibodies against the virus (ex. through vaccination), they will block
the transmission from blood to liver. Without previous vaccination or previous exposure and
resolution, the virus is shed back into the blood from the infected liver and then is excreted into
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various bodily fluids, completing the cycle. Note that a strong cell mediated response can clear the
virus before it is shed from the liver into blood.
Clinical presentation
A. Acute infection with resolution

Acute symptoms include: fever, malaise (95% of patients), nausea (80% of patients), anorexia
(90% of patients), vomiting, abdominal pain (right upper quadrant, 60% of patients) and rash in 15%
of the patients. Classical icteric symptoms (jaundice, yellow sclera, dark urine, pale stool) follow. In
hepatitis, due to inflammation of the biliary ducts there is a defect in secretion of bilirubin into biliary
tract which causes bilirubin accumulation in the blood resulting in jaundice. In healthy individuals, liver
conjugates bilirubin and when conjugated bilirubin is secreted into the bile tract, it is converted to dark
colored stercobulin by intestinal bacteria. This causes stool to have its characteristic dark color. When
liver does not function properly, conjugated bilirubin does not reach the intestines, resulting in pale
stool. Accumulated conjugated bilirubin also gives urine a darker yellow color when the kidneys filter
the bilirubin rich blood.
In few cases (1%) fulminant hepatitis (severe liver damage) may occur. Because of the excessive
amount of HBsAg produced, hypersensitivity reactions caused by immune complex depositions, may
promote rash, arthritis, glomerulonephritis as well as other symptoms of Type III hypersensitivity.
A. Acute Hepatitis B serology

Viral antigens:
1) Surface antigen (HBsAg) is the first
antigen detected and is secreted in excess
into the blood as 22 nm spheres or tubules.
Its presence in serum indicates that virus
replication is occurring in the liver. It can be
detected very early during an infection, even
prior to symptoms.
2) 'e' antigen (HBeAg) is a secreted protein
that is shed in small amounts into the blood.
Since its levels directly correlate to the
amount of viral progeny in that cell, its The serologic events associated with the typical course of acute
hepatitis B disease.
presence in the blood indicates that a high
level of viral replication is occurring in the liver.
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3) Core antigen (HBcAg) – Since core antigen is an internal part of the virion, it is not detected in the
blood. However, during the lysis of the liver cells by CD8 T cells, a small undetectable level of HBcAg
is present in the body to stimulate the anti-HBc immune response.
Viral antibodies:
1) Surface antibody (anti-HBs) becomes detectable late in convalescence, and indicates immunity
following infection. It remains detectable for life and is not found in chronic carriers. Note that in
vaccinated people only HBsAb will be present.
2) e antibody (anti-HBe) becomes detectable as viral replication falls. It indicates low infectivity in a
carrier.
3) Core IgM rises early in an infection and indicates recent infection. Its peak follows symptoms of
hepatitis because the release of core antigen from the liver cell is necessary for the antibodies to be
produced. The killing of liver cell by CD8 T cell causes symptoms.
4) Core IgG rises soon after IgM, and remains present for life in both chronic carriers as well as those
who clear the infection. Its presence indicates exposure to HBV. It usually appears 3-6 months after
infection.
Hepatitis B window period – In early convalescence stage there is no detectable HBsAg nor HBsAb
because these molecules are complexed together. This period is referred to as “window period”.
B. Serological events in chronic hepatitis:
Following acute infection, approximately 5-10% of infected individuals fail to eliminate the virus
completely and become persistently infected. Continual destruction of the liver nodules leads to
scarring, obstructed blood supply to the liver, cirrhosis, liver failure and is some cases cancer. Since
chronic Hepatitis B can also be asymptomatic, it can be discovered accidentally by observing
elevated liver enzymes in a routine blood test. Many chronically infected people are asymptomatic
and are a major source of infection. At a particular risk for chronic hepatitis are:
• Babies (95% chance of developing chronic hepatitis if infected)

• Young children
• Immunocompromised patients
• males > females
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Chronic infection may take one of two forms:

Chronic Persistent Hepatitis - The virus persists, but there is minimal liver damage.
Chronic Active Hepatitis - There is aggressive destruction of liver tissue and rapid progression to
cirrhosis or liver failure.
Antigens in Chronic hepatitis B:

1) HBsAg - When an individual is infected with Hepatitis B for longer than 6 months, he is considered
chronic, and unless the infection is resolved, will continue to synthesize HBsAg which can be
detected in the blood. This time frame is somewhat arbitrary but is important in classifying hepatitis as
acute or chronic. Presence of Hepatitis B surface antigen and simultaneous presence of anti-HBc
IgGs also signifies chronic hepatitis.
2) HBeAg – in chronic infection,
levels of HBeAg may vary and
correlate to viral reproducibility.
3) HBcAg - Core antigen is not
detectable in the blood during either
acute or chronic infection.
Viral antibodies in chronic

hepatitis B:
1) HBsAb - Surface antibody would

Development of the chronic hepatitis B virus carrier state. Routine serodiagnosis
not be found in chronic carriers. Only is difficult during the hepatitis B surface antigen (HBsAg) window, when HBs and
anti-HBs are undetectable.
in patients who have resolved the
infection is HBsAb found.
2) HBeAb - e antibody (anti-HBe) in blood indicates low infectivity of the patient, however if HBsAg is
present, the patient is still considered infectious and is a carrier of the virus.
3) HBc IgM – Core IgM is not present in chronic carriers. Due to isotype switching, 3 - 6 months post
initial infection, all anti-core antibody should be of IgG isotype.
4) HBc IgG – Core IgG is evident in chronic carriers and persists for the rest of the individual’s life.
Hepatocellular carcinoma:
Patients who become persistently infected with HBV are at risk of developing hepatocellular
carcinoma (HCC). Since the virus persists in the hepatocytes, on-going liver damage occurs due to
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the host immune response fighting against the infected liver cells. Continual liver damage introduces
mutations that can immortalize liver cells. Liver cancer leads to death within 6 months. HBV is thought
to be a factor in the development of HCC because:
a) 80% of patients with HCC are carriers of hepatitis B,

b) Virus DNA can be identified in hepatocellular carcinoma cells,
c) Virus DNA can integrate into the host chromosome.
Liver cirrhosis and HCC can lead to portal hypertension:

Increased resistance to blood flow can cause portal vein engorgement.
b. c.
a.
Portal hypertension. Image a: The hepatic portal vein carries the blood from the GI tract and spleen to the
liver before this venous blood enters the inferior vena cava and the general circulation. Images b and c:
Portal hypertension is an increase in the pressure within the portal vein. Increased pressure in the portal vein
causes large veins (varices) to develop across the esophagus and stomach to bypass the blockage. The
varices become fragile and can bleed easily. In cirrhosis, the scar tissue blocks the flow of blood through the
liver and slows its processing functions.
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Prevention/Treatment
1) Active Immunization with recombinant HBsAg is made by genetic engineering in yeast.

The administration of three doses induces protective levels of antibodies in 95% of vaccine
recipients. Universal immunization of infants was introduced in April 1995. Infants receive three doses
before they turn 1 year old.
2) Passive Antibody -Hepatitis B immune globulin should be administered to non-immune
individuals following single episode exposure to HBV-infected blood.
Interpretation of Hepatitis B Serologic Test Results
Another representation of acute and chronic Hepatitis B serology
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LECTURE 15: PICORNAVIRIDAE and ORTHOMYXOVIRIDAE

Learning Objectives
• Describe the structural features of Picornaviridae
• Describe the clinical presentation of various conditions caused by this viral family
• Describe methods of preventions if such exist
• Describe the structural features of Orthomyxoviridae
• Describe the clinical presentation of flu
For all positive strand RNA viruses the

naked genome, if artificially introduced
into a cell, is sufficient for infection.
All negative strand RNA viruses must

first transcribe their genome to positive
strand mRNAsw.
PICORNAVIRIDAE
Examples of viruses that belong to the Picornaviridae family:

• Polio (3 serotypes)
• Coxsackie A > 22 serotypes
• Coxsackie B > 6 serotypes
• Rhinovirus > 100 serotypes
• Hepatitis A (one serotype)
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Unique properties of Picornaviruse:
• These viruses are small naked, icosahedral with a positive RNA genome.
• Genome alone is sufficient for infection.
• Picornaviruses bind to receptors that are members of immunoglobulin superfamily (such as ICAM-
1).
• They replicate in the cytoplasm.
• Most Picornaviruses are cytolytic (except Hepatitis A).
• Viral, rather than immune pathology causes symptoms.
• Antibody is important for control and prevention of future disease. Serum Ab important in
preventing/controlling viremia.
• All but Rhinoviruses are resistant to low pH, detergents, mild sewage treatment and heat.
• Rhinoviruses grow best at 330Celcius and stay in respiratory tract.
• Many Picorna viruses are spread by fecal-oral route via ingestion of contaminated food or contact
with infected hands or fomites.
• Some Picorna viruses are transmitted through inhalation of infectious aerosol.
• Infection is often asymptomatic or mild.
• Picrona viruses that enter via GI tract are shed in feces for a long time.
• Antibodies control infection and block viremia.
Replication cycle (Figure right):
• After entering the cytoplasm and

uncoating, the single RNA is
a. b.
translated into a polyprotein (a)
that is subsequently cleaved (b). c.
• Vpg (“virion protein genome”) is a

d.
protein primer attached to the 5'
e.
end of the RNA.
• After being translated, the RdR
f.
polymerase synthesizes a
negative strand RNA template called replication intermediate (c), which is then copied to positive
RNA (d).
• Virus assembles in cytoplasm (e), and leaves the cell through cell lysis (f).
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Polio:
Like other enteroviruses, polio enters the body through the

gastrointestinal tract (Figure right). The virus replicates efficiently
in the intestinal tract and is typically shed in the stool for two to
four weeks, and sometimes for several weeks longer. Shedding
may be intermittent and is affected by the immune status and
immune competence of the individual. Poliovirus divides within
gastrointestinal cells for about a week. After division the virus
spreads to the intestinal lymphoid tissue including the M cells of
Peyer's patches, and the deep cervical and mesenteric lymph
nodes, where it multiplies abundantly. The virus then enters the bloodstream.
Since virus also replicates in the upper respiratory tract, polioviruses can also be spread through
upper respiratory tract secretions. Virus can be recovered from throat swabs and washings during the
early acute phases of infection. Respiratory tract secretions are infectious and may provide a source
of virus for close contact spread through direct person-to-person contact, large-particle aerosols, or
fomites. From the pharynx the virus can spread to the tonsils (specifically the follicular dendritic cells
residing within the tonsilar germinal centers.
Poliovirus must cross blood brain barrier in order to cause paralytic disease. In the nervous tissue
it has a narrow tissue tropism recognizing receptors on the motor neurons in the anterior horn of the
spinal cord. The main receptor for polio is called CD155 and is believed to be present on most
human cells. Therefore receptor expression does not explain why poliovirus preferentially infects
certain tissues. This suggests that tissue tropism is determined after cellular infection. It is suggested
that Type I interferons (specifically that of interferon alpha and beta) is an important factor that
defines which types of cells support poliovirus replication. If a cell does not bind these interferons,
then it can replicated the virus very effectively.
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Poliovirus infection can have 4 outcomes: Polio outcomes

1. Asymptomatic illness - 90% of cases. Minor
• Limited to the gut and the oropharynx. illness
2. Abortive polio (minor illness) - 5 % of polio cases
• Nonspecific illness, headache, vomiting, fever,
malaise
• Symptoms 3-4 days after exposure
• Lasts a few days
3. Nonparalytic polio (aseptic meningitis) occurs in 1-
2% of people with polio.
• Virus progresses from GI to the blood and into
the CNS (meninges) causing back pain, muscle spasms in addition to minor illness
4. Paralytic polio (major illness) - 0.1 - 2% of cases. 3 - 4 days after minor illness subsided, a
patient may experience more debilitating symptoms in Polio outcomes
addition to meningitis sometime a patient progresses Major
illness
directly to paralytic polio. In paralytic polio the virus travels
from blood to motor neurons in anterior horn cells of
spinal cord and to the primary motor cortex of the brain.
Outcomes of paralytic polio are:
a) Spinal polio (Figure bottom right)
Most commonly there is damage to alpha motor neurons
so there is no stimulation of the skeletal muscle
controlling limb movement. This causes flaccid paralysis
in which limbs are weakened but the respiratory center is not affected. It may take years for
recovery.
Other symptoms include:
• Severe muscle pain

• Spasm of involved muscle
• Hyperesthesia (increased sensitivity to stimuli)
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b) Bulbar polio (Figure right) - polio destroys cranial nerves:
Nerves affected in bulbar polio include glossopharyngeal nerve

(IX), accessory nerve (XI), trigeminal nerve (V), facial nerve (VII)
and cervical nerves (C3, 4, 5). This results in muscle atrophy of
the muscle controlling the eyes, facial muscles, pharynx, vocal
cords, and diaphragm.
• Results in death in 75 % of patients

c) Postpolio syndrome (Figure right) – sequel of
polio that may occur much later in life (30-40
yr. later). People suffer deterioration of the
originally affected muscles. Poliovirus at this
point is not present, but the syndromes is
believed to result from the loss of originally
effected neurons.
Figure: Mechanism of paralytic polio:
A . Three normal motor neurons and the muscle cells they supply;
B. Acute stage of polio. Some motor neuron are invaded by the
poliovirus and get damaged. The muscle cells (shaded) fed by the
middle neuron become “stranded.” In image C the orphaned
muscle cells are re-connected to the surviving motor neurons by
the growth of new terminal axon sprouts (TAS) creating “giant
motor units.” When these muscle cells become re-connected and
start working again, the individual regains lost strength. Note the
enlarged or hypertrophied muscle cells which develop in response
to exercise.
D. PPS. In the two remaining motor neurons, some terminal axon
sprouts are dying leading to new weakness.
Diagnosis:
Polio may be isolated from a patient’s pharynx
in the first few days, or from the feces for as long as 30
days. The virus is rarely found in CSF. Serology (IF, neutralization assays, ELISA) are useful in
diagnosing polio.
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Prevention:
There are two commonly used polio
vaccines:
1. IPV = Inactive Polio Vaccine (killed
vaccine), administered as an injection and
2. OPV = Oral Polio Vaccine (live attenuated
vaccine) administered as drops by mouth.
Both include the three known serotypes of
polio. OPV was developed in 1958 by Dr. Albert
Sabin. IPV, also called the Salk vaccine, was
developed by Dr. Jonas Salk in 1952. The vaccine
is a suspension for subcutaneous injection. IPV
contains strains of the 3 types of polioviruses (Types 1, 2, and 3), originally grown in monkey kidney
cell culture and inactivated by exposure to formaldehyde.
Coxsackie viruses:
• There are two groups of coxsackieviruses: A and B.
• Infected individuals shed the virus in feces and respiratory secretions. These secretions can
contaminate surfaces and remain on them for a long time.
• Most are systemic and depending on the serotype Coxsackie viruses may cause a febrile upper
respiratory tract infection with sore throat, a runny nose, gastrointestinal disease, rashes, and/or
organ infections.
• The key to prevention of coxsackievirus infection is good hand washing and covering the mouth
when coughing or sneezing.
• The virus is not destroyed by the acid in the stomach, and it can live on surfaces for several hours.
• Once a person gets the virus, symptoms develop in a few days.
• Coxsackie A – often associates with systemic infection, accompanied by rash with or without
gastroenteritis.
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Diseases
Vesicular lesions:
• Herpangina –a disease that presents as a fever,

sore throat, lesions in soft palate and uvula
• Hand-foot mouth disease - symptoms include
fever, blister-like sores in the mouth (herpangina),
and a skin rash.
• Non-specific rashes – This virus often causes a
nonspecific generalized red rash or clusters of fine
red spots. The rash appears towards the end of
the disease, and sometimes looks like a sunburn.
Conjunctivitis
People who have infected eyes (conjunctivitis) can spread the virus by touching their eyes and
touching other people or touching a surface. It often presents as red hemorrhages in the
whites of the eye. This infection may appear in both eyes.
Complications
Meningitis- Patients complain of a headache and fever with mild neck stiffness.
Coxsackie B – typically more serious than Coxsackie A and infects deeper tissues.
Diseases:
• Pleurodynia: also known as Bornholm’s disease or devil’s grip, is characterized by thoracic and
chest pain because of the inflammation of the muscles in the chest.
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• Myocarditis: infects myocardium and causes inflammation of the

heart. Symptoms include: chest tightness, heart palpitations,
enlarged heart, arrhythmia, ECG ST-T changes on the ectopic
rhythm.
• Pericarditis is an inflammation of the pericardium, the thin sac
(membrane) that surrounds the heart and the roots of the great
vessels. Chest pain, pleuritic pain that is worse with inspiration,
fever and chills are very common.
Histology of normal heart Histology of damaged heart

Structure of the
Complications:
Meningitis: Patients complain of a headache and fever with mild neck stiffness.
Hepatitis A: - "Infectious Hepatitis"

Incubation period is 3-5 weeks (the mean is 28 days).
Stable to: acid pH 1, ether, chloroform, salt water, drying, temperature (at 4oC for weeks, 56oC for
30 min)
Inactivated by: chlorine, formalin, UV radiation, peracetic acid
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Clinical features:
• The virus is transmitted predominantly by fecal-oral, Hepatitis A spread: Virus enters via the gut; replicates
in the alimentary tract. There is a short viremia stage.
enteric route.
Virus spreads to the liver, where it multiplies in
• Contamination of food or water with sewage is a hepatocytes. It is excreted through bile in stool.
common source of the virus. Shell fish from

sewage-polluted water can carry Hepatitis A.
• Asymptomatic infections are very common,
especially in children
• Adults, especially pregnant women, may develop
more severe disease.
• Symptoms occur abruptly and include fever, fatigue, nausea, jaundice.
• Virus is excreted in the stools for two weeks preceding the onset of symptoms.
• Fulminant hepatitis is rare: 0.1% of cases.
• Although convalescence may be prolonged, there is no chronic
form of the disease.
• Complete recovery occurs in 99% of cases.
• Diagnosis is made based on the presence of HAV-specific IgM
in the patient's blood.
• Prevention is usually through active immunization with a killed
vaccine, although a live, attenuated cell culture-derived vaccine
has recently become available but is not in general use.
• Milder disease than Hepatitis B
Remember the 4 “No’s” of Hepatitis A

1. Not chronic
2. No cancer
3. Not fatal
4. Not lytic (unlike other Picornas)
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Summary for pathogenesis of Picornaviridae enteroviruses
Rhinovirus:
The primary route of entry for human rhinoviruses is the upper respiratory tract (mouth and nose).
There are at least 100 serotypes of rhinoviruses but most share a common receptor ICAM-1 (Inter-
Cellular Adhesion Molecule 1) also known as CD54 repressed on respiratory epithelial cells. Infection
occurs rapidly, with the virus adhering to surface receptors within 15 minutes of entering the
respiratory tract. As the virus replicates and spreads, infected cells release
chemokines and cytokines which in turn activate inflammatory mediators. Cell lysis occurs at the
upper respiratory epithelium.
Rhinoviruses are spread by aerosol and on fomites and are the most common cause of the
common cold. Secretory IgA is transiently protective while interferon and other innate immune
components generated in response to the virus may limit the spread and contribute to symptoms,
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ORTHOMYXOVIRIDAE
Unique properties of Orthomyxovirus

(Influenza A and B)
• Enveloped virion with eight negative-sense RNA

nucleocapsid segments
• Large envelope, easily inactivated (half-life
of a few hours at room temperature
• Likes cool, less humid atmosphere
• The outer surface of the particle consists of a lipid envelope and two prominent glycoprotein
spikes: haemagglutinin (H or HA) and neuraminidase (N or NA).
• Hemagglutinin glycoprotein is the viral attachment protein and fusion protein. Note: this protein is
unlike fusion proteins that fuse at normal pH because it fuses with vesicular membrane at low pH.
Therefor fusion happens within the vesicle, rather than at plasma membrane. The H protein elicits
neutralizing, protective antibody response. Entry and exit of influenza virus
• Neuraminidase is a sialidase that cleaves the sialic acid
off the cell membrane during the budding stage of the
virus. This prevents viral particles from clumping with the
cell and attaching to each other.
• 95% of the surface spikes are hemagglutinin and
only 5% are neuraminidase
• The inner side of the envelope is lined by the matrix
protein. The matrix protein is a structural protein that links
the viral envelope with the virus core. It plays a crucial
role in virus assembly, and interacts with the RNP (RNA
+ nucleoprotein (N)) complex as well as with the viral
membrane.
• The genome segments are packaged into the core. The RNP is in a helical form with the 3
polymerase polypeptides associated with each segment.
• Influenza A infects humans, mammals and birds (mutates frequently due to genetic shift and drift,
see below)
• Influenza B only infects humans and only undergoes genetic drift (see below)
• Killed (injection) and live (nasal) vaccines contain predicted yearly strains of influenza A and B
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• Drugs are developed against the neurominidase protein of Influenza A and B (Tamiflu also
known as oseltamivir)
Replication cycle of Orthomyxovirus
Unique feature: RNA virus that transcribes and replicates its genome in the target nucleus
Figure above:
• (1) Virus binds sialic acid and enters via receptor mediated endoctysis
• (2) Vesivular uncoating step utilizes a well-studies M2 protein (found in Influenza A only) and is
a target of drug Amantadine
• All of the -RNA's have identical 5'ends containing 17-22 nucleotides. In addition, the 3'ends
have a high degree of conservation.
• (3) In the nucleus, the RNA dependent RNA polymerase snatches host’s 5’ CAP which serves
as a primer for RNA synthesis of 8 mRNA segments
• (4) mRNA is transported to the cytoplasm and is translated
• (5) –RNA is replicated to progeny genomes via full length +RNA intermediate
Two classes of (+)sense RNA are made in infected cells:
a. Incomplete (meaning not full-length), 3' polyadenylated mRNA transcripts which are
exported to the cytoplasm and serve as mRNAs. The presence of a specific
polyadenylation sequence ~20nt from the 5' end of the (-)sense vRNA template strand
causes transcription termination.
b. cRNA = complete RNA, non-polyadenylated (+)sense copies of the (-)sense vRNA
(made by read-through of the polyadenylation signal). Full (+)RNA serve as template
for the synthesis of progeny (-) sense vRNAs (viral RNAs)
• (6 and 7). Virus assembles in the cytoplasm and exits by budding.
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Genetic shift and drift
The infidelity of RdRpol results in genetic drift (minor mutation). Additionally, the segmented
genome of Influenza A promotes genetic diversity through genetic shift (major reassortment).
Influenza viruses are changing by antigenic drift all the time, but antigenic shift happens only
occasionally.
Genetic drift refers to small, gradual changes that
occur through point mutations in the two genes which contain
the genetic material to produce the main surface proteins:
hemagglutinin, and neuraminidase. These point mutations
result in minor changes to these surface proteins that often
leaves previous influenza antibodies partially protective.
Antigenic drift produces new virus strains that may not be recognized by antibodies to earlier
influenza strains. This process works as follows: a person infected with a particular influenza virus
strain develops antibody against that strain. As new virus strains appear, the antibodies against the
older strains might not recognize the "newer" virus, and infection with a new strain can occur.
Genetic shift refers to an abrupt,
major change to produce a novel influenza A
virus subtype in humans that is not currently
circulating among people.
Antigenic shift usually occurs through
mixing of human influenza A and animal
influenza A virus genes to create a new
human influenza A subtype virus through
a process called genetic reassortment.
Pigs and birds are believed to be particularly
important reservoirs, generating pools of
genetically/antigenically diverse viruses
which get transferred back to the human population after reassortment. A reassortment of the highly
aggressive and deadly H5N1 avian virus with a human influenza virus is feared to cause a world
pandemic. However, this has not yet taken place.
A global influenza pandemic
(worldwide spread) may occur if three
conditions are met:
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1. A new subtype of influenza A virus

is introduced into the human population.
2. The virus causes serious illness in
humans.
3. The virus can spread easily from
person to person in a sustained manner.
Conditions 1 and 2, but not 3 have been met.
In contrast to H5N1 bird flu virus, the 2009 pandemic with H1N1 swine flu was due to an
emergence of a previously existing swine flu virus (limited to pigs) in a human population. Unlike
H5N1, H1N1 is easily spread, and does not cause as severe of an infection as H5N1. No direct
recombination with H5N1 was shown in swine H1N1, although swine H1N1 does contain some
structural similarities to the avian flu that infected humans in 1918 (Spanish flu).
Influenza A/H1N1
Transmission Symptoms
Reassortment between human H3N2, North Swine flu is an example of several genetic
American avian, and classical swine viruses shifts creating a new swine virus, although
resulted in triple reassortant H3N2. H3N2 and this virus jumped to humans it did not result in
H1N2 swine viruses circulated in North American a very serious flu.
pig populations. A triple reassortant swine virus
and H1N2 reassorted with a Eurasian avian-like
swine virus to produce swine H1N1. 160
Disease mechanism:
• The virus is spread by small aerosol

droplets expelled during talking, breathing
and coughing.
• Influenza usually causes a fever that lasts
3-8 days and unless complications occur,
recovery is complete within 10 days.
• The symptoms of Influenza in adults
include malaise, sore throat, high fever,
muscle aches and fatigue. In children, the
symptoms may also include bronchiolitis,
croup, otitis media (ear infection) and
abdominal pain. Pathogenesis of influenza A virus. The symptoms of influenza are
caused by viral pathologic and immunopathologic effects, but the
• It must first pass through inhibitory infection may promote secondary bacterial infection. CNS, Central
nervous system.
mucoproteins, (secretions), however the
neuraminidase hydrolyses the mucoproteins, rendering them ineffective as inhibitors.
• Virus enters the nasopharynx and spreads to susceptible cells whose membranes contain specific
receptors. It establishes infection in upper respiratory tract and targets mucus-secreting, ciliated
epithelial cells, causing a loss of primary defense system.
• The symptoms of influenza result from infection and destruction of cells lining the upper
respiratory tract, trachea and bronchi. If the virus did not have neuraminidase, the secretory
mucoproteins would inhibit the virus.
• When lower tract is involved, lower tract infection can cause severe shedding of bronchial and
alveolar epithelium. This can often result in pneumonia. Although pneumonia can have a primary
viral cause, it is usually due to a bacterial secondary infection. After flu bacteria can infect the
respiratory tract much easier since the virus has already destroyed the natural barriers of the
lungs.
• Systemic symptoms are caused by interferon and lymphokine response to the virus.
• Facial nerve inflammation is a rare complication of flu.
• Influenza B viruses infect humans only and can cause disease, but generally not as severe as A
types. Unlike influenza A viruses, influenza B viruses do not have distinguishable serotypes.
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• Flu can be prevented by a killed (injection) and live (nasal mist) vaccines that contain predicted
yearly strains of influenza A and B. However these vaccines are only 50% effective.
Time course of influenza A virus infection. The classic

"flu syndrome" occurs early. Later, pneumonia may
result from bacterial pathogenesis, viral pathogenesis, or
immunopathogenesis.
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LECTURE 16: PARAMYXOVIRIDAE, TOGAVIRIDAE AND FLAVIVIRIDAE
Learning Objectives
Describe biochemical and structural features of Paramyxoviridae.
• Describe the structural features of Paramyxoviriade

• Describe the clinical presentation of various
• conditions caused by this viral family
• Describe the structural features of Toga and Flavivirade
• Describe the clinical presentation
Describe biochemical and structural features of Togaviridae and Flaviviridae.
• Describe vector‐transmitted versus human‐to‐human transmitted infections

• Describe clinical features of Dengue fever (explain why second time is worse), yellow fever,
WNE
• Identify how rubella is different from other childhood rashes
• List the differences between Hepatitis A, B and C
PARAMYXOVIRIDAE
Unique properties of Paramyxovirus
• Enveloped, easily inactivated, non-segmented,

negative-sense RNA virus
• Attaches via VAP, enters by cell by fusion
(via F protein which works in a pH
independent manner), exits by budding
• The attachment proteins of the
paramyxoviridae bind to sialic-acid containing
receptors on cells
• Replication occurs in the cytoplasm
• The virion RNA is first copied into the
complementary positive-sense mRNAs
before proteins can be made. Thus, besides
163
needing to code for an RNA-dependent RNA-polymerase, these viruses also need to package
this enzyme into the virion so that they can make mRNAs upon infecting the cell.
• RNAd.RNA polymerase is capable of capping and polyadenylation
• M protein enables assembly between the genome and viral transmembrane proteins
• Virus may exit by budding but can also induce cell fusion, causing multinucleated giant cells
• All members of paramyxoviridae are transmitted by respiratory droplets and initiate infection in the
respiratory tract
• Cell mediated immunity causes symptoms but is also essential for control of the infection
Replication cycle (Figure right)

a
• The VAP protein recognizes receptors on b
cell surface (a, b).
• The F protein facilitates fusion between
membranes at physiological pH
• Because the F protein functions at
physiological pH, this can result in syncytia c
during paramyxovirus infections d
• The viral RNA polymerase uses the f
negative sense RNA in the nucleocapsid g e
as a template for transcription (c).
• Viral mRNAs are transcribed; these are
h
capped, methylated and polyadenylated
(d). i
• The viral mRNAs are translated to give
viral proteins (e).
• There is no distinction between early and j
late functions in gene expression.
• Viral RNA replication involves full length
positive strand synthesis (f).
• The positive RNA intermediate is used as
a template for full length negative strand
(g). New full length minus strands may
serve as templates for replication, or
templates for transcription, or they may be packaged into new virions.
• Since this is a negative-strand RNA virus, RNA polymerase and RNA modification enzymes
are packaged in the virion (h).
• The viral glycoproteins are translated as transmembrane proteins and transported to the cell
plasma membrane (i).
• M (matrix) protein enables nucleocapsids to interact with the regions of the plasma membrane
which have the viral glycoproteins inserted.
• The virus buds out through membrane (j).
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Disease mechanism
• The virus initially replicates in the epithelial tissues of the upper or the lower respiratory tract. In
the case of measles and mumps, this is followed by replication of the virus in the lymphoid tissues
leading to viremia and growth in a variety of other sites.
• Parainfluenza or RSV replicate predominantly in the respiratory tract and are not systemic.
• Infections with measles and mumps viruses are systemic. Mumps usually causes a benign
systemic febrile illness with swelling of salivary glands. Measles causes a childhood rash.
• Measles and mumps exist as a single serotype. MMR (mumps, measles, rubella) vaccine contains
live, attenuated forms of all three of these viruses.
• One important and unique feature of measles virus infection, in particular, is the virus’ ability to
persistently infect brain cells, which has been implicated in subacute sclerosing panencephalitis
(SSPE).
Specific human diseases:
Measles
• Highly contagious disease spread by

respiratory droplets
• Most serious childhood exanthem
• Infects epithelial cells of the respiratory
tract. Can infect conjunctiva, respiratory
tract, urinary tract, small blood vessels,
and CNS
• Prior to rash patients experience high fever, three C’s

(cough, coryza, conjunctivitis) and photophobia
• First rash appears in the mouth, it is called Koplik
spots (small while lesions, surrounded by red halo)
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• A day later rash appears behind the ears, and spreads all
over the body, sometimes becoming confluent
• Rash is caused by T-cell response to virus-infected epithelial
cells lining the capillaries
• Cell-mediated response is necessary for resolution
• Antibody is not sufficient because measles can spread from
cell to cell
• Sequelae in central nervous system may result from
immonopathogenesis
• Vaccine: Measles/Mumps/Rubella live vaccine
166
Mumps
• Enters epithelial cells of respiratory tract

• Spreads systemically by viremia (right figure)
• Up to 1 in 5 people infected with the mumps
virus have no signs or symptoms
• When signs and symptoms do develop, they
usually appear about two to three weeks after
exposure to the virus and may include:
• Fever, fatigue, swelling of parotid gland (due
to the viral infection of parotid gland, right
bottom)
• Rarer complications are: orchitis (infection of the testes) and
meningitis
• Cell mediated immunity is essential for control of infection and is
responsible for causing a portion of the symptoms
• Antibody is not sufficient because mumps can spread cell to cell
• Vaccine: Measles/Mumps/Rubella live vaccine
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Mumps diagnosis
• Serologic assays
• IgM test using EIA and IFA
• Molecular techniques
• Real time RT-PCR to detect mumps viral RNA.
• Virus isolation and growth in tissue culture
• Urine, saliva, pharynx
• Syncytia will be observed
• Infected cells will be able to hemadsorb rbc (due to viral hemagglutinin)
Parainfluenza
• There are 5 serotypes of Parainfluenza
• Infection is limited to the respiratory tract, no viremia.
• The incubation period is 1-7 days.
• Pathophysiology includes colonization of the nose
and the nasopharynx, invasion of the respiratory
epithelium, cell damage, inflammation, downward
spread of fibrinous exudate, excessive mucus
production, edema, and loss of cilia.
• Respiratory epithelium is sloughed off, which causes
interstitial infiltration of the lung.
• Patients present with cold-like symptoms,
nonproductive to minimally productive barking cough, coryza, inspiratory stridor, fever, nasal
congestion, pharyngeal erythema and more rarely bronchitis and wheezing.
• Inflammation of the larynx and laryngeal muscle spasm account for lost voice.
• In children, between ages 6 months and 3 yr. the disease is called laryngotracheobronchitis
(croup), although bronchiolitis and pneumonia may occur.
• Tachycardia, tachypnea (when lower airways become involved) is stimulated by excess CO2.
• Protective immunity is of short duration.
“Steeple sign” in Parainfluenza croup. Steeple sign on CXR results
from subglottic narrowing of the trachea (arrow) and is indicative
either of laryngotracheobronchitis (croup). Viral infection causes
croup which leads to the swelling of the larynx, trachea, and large
bronchi due to infiltration of white blood cells. Swelling produces
airway obstruction which, when significant, leads to dramatically
increased work of breathing and the characteristic turbulent, noisy
airflow known as stridor.
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Respiratory syncytial virus
(RSV)
• RSV is most serious in infants

from birth to 12 months of age and
causes 90,000 hospitalizations
and 4500 deaths each year in the
US.
• Premature babies infected with
RSV are at risk for respiratory
distress.
• RSV infection is limited to the respiratory tract
• There is no viremia
• In adults RSV typically causes a mild upper respiratory infection but in children lower respiratory
infections are more common.
• Spread of the virus down the respiratory tract occurs through cell-to-cell transfer of the virus along
intracytoplasmic bridges (syncytia from the upper to the lower respiratory tract).
• RSV is the most important viral cause of lower respiratory disease in infants and small children.
• Narrow airways of young infants are readily obstructed by virus-induced and immune pathology.
• Most common disease, bronchiolitis, is likely mediated by host’s immune response.
• Maternal antibody declines rapidly in infants and does not offer long protection in the newborn.
In infants, most common symptoms include:
 Fever (typically low-grade)
 Cough
 Tachypnea
 Cyanosis
 Retractions
 Wheezing sound on expiration
 Bronchiolitis
 Pneumonia (results from cytopathological spread of virus)
• Natural infection does not prevent re-infection.
169
Treatment for RSV
• Synagis (Palivizumab)
• Humanized IgG1 against F protein for high-risk babies
Pathophysiology of RSV
Comparing and contrasting various diseases of the respiratory tract
170
TOGAVIRIDAE and FLAVIVIRIDAE
• Toga and Flaviviridae are enveloped positive

RNA viruses consisting of more than 400
members, which are transmitted primarily (but
not exclusively) by arthropod vectors such as:
mosquitoes, sand-flies, fleas, ticks, lice, etc.
These viruses were previously grouped
together under the name 'Arboviruses'
(arthropod-born-viruses). They are relatively
fragile viruses and therefore many are reliant
on vector for transmission.
• Both replicate in the cytoplasm.
• Togaviruses: Single-stranded, (+)sense, non-segmented RNA, resembles cellular mRNAs: i.e. 5'
cap, 3' poly-A.
• Flaviviruses: Single-stranded, (+)sense non-segmented RNA, 5' cap but not polyadenylated.
• Toga - bud at the plasma membrane, Flavi –
bud at the internal membrane.
• These viruses can be asymptomatic, or
cause nonspecific symptoms (fever, chills),
but could also lead to encephalitis, arthritis
and hemorrhagic fever.
• Rubella (Toga) and Hepatitis C (Flavi) have a
strictly human-to-human transmitted.
• Can survive for long periods in hosts such as
ticks by replicating in this host (without damage
to the insect).
• Although these are enveloped viruses they
usually cause lysis of the cell because they
change the permeability, and introduce several
other insults to the cell that ultimately lead to
cell death.
• There are live attenuated vaccine for yellow fever and Japanese encephalitis (but not routinely
administered).
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Replication cycle of Flaviviridae (note the difference between exocytosis, shown here, and
budding, shown earlier)
A. Virions attach to the

surface of a host
cell
B. Enter the cell by

receptor-mediated
endocytosis.
Endosomal vesicle
triggers
conformational
changes in the
virion, and results
in particle
disassembly.
C. In the cytoplasm,
the positive-sense
RNA is translated into a single polyprotein that is processed co- and post-translationally by
viral and host proteases.
D. Genome replication occurs on intracellular membranes.
E. Virus assembly occurs on the surface of the endoplasmic reticulum (ER) when the structural
proteins and newly synthesized RNA buds into the lumen of the ER.
F. The resultant non-infectious, immature viral and subviral particles are transported through the
trans-Golgi network (TGN).
G. Mature virions and subviral particles are subsequently released by exocytosis.
172
Most human-disease causing Togaviridae and Flaviviridae are transmitted by arthropods such
as mosquito
Mosquito life cycle
• Mosquitoes acquire virus by blood meal from

a viremic host.
• The virus then infects the epithelial cells of
the mosquito’s gut, goes to circulation and
infects the salivary glands. Only female
mosquitos support replication cycle.
• It then persists in the salivary glands which
release the virus in to the viral saliva.
• Only those species that can get infection of
the salivary glands, will transmit the virus to
humans. This happens when the mosquito
bites its host and regurgitates virus-
containing saliva into the host’s bloodstream.
On the left are examples of common arboviruses.
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Disease mechanism
Togaviruses and Flaviviruses – Mosquito transmitted examples
• Most are arboviruses have a broad host range including vertebrates and invertebrates.
• Most viral infections do not progress beyond the initial viremia in the blood.
• If sufficient virus is produced during initial replication in the WBC of the blood (usually in
monocytes) then other organs such as brain, liver, skin and vasculature can be effected.
• Although these viruses exit by exocytosis, they usually lyse the cell (even being enveloped)
because they change the permeability of the cell.
• Both cell mediated and humoral immunity are important for to control infections.
Typical transmission of arboviruses that cause meningitis/

Togaviruses and Flaviviruses: encephalitis
Encephalitis
• Virus is transmitted from the salivary
glands of the mosquito to the
bloodstream of the vertebrate host.
• From circulation, virus travels to the skin
and reticuloendothelial system (spleen
and lymph nodes), where the primary
infection occurs. Replication in these
organs leads to a more pronounced viremia leading to systemic infection.
• Acute onset of fever, headache, and vomiting progress to signs of meningeal involvement (stiff
neck and back)
• More pronounced CNS involvement than meningitis alone
• After acute symptoms, evidence of neuronal damage such as: drowsiness, coma, paralysis,
convulsions, ataxia and organic psychoses may occur.
• CSF pleocytosis is usual, with up to 1,000 leukocytes/mm3.
• In CSF, mononuclear cells usually predominate, although early in fulminant encephalitis,
polymorphonuclear leukocytes predominate, glucose concentration in the CSF is normal, and the
protein is increased.
• The peripheral blood shows a moderate polymorphonuclear leukocytosis.
• With recovery from acute viral encephalitis, evidence of neuronal injury and death becomes
apparent as residual neurologic defects, impairment of intelligence, and psychiatric disturbances.
• The severity of these sequelae varies according to the causative virus.
174
• Encephalitis can be caused by Togaviridae (Venezuelan equine (VEE), Western equine (WEE) and
Eastern equine (EEE)), and by Flaviviridae (West Nile and Japanese encephalitis)
• Infection with these viruses causes mild symptoms in 20% of the cases that last a few days and will
cause encephalitis in 1% of WNE-infected people
Flavivirus infections:
Hemorrhagic fevers
Common symptoms:
• Flu-like
• Chills
• High fever
• Malaise
• Myalgias
• Headache
• Nausia and vomiting
• Possible Hemorrhagic diathesis, hypotension, shock
Lab findings:
• Leukopenia (often neutropenia)

• Thrombocytopenia
• Increase in hematocrit
• Proteinuria
• +/- increase in AST/ALT
Dengue fever
• Endemic to North and Central Africa, S.E. Asia and some

areas in Caribbean
• There are 100 million Dengue cases per year. 300,000
cases per year are of a more serious infection called
Dengue hemorrhagic fever (DHF) or Dengue Shock
Syndrome (DSS).
• Dengue fever is also known as break-bone fever, the
symptoms of dengue include:
o fever, headache, rash, platelet loss, bleeding from mucous membranes, and serious
bone pain.
• Severe disease is marked by two problems: dysfunction of endothelium and disordered blood
clotting.
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• If a person with Dengue fever is re-challenged with another strain of Dengue fever, he will develop
dengue shock syndrome (DSS)/ DHF which results in severe weakening and rupture of
vasculature and internal bleeding.
• Because the principal targets of dengue are cells of the monocyte/macrophage lineage, a
newly acquired serotypes of Dengue virus can be opsonized by antibodies that were produced
against the
previous
dengue virus
serotype (which
bind to the
virion but do not
neutralize its
infectivity);
hence, this virus is more avidly taken up via Fc receptors into the very cell in which it replicates
best (see below).
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Above: Antibody dependent enhancement in Dengue fever. Opsonizing antibodies against Dengue
facilitate its entry into target macrophages.
Diagnosis:
• Test blood using RT-PCR for the presence of the viral genome
• Test blood using ELISA to detect viral antigens or patient’s antibodies
• Test CSF for antibodies to the virus
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Yellow fever:
• Yellow fever (YF) was the first arthropod-borne viral

disease identified.
• Transmission is human-mosquito-human. Thus, like
dengue fever and unlike most other arboviruses, YF
reaches high titers in human blood. The major organ
target is the liver, and massive necrosis of
hepatocytes leads to a decrease in the rate of
formation of prothrombin as well as to jaundice.
• After a brief remission a minority of patients progress
to severe jaundice, massive gastrointestinal
hemorrhages, hypotension, dehydration, proteinuria, and kidney failure.
• Infection of the skin, bone marrow and blood vessels is noted. Heart damage is also seen.
• Massive gastrointestinal hemorrhages are called “black vomit”.
• Mortality from this severe form of the disease is 20 - 50%.
Figure left: Phase 1 (period of infection) - corresponds to

the host's initial viremia and typically presents with nausea
and vomiting, fever, headache, dizziness, and myalgia.
Leukopenia may be identifiable at the beginning of the acute
symptoms, while liver enzymes typically increase later. The
viral load will typically reach its maximum 2 to 3 days after the
initial infection. Importantly, those cases that end in fatality will
often have a higher and extended duration of viremia relative
to non-fatal cases.
Phase 2 is known as the remission, can be identified by a

break in the fever and relief from the clinical symptoms. The
remission can last up to 2 days, but is not a necessary part of
the natural history of disease. Some persons do
not experience any remission in their clinical course. On the
other hand, some infected persons will recover entirely during
this remission and are referred to as abortive infections.
These persons also do not demonstrate the jaundice
associated with the severe third phase, from which the
disease and the virus derive their names.
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Phase 3 is referred to as the intoxication phase, and is the period of greatest clinical severity. Approximately 15% of
infected individuals enter the intoxication period, when more advanced liver dysfunction begins to manifest. Jaundice is
typically present, along with the return of nausea, vomiting and fever. Due to the associated coagulopathy, bleeding
diathesis is also common at this stage. Multiple organ failure is common, with kidney, heart and neurologic involvement,
depending on the extent of liver damage. As mentioned above, the case-fatality is 20% among endemic populations.
Diagnosis
Similar to Dengue fever:
For yellow and dengue fevers patient blood can be directly examined for viral genome by RT-PCR and
for viral antigens by monoclonal antibodies that are directed against the individual strains of viruses.
Serological methods including ELISA can be used to diagnose a recent infection, but serological cross-
reactivity among viruses limits the distinction of the actual viral species.
In the cases of encephalitis, the measurement of IgM in a patient’s CSF can be used for diagnosis.
Both Toga and Flavi viruses are difficult to grow and analyze in tissue culture.
Togavirus and Flavivirus disease exceptions (not transmitted by vectors):
Rubella
• Has a limited host range – infecting mammalian cells only.

• Rubella is transmitted by aerosols and is
highly contagious.
• After early viremia, virus multiplies in many
organs, particularly lymph nodes
(lymphadenopathy), including the placenta.
• In children, it causes a mild febrile illness
characterized by maculopapular rash and
swollen glands (less severe than measles).
 The rash is a continuous maculopapular
and pink. It is seen 14-25 days after
infection in 95% of infected patients.
 Patients are infectious for most of this
time as well as several weeks after the
onset of the rash.
• In adults, in addition to the rash, rubella may cause more severe disease including bone pain and
arthralgia (joint pain). Rash is mediated by immune complexes rather than direct viral damage.
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• The most serious outcome of rubella is the fact that it

crosses the placenta and causes congenital defects
(most risk prior to week 20 post gestation) which include
cataracts, mental retardation, deafness, and heart
defects.
• Studies have demonstrated that cells infected with
rubella in the early fetal period have reduced mitotic
activity.
• Live attenuated MMR vaccine prevents Rubella.
• For women infected during first trimester of pregnancy, therapeutic abortion may be
recommended.
Spread of rubella virus within the host (right). Rubella enters

and infects the nasopharynx and lung and then spreads to the
lymph nodes and monocyte-macrophage system. The resulting
viremia spreads the virus to other tissues and the skin.
Circulating antibody can block the transfer of virus at the
indicated points (X). In an immunologically deficient pregnant
woman, the virus can infect the placenta and spread to the fetus.
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Hepatitis C - (formerly known as Non-A, Non-B hepatitis)
• Hepatitis C is a disease unique to other Flaviviruses because it is not spread by arthropods.

• Hepatitis C is a worldwide problem. The hepatitis C virus (HCV) is a major cause of both acute
and chronic hepatitis. WHO estimates that 170 million individuals worldwide are infected with
HCV.
• Transfusion of blood contaminated with HCV was once an important source of transmission. Since
1990, all blood products are screened for Hepatitis C. Since the screening, needle-stick
transmission via non-sterile needles or needle stick injuries are the predominant ways to acquire
HCV. HCV may also be transmitted via tattooing, sharing razors, and acupuncture
Disease progression:
• Incubation period is 6-8

weeks
• Infection with HCV is self-
limited in only a small
minority of infected
persons.
• In most infected people,

viremia persists and is
accompanied by variable
degrees of hepatic
inflammation and fibrosis.
Findings from studies
suggest at least 50% of
hepatocytes may be
infected in HCV patients
with chronic hepatitis.
• In 70% of affected people acute illness lasts 2-3 years, followed by a silent period of 10-15 years,
followed by chronic illness.
• Development and persistence of strong virus-specific responses by cytotoxic T lymphocytes and

helper T cells is important for viral clearance.
Treatment:
• Depends on the genotype:
• Combination therapy with interferon-alfa (called PEG-IFNα) and nucleoside analogue ribavirin
• Combination of PEG-IFN, ribavirin and protease inhibitors
• Newer drugs sofobuvir + ledipasvir approved in 2014. Sofosbuvir inhibits RNAdRNA pol.
Ledipasvir is an inhibitor of HCV’s transcription activator NS5A.
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LECTURE 17: RHABDOVIRIDAE

Describe biochemical and clinical features of Rhabdoviridae.
• Describe the structure and unique features of rabies

• Characterize rabies infection
• Describe the roles of passive and active immunizations
Unique properties of Rhabdoviridae
• Bullet shaped, Enveloped

• Single stranded negative RNA with 5 genes
• As other RNA viruses rabies brings its own RNA
dependent RNA polymerase into the cell in order to
make short mRNAs
• RNA dependent RNA polymerase makes single gene-
unit length mRNAs (i.e., each mRNA encodes only a
single protein)
• This is achieved by the use of transcriptional start and stop signals, which are located at the
boundaries of all of the viral genes.
• mRNAs are capped at the 5' end and polyadenylated at the 3' end
• Replication occurs in the cytoplasm
• The release of the virus does not result in cell death
Mechanism of disease of rabies
Zoonotic with reservoirs being wild carnivorous animals including skunks, raccoons, bats and
unvaccinated dogs and cats
o Transmitted by saliva, acquired from the bite of a rabid animal.
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o Can also be spread by aerosol (in bat caves), and transplantation of infected tissue
Pathogenesis of rabies - see figure on the right:
Incubation phase (1-2)
• The virus binds to the muscle cells at the site

of the inoculation via nicotinic acetylcholine
receptors. Here the virus replicates and can
remain for a prolonged period of time (up to
several months). The length of the incubation
phase depends on the infectious dose and
proximity to CNS.
Prodrome phase
• After weeks to months, the virus infects the

peripheral nerves through neurotropin (3-5)
and travels up the spinal cord to the brain (6).
• When the virus multiplies in the brain (7) it
produces Negri bodies (inclusion bodies) that
are visible in stained brain sections using a
Rabies transmission within the body
light microscope.
• Symptoms at this stage of rabies include fever, headache, itching, burning at the bite site
Neurological phase
• During the neurological phase, the virus spreads from the brain to the skin, eyes, and salivary
glands from where it is transmitted (8).
• Infection of the brain leads to encephalitis and subsequent neural degeneration
• Symptoms before death include extreme irritability, sensitivity to light and sound, depression,
fatigue, fever and eventually paralysis. Hydrophobia sets off spasms that are characteristic of the
disease in humans and animals in later stages
• Involvement of the brain leads to coma and death
• The virus causes little cytopathic effect except Negri bodies in the infected neurons
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• There are various factors that determine the

timing of the onset of symptomatic rabies but
most important are the number of virus
particles in the infection and how close the
bite is to the brain.
• The immune response to naturally

acquired virus is slow and a good
neutralizing response is not seen until the
virus has reached the brain which is too
late for survival.
• Cell-mediated immunity plays little role in a

rabies infection.
• Rabies infection does not elicit an antibody
response until the late stages
Treatment:
• Because the virus has a long incubation

period, a good immune response that Rabies disease progression
eliminates the infection can be achieved
using a killed vaccine even after infection.
• Post-exposure rabies treatment usually involves passive immunization administered into the
wound followed by active immunization with a killed rabies vaccine administered at a different
site.
• Since the pathophysiology of rabies virus infection appears to be primarily neuronal dysfunction
rather than inflammation and cell death, the clinical syndrome of rabies encephalitis is theoretically
reversible. A fundamental requirement for recovery would be viral clearance and development of a
protective immune response.
• After symptom onset, rabies is almost always fatal and only three survivors of symptomatic rabies
have been documented.
Diagnosis
• Unfortunately, due to the lack of evidence for infection during asymptomatic phase, the lab
diagnosis usually occurs too late, and is mainly used to confirm the diagnosis postmortem.
• Viral Ag can be detected in CNS, skin, saliva, and blood using direct IF or RT-PCR.
• Antibodies to rabies can also be detected too late in the disease in CSF, and serum using
ELISA.
• Brain biopsy can also be assayed for Negri bodies (the only CPE that can be detected in
infected cell).
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LECTURE 18: RETROVIRIDAE
Learning Objectives
Describe in detail the replication cycle of HIV.

• Detail the entry, reverse transcription, integration, mRNA synthesis, replication, protein
• synthesis, exit, and the role of protease in HIV
• Describe the disease mechanism and stages of HIV
• Be able to read and Describe HIV serology as it relates to various stages of HIV
• Describe briefly the stages of HIV replication targeted by drugs
Retroviruses
The three subfamilies of human retroviruses are the Oncovirinae (HTLV-1, HTLV-2, HTLV-5),
the Lentivirinae (HIV-1, HIV-2), and the Spumavirinae. The retrovirus genome is positive RNA (but
not infectious) and has a 5′-cap and is polyadenylated at the 3′-end.
Unique features of HIV
• Enveloped with a capsid, contains 2 copies of ss RNA (positive-strand) genome

• Reverse transcriptase, integrase, and protease enzymes are carried in the virion
• Replication proceeds through a dsDNA intermediate, termed a provirus
• Provirus integrates into the host chromosome and becomes a cellular gene
• Transcription of the genome is regulated by host transcription factors interacting with promoter
and enhancer elements in the LTR portion of the genome
• HIV can also spread from cell to cell through the production of multinucleated giant cells, or
syncytia
• HIV is easily inactivated and must be transmitted in bodily fluids
HIV virion
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There are two general types of retroviruses: simple ones and complex ones.
• Simple retroviruses contain a core genome, consisting of three genes:

o Gag (encodes structural proteins)
o Pol (encodes enzymes)
o Env (encodes envelope proteins).
o Examples of simple retroviruses are murine leukemia virus (a virus which causes
cancer in mice) and human T-cell leukemia virus (the first human retrovirus
discovered in 1982 and can cause cancer in the body’s developing T cells).
• Complex retroviruses have additional accessory genes that carry out diverse functions
inside an infected cell. HIV is one example of a complex retrovirus, consisting of the
three essential genes (Gag, Pol, and Env) and six accessory genes: Vif, Vpr, Vpu, Rev,
Tat, Nef.
• In HIV, each gene can produce more than one final protein through the mechanism of
alternative RNA splicing, post translational modifications and frame shift mutations
during translation.
Simple retrovirus Complex retrovirus (HIV)
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Replication of HIV
Entry of HIV into cells
1.
2.
3.
The initial co-receptor used by the virus is CCR5, which is expressed on myeloid cells
(macrophages, DC and monocytes). The viruses attaching to CCR5 are called [M]-tropic.
Later, during chronic infection of a person, the envelope gene mutates so that the gp120 binds to a
different chemokine receptor called CXCR4, which is expressed primarily on T cells. Such viruses are
considered T-tropic.
Reverse transcription/Integration
• RdDpol activity of RTase makes DNA strand complementary to genomic RNA

• RNAse H of RTase destroys RNA
• DdDpolymerase activity of RTase allow formation of double-stranded viral cDNA
• Viral cDNA is integrated into host chromosome
Transcription/Replication/Translation of HIV genome
• Usually takes place in activated T cells
• T cells are activated by HIV or by being primed for infection
• Viral mRNAs and genomic RNA are made and are transported to the cytoplasm
• Late proteins, Gag, Env and Pol are translated as polyproteins
Assembly and Budding
• Env polyprotein (gp160) is cleaved into gp120 and gp41 in the host cell by host proteases
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During budding and release:

1.
• Gag and Gag-Pol are translated as
polypeptides
• Many identical Gag polyproteins are
cleaved by the viral protease into 4.
individual matrix and nucleocapsid
proteins. 5. 3. 2.
• Many nucleocapsid proteins assemble
together to form a nuncleocapsid and 6.
many matrix proteins together form the 6.
matrix.
• Pol polyprotein is cleaved into protease,
reverse transcriptase and integrase
• Virus can cause syncytia formation with 7. 7.
subsequent lysis of these cells
Viral lifecycle:
http://www.youtube.com/watch?v=RO8MP3wMvqg 8.
9.
Summary:
1-3. Attachment /fusion/uncoating
4. Viral RT converts ssRNA to dsDNA
5. Viral dsDNA integrates 10
6. Cellular transcription/genome replication
7. mRNA export (both short mRNA and viral
genome)
8. Cellular translation, protease cleavage
9. Viral assembly
10. Viral budding (After envelopment and release
from the cell, the viral protease cleaves the Gag
and Gag-Pol polyproteins to release the reverse
transcriptase and form the virion core, thus
ensuring the inclusion of these components into
the virion. The protease step is required for the
production of infectious virions)
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HIV infection
Sexually active people (homosexual and
heterosexual), intravenous drug abusers and their
sexual partners as well as the newborns of HIV- 1.
positive mothers are at highest risk for HIV infections. 3.
2.
1) During sexual transmission, HIV infects a mucosal
surface, enters and rapidly infects cells of the 4.
mucosal-associated lymphoid tissue (MALT). HIV

enters the body mainly through the vagina,
penis, colon-rectum, or blood.
2) Mucosal APC’s will pick up the virus and transfer it 5.
to the regional lymph nodes, where the virus will

infect many lymph node CD4+ T cells and replicate in
them.
3) High viremia results from the increased virus replication in the lymph nodes and infected CD4+ T
cells leaving the lymph nodes to enter the blood. At some point in infection, the virus shifts its tropism
from M-tropic (R5) to T-tropic (X4 virus). It now mainly infects CD4 T cells via CD4 and CXCR4
chemoreceptor.
4) CD4+ T cells are gradually destroyed by the pathological effects of the virus.
5) Loss of CD4+ T cells leads to immune system dysfunction and rise of opportunistic infections
Infection of cells of the brain including microglial cell and neurons eventually causes the release of
neurotoxic substances and promote inflammatory response in the brain.
CD8 T cells rather than antibodies are critical for controlling HIV disease progression. CD8 T
cells can kill infected cells by direct cytotoxic action and can produce suppressive factors that restrict
viral replication.
However because CD8 T cells require CD4 T cells to help them divide, CD8 T-cell numbers
decrease with decreasing CD4 T-cell numbers, and their reduction correlates with and is an indicator
of disease progression to AIDS.
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Disease Progression
HIV infection and disease progression

are divided into 3 stages based on virological,
immunological, and clinical characteristics:
Stage 1 = Acute phase (first 3 months)
• Initial HIV symptoms occur 2-4 weeks

after infection, resembling influenza or
mono, and usually subside after several
weeks. 95% of people seroconvert by 4
weeks (i.e. develop anti-HIV antibodies).
• During acute stage, HIV replication
explodes as the virus gains access to
many target cells in the lymph nodes.
• This surge in viral replication is eventually
partially controlled by the adaptive
immune response (mainly CTL’s and to some extent plasma cells making anti-viral Ab’s).
Stage 2 = Chronic phase (lasts years)

• This stage is also termed “asymptomatic” or “latency” because many infected individuals don’t
present with overt clinical symptoms. During this time, HIV replication is counterbalanced by
HIV destruction via the immune system. Nevertheless, CD4+ T cells continue to decline and
HIV continues to grow in lymph nodes. Antibody to HIV is readily detected at this stage. When
CD4 T cell count drops below
450-500 cells per microliter,
opportunistic infection may Opportunistic infections seen in HIV
begin to appear, and

increased number of viral
RNA appears in the blood.
Stage 3 = AIDS
When CD4+ T cells drop
below 200 cells/µl, life-threatening
opportunistic infections appear
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(i.e., Kaposi’s sarcoma, CMV, toxoplasmosis, Pneumocystis pneumonia). Most of these infections
DO NOT cause disease in immunocompetent hosts, but can cause severe infections in people with
compromised immune systems, such as AIDS patients.
Why doesn’t the immune system control HIV infection?
1) Subversion: HIV infects and destroys the very immune cells needed to help control the virus.
2) Mutations: HIV acquires numerous mutations

during reverse transcription that lead to antigenic
drift of viral proteins, thereby making it harder for
the immune system to recognize viral antigen.
Specifically the genes coding for the reverse
transcriptase and gp120 are heavily mutated.
Additionally, the gp120 of HIV is extensively

glycosylated which changes its antigenicity.
3) Latency: If HIV infects resting memory CD4+

T cells, the virus will remain latent or quiescent
with viral replication minimal in these cells. The
immune system will not be able to recognize
these infected cells. At some later point, these
resting memory cells can be “awakened” and
begin producing substantial amounts of virus.
4) Immune exhaustion: Persistent exposure of immune cells

to HIV antigen causes the immune system to prematurely
shut down due to upregulation of inhibitory receptors, inducing
cell anergy.
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CD4 T cells have a

critical role in activating
and regulating cell-
mediated immune
responses, especially
toward intracellular
pathogens. Human
immunodeficiency
virus (HIV)-induced loss
of CD4 T cells results in
loss of the functions shown, especially the delayed-type hypersensitivity responses and the cytokine
control of immune responses.
The destruction of immune function as a result of HIV infection leads to increased

susceptibility to opportunistic infection and eventually to death
AIDS affects most systems within the body including CNS, lungs, GI and skin. HIV transmission to the fetus
• In the absence of treatment, the

risk of HIV transmission from
mother to child is 25-30%.
• Antiretrovirals medication lower
mother-to-infant transmission to
less than 2%.
• The vast majority of transmission
of HIV from mother to child
occurs at the time of birth when
the membrane is ruptured and
the blood systems can mix
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A number of diagnostic tests can be used to determine whether a person is infected with HIV:
• Blood tests are the most common way to diagnose HIV. The most sensitive of these tests is a
PCR-based method to detect HIV RNA (2 above) in the blood.
• Depending on the estimated stage of disease, either an indirect ELISA to detect patient’s anti-
HIV Ab’s (1 above) or a sandwich ELISA to detect HIV Ag (4 above) can also be used. A
positive ELISA test is usually confirmed by Western Blot.
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AIDS diagnosis occurs when certain criteria are met:
1. Evidence of HIV virus infection & < 200 CD4+ T cells/µl or

2. Evidence of HIV virus infection & one AIDS-defining illness.
Treatment
For years, most AIDS experts have recommended starting antiretroviral drugs when the CD4
count fell below 350. One reason for waiting was that HIV-fighting drugs can cause unpleasant side
effects. The new guidelines say treatment should begin as soon as possible, regardless of the CD4
count. Drug treatment can’t totally get rid of the virus. But it can keep the amount of virus in the body
low enough to slow down and often prevent destruction of the immune system. It will also limit the
spread of HIV from person to person. Some exciting new data indicate that the use of one or more of
these drugs can prevent infection in individuals who do not have HIV but who are high risk for it. In
fact, the FDA recently approved the use of Truvada (a combination of two antiretroviral drugs) to
reduce the risk of becoming infected with HIV by sexual transmission of the virus.
There are 25 anti-HIV drugs clinically available today to treat HIV infection, and it is highly
recommended that a combination of drugs (encompassing different classes) be used to minimize the
emergence of drug-resistant virus.
Classes of anti-HIV drugs:
1) nucleoside RTase inhibitors (NRTI) = inhibit HIV RT and the process of reverse transcription
2) non-nucleoside RTase inhibitors (NNRTI) = inhibit HIV RT and the process of reverse
transcription
3) integrase inhibitors (II) = inhibit HIV integrase and the process of proviral integration into host
DNA
4) fusion/binding inhibitors = inhibit HIV binding to CD4 or gp41-mediated fusion
5) protease inhibitors (PI) = inhibit HIV protease and the process of HIV protein maturation
6) CCR5 antagonists = prevent interaction of gp120 with CCR5R
HAART = 2 NRTIs + a PI or NNRTI or II
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Virology practice questions:
1. A patient presents to your office with chest pain and fever. You suspect a viral cause for his
condition. Which of the following would differentiate the suspected virus from Rhinovirus?
a. A gene for RNA dependent RNA polymerase

b. Presence of envelope
c. Ability to replicate in the nucleus
d. Resistance to acidic environment
e. Stability inside the cell
2. Which of the following is true regarding viral pathogenesis?
a. Viruses that are acquired through respiratory do not cause viremia

b. Humoral immunity but not cell mediated immunity (CMI) acts
against viruses
c. Viruses that are acquired via skin contact can cause viremia
d. RNA viral mutations can only occur in the cytoplasm
e. RNA viruses are more likely than DNA viruses to cause persistent infections
2. Which of the following is a major difference between Hepatitis B and Hepatitis C?
a. Tissue tropism
b. Polymerase
c. Symptoms
d. Immune response
e. Transmission
4. A virus was isolated from the stool of a patient with flaccid paralysis. What else is a likely
characteristic of this virus?
a. Two versions of oral vaccines are available for public use

b. Viral infection of the motor neurons can lead to latency
c. Immunity can be transferred by serum from recovered person
d. Sub-clinical infection is uncommon
e. Relatives of the patient are likely to develop paralysis
5. Regarding viral hepatitis which is the wrong answer?
a. HBV-Abs can be diagnosed in the lab by ELISA

b. Hepatitis B vaccine will prevent HepB-induced hepatocellular carcinoma
c. Hepatitis B can cause liver cancer and integrate into host genome
d. Blocking RNAseH activity would prevent the spread of Hepatitis B virus
e. In chronic Hepatitis B one finds HBsAg and pentameric anti-HBc antibodies
6. Which of the following is correct regarding varicella and zoster diseases?
a. They are two diseases caused by one virus

b. They have the same clinical picture
c. Zoster is a disease of children, whereas varicella is a disease of
195
elderly and immunosuppressed patients

d. Varicella and zoster can be prevented by killed vaccinate
e. Drugs that function as DNA polymerase inhibitors are toxic to host cells
7. A 31-year-old man presents to your free clinic complaining that his urine is dark and his stools are
pale. On physical examination, you note icteric sclera and conclude that he likely has hepatitis. A
hepatitis serology panel is performed:
Lab Test Result

HBsAg positive
anti-HBcIgM positive
HBV DNA 1010 copies/mL
You see the patient three months later, at which time a second hepatitis serology panel is performed:
Lab Test Result

HBsAg negative
anti-HBs IgG positive
anti-HBcIgG positive
HBV DNA not detectable
What is the most likely interpretation?
a. The man was vaccinated for HBV

b. The man has chronic HBV
c. The man has acute HBV and will become chronically infected
d. The man had acute HBV but cleared the infection
e. These results are inconclusive
f. The man had chronic HBV but cleared the infection
8. Refer to the serological data below, obtained from a patient recovering from Hepatitis B. The x-axis
indicates the number of months before and after acute symptoms (acute symptoms are marked by
month “0”). The y-axis indicates Hepatitis B specific antigens and antibodies detected in the patient.
Which of the following letters point
to the curve(s) that represent(s) the
antibody(s) one use(s) as a marker
of Hepatitis B vaccination?
a. A
b. B
c. C and F
d. C and D
e. E
f. F
9. The replication cycle of which of

the following virus is illustrated
below?
196
a. Hepatitis A
b. HIV
c. Hepatitis B
d. Rubella
e. Herpes simplex 1
10. Pick the correct match for the following rashes and their description:
Measles A. Starts with Koplik’s spots in the mouth

Chicken pox B. Infects erythroid precursors
Erythema infectiosum (B19) C. Uses reverse transcriptase
Roseola D. Rash follows fever, spares the face
E. can be reactivated from neurons as shingles
F. caused by a naked RNA virus
a. Measles→A; chicken pox→F; B19→C; roseola→B

b. Measles→C; chicken pox→B; B19→A; roseoa →E
c. Measles→E; chicken pox→F; B19→B; roseola→A
d. Measles →F; chicken pox→B; B19→E; roseola→D
e. Measles→A; chicken pox→E; B19→B; roseola→D
11. Each of the following clinical symptoms is associated with infection by picornaviruses except :
a. Myocarditis
b. Hepatitis
c. Paralysis
d. Childhood rash
e. Common cold
12. What is the first virus-specific synthetic event that occurs in cells infected with the HIV virus?
a. DNA syntheses
b. Synthesis of –RNA strand
c. Synthesis of + RNA strand
d. Translation to generate viral proteins
e. Synthesis of mRNA
197
13. A virus isolated from blood is RNAse and detergent sensitive. Which of the following is a virus that
was isolated?
a. Hepatitis A
b. Hepatitis B
c. Coxsackie A
d. Coxsackie B
e. Polio
f. Small pox
g. Measles
14. Which of these viruses is not transmitted via a blood transfusion?
a. HIV
b. Hepatitis B
c. CMV
d. Hepatitis C
e. HPV16
15. You identify a new dsDNA, enveloped virus that is persistent and is found in all organs of the
body, including the basolateral lining of the GI tract. Which of the following means of transmission
was most probably not utilized by this virus?
a. Sexual contact
b. Fecal/oral
c. Contact with spit
d. Respiratory secretions
e. Blood
198
MERP Medical Bacteriology
199
BACTERIOLOGY MODULE
LECTURE 19: INTRODUCTION TO BACTERIOLOGY: STRUCTURE AND METABOLISM
Describe the differences between the gram-positive and the gram-negative bacteria.
• Describe various bacterial structures and know detail function these structures serve
• Describe the differences in transcription mechanism of eukaryotes vs. prokaryotes (Describe
lac operon)
• Describe the differences between exotoxins and endotoxins
Describe the difference between aerobic and anaerobic bacteria in terms of the enzymes they encode
and growth conditions they prefer.
• Detail the role of oxygen in the process of glycolysis, anaerobic fermentation, aerobic
• fermentation
• Describe the mechanisms by which bacteria are able to disarm oxygen radicals
• Identify organisms as strict anaerobes, strict aerobes, facultative anaerobes or air‐tolerant
Describe the various stages of the growth curve of bacteria in liquid media.
• Calculate the number of bacteria, generation times etc. based on information given
Bacterial shapes
Bacteria are the smallest living things and lack mitochondria, nucleus, Golgi, ER and lysosomes. This
structure is distinguished from eukaryotic cells and is called prokaryotic.
1. There are several common shapes of bacteria: coccus (spherical), coccibacillus, vibrio
(comma shaped), bacillus (rods), spirillum, and spirochete.
2. In addition to shape, bacteria can arrange in aggregates (due to their “stickiness”). For cocci,
these include diplococci (pairs) streptococci (chains), and staphylococci (irregular or grape-
like).
The basic structure of bacteria can be divided into two main parts: the envelope which containing
many complex and unique molecules and the interior which nuclear body and cytosol.
200
Envelope: cell membrane, cell wall, capsule, flagella, pili
• Protects bacteria against chemical and biological threats

• Carries out many bacterial processes
• Contains appendages that make it possible for bacteria to colonize surfaces
• All bacteria have a cell membrane and cell wall, the other components are optional
CELL MEMBRANE:
• An electron transport system of the cell that is vital for cell growth.
• Cell membrane is similar to eukaryotic cells except that it has many more proteins and no
sterols (except Mycoplasma which has sterols).
• The bacterial chromosome is attached to cell membrane.
• The membrane has proteins that control the entrance and exit of solutes and proteins.
CELL WALL:
Structure essential for bacterial life
• Almost all bacteria have a rigid peptidoglycan

(murein) layers that surround the cytoplasmic
membrane. This structure is totally unique to
prokaryotes.
• Cell wall consists of alternating sugars N-

acetylglucosamine (NAG) and N-acetylmuramic acid
(NAM) connected via β (1,4)-glycosidic bond. The
NAM, NAG layers may be cross-linked into sheets by
amino acids.
• Cell wall is the main feature that distinguishes Gram

positive and gram negative bacteria.
• Gram-positive bacteria have a thick multilayered

cell wall (figure right) with embedded teichoic acid.
The teichoic acids consist of polymers of glycol
phosphate or ribotol phosphate with extra moieties
such as amino acids. A type of teichoic acid called
lipoteichioc acid also has a fatty acid that anchors it
into the cytosolic membrane. Both of these elements
promote bacterial adhesion to the host.
• Beta-lactams prevent bacteria from the final stages

(cross-linking of the peptide subunits) of constructing a cell wall, creating an osmotically
imbalanced bacteria
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• Gram-negative bacteria have a

more complex outer structure.
o They have a thin peptidoglycan

layer, no teichoic acid, BUT they
have an additional outer
membrane (OM) external to the
peptidoglycan.
o The space between cytoplasmic

(or inner) membrane (IM) and OM
is called the periplasmic space
and contains peptidoglycan wall
(not extensively cross-linked), as
well as many metabolic enzymes
and virulence factors.
o Outer leaflet of OM contains lipopolysaccharide (LPS), also called endotoxin. LPS consists
of toxic Lipid A, core polysaccharide, and O antigen. Lipid A is made of hydrophobic
fatty acid chains that anchor the LPS into the bacterial membrane, the core domain
contains an oligosaccharide component that attaches directly to lipid A and commonly
contains sugar heptose, the O antigen is a repetitive glycan polymer. The lipid A domain is
responsible for much of the toxicity of Gram-negative bacteria.
o OM serves as a barrier to organic molecules and hydrophobic molecules. Active transport

and diffusion of nutrients is facilitated by proteins called porins that traverse the entire OM.
• G(+) or G(-) are distinguished based on a gram stain.
The gram stain is called a differential stain since it differentiates between gram-positive and
gram-negative bacteria. Bacteria which stain purple with the gram staining procedure are termed
gram-positive; those which stain pink are said to be gram-negative. The terms positive and
negative have nothing to do with electrical charge, but simply designate two distinct morphological
groups of bacteria.
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The gram staining procedure involves four basic steps (figure bottom):
1. The bacteria are first stained with the basic dye crystal violet. Both gram-positive and gram-
negative bacteria become directly stained and appear purple after this step.
2. The bacteria are then treated with gram's iodine solution.
This allows the stain to be retained better by forming an
insoluble crystal violet-iodine complex. Both gram-positive and
gram-negative bacteria remain purple after this step.
3. Gram's decolorizer, a mixture of ethyl alcohol and acetone,
is then added. This is the differential step. Gram-positive
bacteria retain the crystal violet-iodine complex while gram-
negative are decolorized.
4. Finally, the counterstain safranin (also a basic dye) is applied.
Since the gram-positive bacteria are already stained purple,
they are not affected by the counterstain. Gram-negative
bacteria, which are now colorless, become directly stained by
the safranin. Thus, gram-positive appear purple, and gram-negative appear pink.
With the current theory behind gram staining, it is thought that in gram-positive bacteria the crystal
violet and iodine combine to form a larger molecule that precipitates out within the cell. The
alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, thus decreasing
the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex
within the cell. In the case of gram-negative bacteria, the alcohol/acetone mixture, being a lipid
solvent, dissolves the outer membrane and the cell wall (and may also damage the cytoplasmic
membrane to which the peptidoglycan is attached). The single thin layer of peptidoglycan is unable to
retain the crystal violet-iodine complex and the cell is decolorized.
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CAPSULE:
Generalized structure of bacteria (is this a gram-positive or a gram-negative?)
• Capsule is a polysaccharide
slime layer which serves as a
major virulence factor, but is
NOT essential for survival.
• Capsules are found on both
G(+) and G(-) bacteria.
• Capsules protect against
phagocytosis and resist
complement binding.
• Several bacteria including
Streptococcus pneumoniae,
Haemophilus influenzae and
Klebsiella pneumoniae, produce
a glycocalyx capsule that inhibits phagocytosis.
In addition to, or sometimes instead of the capsules, other bacteria use aletrnative structures to
protect themselves against phagocytosis. Mycobacterium tuberculosis accomplishes this by the
insertion of lipids into its cell wall and Streptococcus pyogenes has the M protein in its cell wall to
decrease phagocytosis. Staphylococcus and Streptococcus use leukocidins to destroy leukocytes
and macrophages and hemolysins to disrupt erythrocytes.
FLAGELLA
• These structures are responsible for locomotion: bacteria may have none, one, or many flagella.
These structures are found on certain G(+) and G(-) bacteria and are made of helically coiled
proteins that are anchored into bacterial inner membrane.
• Proteins called flagellins differ between strains and therefore can be used for differentiation of
bacteria.
PILI
• Pili are made of proteins called pilin that surround a hollow core.
• They are much smaller than flagella in diameter, and are all over the surface of the bacteria. Their
main function is to promote attachment to host cells often in a specific manner. Pili that serve for
attachment are called fimbriae.
• Pili can also function in genetic exchange of DNA between bacteria; these are called F pili or sex
pili. Sex pili are usually found one per cell.
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• Pili can be found on certain G(+) and G(-) bacteria.
ENDOSPORE/SPORE
• Some gram-positive bacteria may form

an endospore (right diagram) within the
cytoplasm. This is a structure that allows
the organism to resist adverse conditions
and is the dormant state of bacteria.
• Spores are made of a material called
“calcium dipicolinic acid” several layers of
peptidoglcycan and keratin.
• Spores are dehydrated structures. They
resists chemicals, boiling and radiation,
conditions that would normally kill the
vegetative cells from which they formed.
• Nearly all household cleaning products, alcohols, and detergents have little effect on spores.
• 10% bleach is effective against endospores when they are left in the bleach for at least 10
minutes. Endospores can also be destroyed by autoclaving. Some endospores are able to survive
boiling at 100°C for hours, although the longer the number of hours the fewer that will survive.
• The presence of water, glucose and alanine often cause the germination for the spore.
Interior core; cytosol, nucleoid, plasmids

Cytosol:
The cytosol contains many ribosomes which are made of 5 S rRNA, 16 S rRNA, 23 S rRNA, as
well as a single 50 S and a single 30 S protein subunit. There is little structural but considerable
functional homology between prokaryotic and eukaryotic ribosomes. Most ribosomes at any given
time are involved in the translation of mRNA.
Nucleoid:
Bacterial cells lack a membrane defined nucleus. However a discrete region in the bacterial
cytoplasm seems to contain the genetic material and this nucleiod region can often be distinguished
on EMs of cells. Most cells have only one main chromosome which consists of a single, circle of
deoxyribonucleic acid.
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Plasmids:
Bacteria may have pieces of genetic material other than chromosome. These smaller pieces of
DNA are known as plasmids and are defined as extrachromosomal pieces of DNA which are capable
of autonomous (or regulated) replication.
Examples of roles of plasmids:
• Antibiotic resistance - Some plasmids code for proteins that degrade antibiotics-a big
advantage for pathogens.
• Some encode for proteins which confer virulence factors on the host. For example- E. coli
plasmid Ent P307 codes for an enterotoxin which makes E. coli pathogenic.
• Conjugative plasmids - These allow exchange of DNA between bacterial cells
DNA replication, transcription and translation
Similar to eukaryotic cells, bacteria need to carry out the essential processes of 1) DNA replication,
2) transcription and 3) translation.
1) Replication of DNA:
Replication starts at replication forks and proceeds bi-directionally similar to eukaryotic DNA.
Bacterial replication involved an RNA primer and Okazaki fragments, helicase, and primase. Since
bacterial chromosome is circular, replication introduces a lot of torsion in the strands, which is
relieved by the enzyme called gyrase. Quinolones are antibiotics that block bacterial DNA
replication, many through blocking gyrase.
2) Transcription: Bacteria use DNA dependent RNA polymerase for transcription. This enzyme is
composed of several subunits:
• Sigma factor – a protein within DdRpol that
Structure of bacterial
recognizes a particular nucleotide sequence in DNA dependent RNA polymerase
the promoter of a gene and then serves to initiate
transcription. Different bacteria will have a
different number of sigma factors. E. coli, for
example, has seven sigma factors.
• Alpha and beta subunits – are enzymes that
carry out DNA polymerization.
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Differences between eukaryotic and prokaryotic transcription:

1. In bacteria a single RNA polymerase makes all RNA. In humans there different RNA
polymerases for mRNA, tRNA, rRNA.
2. Bacterial mRNA has a much shorter half-life
Bacterial transcription
than eukaryotic mRNA.
3. Organization of genes is different in
prokaryotes and eukaryotes. In bacteria
many genes are organized into multicistronic
operons (Figure right).
An Operon is a unit of transcription containing

several genes (cistrons) that are transcribed as a
single mRNA. The operon usually consists of a
promoter, operator and terminator.
• The operator can specifically regulate
transcription by binding a repressor which
inhibits transcription or binding an activator to initiate transcription.
• The repressors and activators are themselves products of other genes that are outside the
operon and may not be part of multicistronic sequences.
• Operons can be either:
1) inducible - which means they are normally in the OFF state because they are
repressed by a repressor, unless something in bacteria turns them ON or
2) repressible: which means that the operon is usually ON and is transcribed,
unless something shuts it OFF. An example of an inducible operon is the LAC
operon and an example of a repressible operon is the tryptophan operon.
LAC OPERON:
Bacteria prefer to use glucose rather than other less well-metabolized sugars as carbon
source.
When growing in an environment containing both glucose and lactose, bacteria metabolize
glucose and at the same time prevent the expression of the lac operon, the products of which
transport and metabolize lactose.
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Regulation of the LAC operon:

• Negative regulator: The operon is normally in a repressed state because it is normally subject
to negative regulation by the lac operon repressor protein called LacI.
 LacI is encoded by the lacI gene, which is located immediately upstream of the lactose
operon and transcribed by a separate promoter.
 In the absence of lactose, LacI binds specifically to the operator region of the lac
promoter and blocks transcription.
 Presence of lactose releases LacI from the operator, thereby alleviating the
repression. Therefor the lac operon is switched on only if lactose is available as a
carbon source for cell growth, but remains unexpressed if glucose, the cell's preferred
carbon source, is also present.
• Positive regulator: The transcription factor cAMP-dependent catabolite activator protein

(CAP) is a positive regulator of the LAC operon. It is only active when cAMP is bound to it.
 Because glucose inhibits adenylate cyclase, when bacteria grow in glucose the
cytoplasmic levels of cAMP are low and so CAP is not activated.
 When the glucose is depleted, the cAMP concentration rises, resulting in the formation
of activated cAMP-CAP complexes, which then binds the DNA sequence within the
promoter, recruiting RNA polymerase binding and initiating transcription. CAP is an
example of a global regulatory protein that controls the expression of multiple genes; it
controls the expression of over 100 genes in E. coli.
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3. Translation
The process of translation is very similar for eukaryotes and prokaryotes, except that prokaryotes
have structurally different ribosomes and rRNA molecules. Antibiotics such as macrolides,
aminoglycosides and tetracyclines block translation
Virulence factors:
Virulence factors are genetic traits which enhance the ability of bacteria to cause disease.
Virulence factors include: adhesion molecules, structural molecules and secreted factors. Many
bacteria use virulence factors to cause disease by directly destroying tissue; some others release
toxins, which are then disseminated by the blood to cause system-wide pathogenesis.
Certain surface structures of bacteria are powerful stimulators of host responses such as
cytokines which can be protective but are often the significant causes of the disease symptoms (e.g.,
sepsis). For example, on infection with gram-positive bacteria, peptidoglycan and its breakdown
products, as well as teichoic and lipoteichoic acids are released and can stimulate endotoxin-like
pyrogenic acute-phase response. Acute-phase proteins are a class of proteins whose plasma
concentrations increase (positive acute-phase proteins) or decrease (negative acute-phase proteins)
in response to inflammation. Cytokines such as IL1, IL6 and IL8, and TNFα act on the liver to secrete
these proteins. Acute phase proteins include pro-coagulants and bacteria binding proteins that ihibit
bacterial growth but also add to inflammation. Production of disease results from the combination of
damage caused by the bacteria and the consequences of the innate and immune responses to the
infection.
Bacterial toxins are very important virulence factors and are generally divided into two
categories:
• Exotoxins which are secreted proteins produced by some gram-positive and some gram-
negative bacteria
• Endotoxins (ie LPS), produced only by only by gram-negative bacteria.
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The figure on the right describes

the differences between exo and
endotoxins
Exotoxins include degradative

enzymes that cause lysis of cells
or specific receptor-binding
proteins that initiate toxic
reactions in a specific target
tissue. Below are some features
of exotoxons:
• In many cases, the exotoxin is
completely responsible for
causing the characteristic
symptoms of the disease.
• Because an exotoxin can be

spread systemically through
the bloodstream, symptoms may arise at a site distant from the site of infection.
• A specific type of secreted exotoxins called cytolytic toxins include membrane-disrupting enzymes
which break down sphingomyelin and other membrane phospholipids. Hemolysins insert into and
disrupt erythrocyte and other cell membranes.
• Pore forming exotoxins can promote leakage of ions and water from the cell and disrupt cellular
functions or cell lysis.
• Many exotoxins are dimeric and complesed of A and B subunits. The B portion of the A-B toxins
binds to a specific cell surface receptor, and then the A subunit is transferred into the interior of
the cell, where cell injury is induced.
Endotoxin: The lipopolysaccharide (LPS) produced by gram-negative bacteria is a powerful

activator of acute-phase and inflammatory reactions and is termed endotoxin.
• Gram-negative bacteria release endotoxin during infection.

• Endotoxin binds to specific receptors (CD14 and TLR4) on macrophages, B cells, and other cells
and stimulates the production and release of acute-phase cytokines such as IL-1, TNF-α, IL-6, and
prostaglandinds.
• At low concentrations, endotoxin stimulates the mounting of protective responses such as: fever,
vasodilation, and the activation of immune and inflammatory responses.
• High concentrations of endotoxin can activate the alternative pathway of complement and
production of anaphylotoxins (C3a, C5a), contributing to massive vasodilation and capillary leakage.
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• In combination with TNF and IL-1, this can lead to Effects of LPS
hypotension and shock.
• Disseminated intravascular coagulation (DIC)
can result from the activation and dysregulation of
blood coagulation pathways.
• Therefore, high levels of gram-negative bacteria
are very dangerous and can lead to gram-
negative bacterial sepsis.
Energy production:
Fueling reactions provide bacteria with energy
and precursor metabolites used to make amino
acids, nucleotides, sugars, fatty acids and other
building blocks. Essential minimal nutrients of
bacteria include: source of carbon, nitrogen, water
and ions.
Sources of energy:
All bacteria (aerobic and anaerobic) use Embden-Meyerhof-Parnas (EMP) glycolytic pathway
to make pyruvate from glucose. Then pyruvate can be metabolized by: (1) fermentation, (2) aerobic
respiration, or (3) anaerobic
respiration. Energy production by bacteria
1. Fermentation: large
amount of organic acids or
alcohols are produced from
pyruvate which receives
electrons from NADH. This
pathway has low ATP-
generating efficiency,
because ATP only comes
from the EMP pathway.
2. Aerobic Respiration: In the
presence of oxygen,
pyruvate is completely
oxidized to water and CO2 by the Tricarboxylic Acid Cycle (TCA). Electrons from NADH and
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FADH are transferred through a chain of carriers to an ultimate acceptor O2.This pathway is
very efficient at generating ATP because ATP is made not only during EMP pathways but also
during oxidative phosphorylation. Bacteria that grow aerobically and thus utilize respiration
must have protective enzymes to protect themselves from being destroyed by toxic radicals.
3. Anaerobic respiration is a membrane-bound biological process coupling the oxidation of
electron donating to the reduction of suitable external electron acceptors (other than molecular
oxygen). In contrast to anaerobic respiration, in fermentation, the oxidation of molecules is
coupled to the reduction of an internally-generated electron acceptor, usually pyruvate.
Depending on bacterial oxygen requirement bacteria can be classified as:

1. Obligate anaerobes
2. Obligate aerobes
3. Facultative anaerobes
4. Oxygen tolerant (Airtolerant)
Obligate anaerobes:
Bacteria that are not able to utilize molecular oxygen and are harmed by it are called obligate
anaerobe. These organisms cannot tolerate atmospheric oxygen pressure. In order to make ATP,
they usually ferment or undergo anaerobic respiration.
Aerobes:
An aerobe is a microorganism that can survive on oxygen and may utilize molecular oxygen as its
final electron acceptor, i.e., as in cellular (aerobic) respiration.
1. Obligate aerobes:
An obligate aerobe is a microorganism that cannot live in the absence of molecular oxygen. This
basically means that they cannot obtain energy via fermentative processes. More precisely, obligate
aerobes are organisms that have an electron transport system and are able to grow in the presence
of atmospheric oxygen concentration. These organisms use O2 as a final electron acceptor and
cannot ferment.
212
2. Facultative anaerobe:
A facultative anaerobe is an aerobic microorganism that can utilize fermentation or anaerobic
respiration when molecular oxygen is absent, and utilizes aerobic cellular respiration when O2 is
present.
3. Airtolerant:
Flow chart for figuring out bacterial oxygen requirements
Aerotolerant
bacteria have
an exclusively
anaerobic
(fermentative)
type of
metabolism but
they are
insensitive to
the presence of
O2. They live
by fermentation
alone whether
or not O2 is
present in their
environment.
The response of an organism to O2 in its environment depends on how well the cell is able to
deal with the various oxygen radicals generated by bacteria. All cells contain enzymes capable of
reacting with O2. For example, oxidations of flavoproteins result in the formation of H2O2 (peroxide) as
one major product and small quantities of an even more toxic free radical, superoxide or O2.-. In
aerobes the accumulation of superoxide is prevented by the enzyme superoxide dismutase. All
organisms which can live in the presence of O2 (whether or not they utilize it in their metabolism)
contain superoxide dismutase and catalase or another less efficient enzyme called peroxidase.
Obligate anaerobes lack these enzymes and undergo lethal oxidations by oxygen radicals.
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Enzymes necessary to disarm oxygen radicals
The presence of catalase can be quickly measured on either directly

isolated specimen or bacteria grown culture. In this test, H2O2 is
added to the bacteria, if catalase is present, H2O2 is broken down
and the generation of oxygen bubbles (O2 gas) will be evident.
Identifying bacteria in the laboratory Catalase test
There are often consecutive steps in bacterial diagnostics.
These steps include: 1. Specimen staining, 2. Culture of specimen, 3. Performing specific

biochemical and antigen specific test.
1. Collected specimen can be evaluated directly by various methods (prior to obtaining results
from the culture):
a. Light microscopy (but too difficult to distinguish between bacteria)
b. Gram stain followed by microscopy
c. Acid-fast stain
d. Fluorescent microscopy
e. Nucleic acid tests (NAT)
2. Growing bacteria from the collected specimen prior to its identification is a very sensitive
and commonly used technique for diagnosis. Bacteria can be grown in liquid or solid media. For
solid media agar must be used.
• Agar- acidic polysaccharide extracted from algae that is in a liquid state at 100oC and solidifies into
a gel-like substance at 45oC. Used in most common solid culture preparations.
o Bacterial colonies grown on agar plates may have different morphologies which is
useful for identification of bacteria.
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There are various types of media that can be used to grow bacteria. These media can be classified
as:
• Nutrient media – contains all the ingredients necessary to support the growth of most
bacteria (usually made of digests of animal or plant products such as milk, beans), it may
also contain vitamins and salts depending on the bacteria isolated.
• Selective
Nutrient agar Selective agar Differential
media-
1. agar 2. 3.
inhibits
growth of
unwanted
organisms
from the normal flora. Usually contains chemical additives such as anti-microbial agents that
inhibit contaminating flora but not the pathogen of interest.
• Differential/ Indicator media- Demonstrates biochemical features of the organism: Most
often a carbohydrate and a pH indicator are added to see whether the organism can ferment
that carbohydrate to produce acid which then changes the color of the pH indicator and
therefore the color of the colony.
3. Biochemical test: After growing bacteria, the colonies are often subjected to various biochemical
tests, which allow the differentiation of bacteria.
Bacterial Growth Curve

Bacterial growth in liquid medium is characterized by the standard bacterial growth curve:
Bacteria added to fresh media typically go through four distinct phases of growth
a. Lag phase
b. Log (logarithmic or exponential) phase
c. Stationary phase
d. Decline (death) phase
Lag phase
Transfer of bacteria from one medium to another, where there is a chemical difference
between the two media will cause a lag in cell division. This lag in division is associated with a
physiological adaptation to the new environment, by the cells, prior to their resumption of division
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That is, cells may increase in size during this time, but
simply do not undergo binary fission.
Log phase (exponential phase)

Lag phase is followed by log phase during
which binary fission occurs. This phase of growth is
called exponential because the rate of increase in cell
number is a multiplicative function of cell number.
This can be seen in a graph of cell number versus
time where cell numbers increase at ever increasing
rates with time or generation; that is, the rate of
increase is a function of absolute cell number such that the more cells present, the faster the
population of cells increases in size. When considering the log phase, a formula can be used to
calculate the total number of bacteria generated after a certain amount of time.
This formula is: A(f)= A(i) x 2n, where A(f) is the final number of bacteria, A(i) is the initial number of
bacteria, and n is the number of generations in a given time.
Stationary phase
Stationary phase is a steady-state equilibrium where the rate of cell division is exactly balanced by
the rate of cell death. Cell death (or, at least, lack of cell growth) occurs because of a loss of limiting
nutrients (due to their incorporation into cells during log-phase growth) or a build-up of toxic products
such as fermentative products released during log-phase growth.
Decline phase (death phase)
Stationary phase, in a standard bacterial growth curve, is followed by a die-off of cells. Cell death in
bacteria cultures basically means that the cells are unable to resume division. This death occurs
because vegetative cells can survive exposure to harsh conditions (few nutrients or too-many toxins)
for only so long.
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Practice questions:
1. Characteristics of a bacterial capsule include:
a. Only gram positive bacteria have one

b. It is composed of poly-D glutamic acid
c. It protects bacteria against ingestion by PMNs
d. It is what causes the gram stain reaction
2. How would gram-negative bacteria appear under the microscope if you accidentally forgot to add crystal violet during
the staining procedure, while doing everything else correctly?
a. Clear
b. Red
c. Yellow
d. Purple
e. Blue
f. White
3. Which of the following is true regarding endotoxins?
a. They are primarily made of proteins

b. Most endotoxins have very specific, rather than generalized effects
c. Endotoxin-mediated disease may be prevented by toxoid vaccination
d. Endotoxins are made equally well by both gram (+) and gram (-) bacteria
e. Endotoxin expressing bacteria are unlikely to ever form spores
4. How would you describe an organism that can respire in the absence of oxygen but not in the presence of oxygen?
a. Obligate fermenter
b. Facultative anaerobe
c. Strict aerobe
d. Strict anaerobe
e. Facultative respirator
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LECTURE 20: STAPHYLOCOCCAL INFECTIONS
• Characterize the structure of S. aureus

• Describe the media used to isolate S. aureus
• List the virulence factors for S. aureus
• Describe the infections causes by S. aureus
• Describe the steps in diagnosing S. aureus
Staphylococcus aureus
Background:
• Carried in the normal flora (human skin and Morphology (top) and membrane
mucosal surfaces) structure (bottom) of S. aureus
• Breaking the natural barriers (like skin) can lead to
disease.
• The anterior nares are the main site of colonization
• Other sites of colonization include:
– axilla, rectum, and perineum
– The vaginal carriage rate is approximately
1-10% in premenopausal women
• The rate is higher during menses
Morphology and Physiology
• Staphylococci are gram-positive cocci, whose bacterial

cells arrange in clusters
• Staphylococci are facultative anaerobes that grow by
aerobic respiration or by fermentation that yields mainly
lactic acid.
• The bacteria are catalase-positive and oxidase-
negative.
• Nearly all strains of S. aureus produce the enzyme coagulase
• They are non-motile and do not form spores
• On nutrient again Staph aureus appear as golden yellow colonies (right).
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MSA
• When grown on Mannitol-Salt agar (MSA), S. aureus ferment mannitol,
and grow on high (7.5 %) salt concentration (right figure)
• Resistance to drying
• S. aureus is beta hemolytic on blood agar (see below)
Hemolysis of sheep red blood cells is an in vitro rupturing of RBCs in

culture which may be used as a tool to determine the species and
strains of various bacteria.
Examples of hemolysis
by various bacteria (left)
219
Virulence Factors
S. aureus causes a wide range of

suppurate skin infections, food
poisoning and toxic shock syndrome.
S. aureus expresses many potential

virulence factors:
Structural components:
• Surface proteins that promote

colonization of host tissues
• Surface antigens that inhibit
phagocytic engulfment (capsule,
Protein A)
Secreted toxins:
• Membrane-damaging toxins that lyse eukaryotic cell membranes (cytotoxins such as alpha
toxin hemolysin, leukotoxin, leukocidin);
• Exotoxins that damage host tissues or otherwise provoke symptoms of disease (exfoliative
toxin, TSST-1, Staph enterotoxin)
Specific examples of Virulence Factors
Adherence to Host Cell Proteins

• S aureus express on their surface proteins that promote attachment to host proteins such as
laminin and fibronectin that form the extracellular matrix of epithelial and endothelial surfaces.
• Certain strains express a fibrin/fibrinogen binding protein (clumping factor) which promotes
attachment to blood clots and invasion of traumatized tissue.
• Interaction with collagen may also be important in promoting bacterial attachment to damaged
tissue where the underlying layers have been exposed.
Membrane-damaging toxins and other exotoxins
• Alpha-toxin is the best characterized cytotoxin and most potent membrane-damaging toxin of S
aureus. Subunits oligomerize to form hexameric rings with a central pore through which cellular
220
contents leak. Susceptible cells have a specific receptor for alpha-toxin which allows the toxin to
bind causing small pores through which monovalent cations can pass. In humans, platelets and
monocytes are particularly sensitive to alpha-toxin. After binding the toxin, a complex series of
secondary reactions ensues, causing release of cytokines that trigger production of inflammatory
mediators. These events cause the symptoms of septic shock that occur during severe infections
caused by S aureus.
 Exfoliative toxin is a serine protease that that split the intracellular bridges in the stratum
epidermis. This toxin is the cause of Staphylococcal Scalded Skin Syndrome (see below for toxin-
mediated skin infection).
 Enterotoxin (Staph enterotoxin A – SEA, Staph enterotoxin B) causes direct effect in the gut-intestinal
fluid loss with diarrhea and vomiting.
 Superantigen (TSST-1) toxin is an exotoxin that stimulates T cells, causing fluid loss, nausea and
vomiting. Up to one in five T cells may be activated, whereas only 1 in 10,000 is stimulated during
a usual antigen presentation. Cytokines are released in large amounts, causing the symptoms of
TSS. Superantigens bind directly to class II major histocompatibility complexes of antigen-
presenting cells outside the conventional antigen-binding grove.
Avoidance of host defense

• Coagulase is an extracellular protein which binds to prothrombin in the host to form a complex
called staphylothrombin. The protease activity characteristic of thrombin is activated in the
complex, resulting in the conversion of fibrinogen to fibrin. Coagulase is a traditional marker for
identifying S aureus in the clinical microbiology laboratory. Bacteria is protected from phagocytic
and immune defenses by causing localized clotting.
• Protein A is a surface protein of S
The effect of protein A
aureus which binds IgG molecules
by their Fc region. In serum,
bacteria will bind IgG molecules in
the wrong orientation on their
surface which disrupts
opsonization and phagocytosis.
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Pathogenesis
222
Non toxin-mediated skin infections:
• Staphylococcus aureus causes many

superficial and deep skin lesions. The
severity of infection depends on the
depth of the tissue infected.
o Epidermis: Impetigo (superficial
infection that causes the production
of pus-filled vesicle)
o Superficial dermis/Deep Dermis:
• Furuncles=Boils=Folliculitis are
painful raised nodules that have
underlying collection of necrotic
tissue/pus. These may extend deeper than hair follicle.
Folliculitis
• Carbuncles are coalescence of furuncles with invasion of
deeper subcutaneous tissue; patients have chills and fever
which suggests a systemic infection.
• Erysipelas are lesions with sharply demarcated raised edge.
They are red, swollen, warm, hardened and painful.
Erysipelas does not affect subcutaneous tissue. It does not
release pus, only serous fluid. In erysipelas, the infection rapidly invades and spreads
through the lymphatic vessels. This can produce overlying skin "streaking" and regional
lymph node swelling and tenderness.
o Subcutaneous layer:
• Cellulitis is inflammation of the connective tissue below dermis as well as other
subcutaneous tissue. Cellulitis results in fever and chills in addition to skin infection.
• Fasciitis is an infection of the fascia surrounding the muscle.
Impetigo
Cellulitis
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Carbuncles
Erysipelas
Toxin-mediated skin infections:
o Staph Scalded skin syndrome (SSSS, Ritter’s disease) is usually in

babies and results from production of exfoliatin in Staph lesions. Skin
gets blistered but these lesions contain very little bacteria because they
are caused by a toxin. Also few leukocytes are present in the lesions.
A slight pressure on the skin displaces the skin. Although the disease
looks awful, it usually does not leave any scarring on the skin. The lack
of SSSS in adults is explained by the presence of antibodies specific for
exotoxins and improved renal clearance of toxins.
Non-toxin mediated, non-cutaneous infections:
o Bacteremia and Endocarditis are often hospital acquired, and is associated with bacteria from
the skin getting into the blood stream (as a result of surgical procedure or contaminated
intravascular catheter). Endocarditis (inflammation of the inner layer of the heart, damage to
endothelial lining of the heart) needs to be treated promptly, otherwise the patient has very
poor prognosis.
o Osteomyelitis is an infection of the bone with Staph which can result from trauma that spreads
the bacterial infection to the bone esp. in children where growing bones are highly
vascularized. The disease is characterized by localized pain, high fever and purulent discharge
from the sinus tract overlying the infected bone.
o Pneumonia is consolidation and abscess formation in the lungs seen in very young, elderly
and in patients with underlying pulmonary disease.
Toxin mediated, non-cutaneous infections:
o Staph food poisoning (right) is the most common

form of food poisoning in US. It is caused by
ingestion of the poison, which is not readily
destroyed by the gut. The foods that facilitate
the growth of this organism and the release of
the toxin include ham, pork, potato salad, ice
cream. The food is contaminated by someone
who has a Staph skin infection or carries the
organism in the nasopharyngeal fluid. If the food
remains at room temperature, bacteria can grow
and release the toxin, the bacteria can be killed
by reheating, but the toxin is heat resistant
and is therefore not destroyed by reheating.
o Toxic shock syndrome was first noticed in Australia in

children receiving vaccine contaminated with Staph
aureus. Then, in 1980’s, there was a dramatic
increase of disease in menstruating women.
Menstruation appeared to facilitate TSST-1 toxin
production. This was associated with the use of
hyperadsorbent tampons that were made from
different material than previous tampons. These
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tampons helped Introduce more oxygen (toxin production requires an aerobic atmosphere and
neutral pH) into vaginal tract and blossomed into cup-like shape which helped filter toxins and
encouraged growth of S. aureus. These tampons were later recalled, decreasing the incidence
of disease.
Less than 1% of women carry Staph

aureus strain containing TSST-1 in vaginal
flora. When secreted the toxin stimulates
the release of large amounts of
interleukin-1, interleukin-2 and tumor
necrosis factor. It does so by stimulating
human T cells that express Vbeta 2, which
may represent 5-30% of all host T cells.
The toxin binds primarily to the alpha-
chain of class II MHC and the beta chain
on a TCR stimulating T cells to become
activated and subsequently activate the
antigen presenting cell also bound to the
toxin.
The disease is characterized

by the release of toxin in to
the blood which causes
abrupt onset of:
o Fever
o Hypotension
o Diffuse macular
erythematous rash
o Multiple organ
systems (e.g., central
nervous,
gastrointestinal,
hematologic, hepatic,
musculature, renal)
are also involved, and
the entire skin,
including the palms
and soles, desquamates or peels off. The rash is similar to a sunburn.
In menstruating TSS cases, blood is usually negative for bacteria. With better understanding of the
disease, the death rate dropped to 5 %.
TSS can also occur post various Staph infections including pneumonia, abscess, skin/wound
infection, septicemia, osteomyelitis. In many cases a toxin other than TSST-1 causes the
manifestation of toxic shock.
Diagnosis
Stain
o Gram staining of the lesion illustrates G(+) cocci in clusters

225
Culture
o Lesions usually contain many bacteria that can be

easily cultured overnight in aerobic environment.
When lesions cannot be accessed directly (ex. deep
tissue infections) blood cultures can be done.
Lesions also often contain numerous neutrophils.
o Blood agar - Staph. produces hemolysin which
causes beta (complete) hemolysis of RBC.
o MSA agar - To select the growth of Staph aureus, the agar should be supplemented with
7.5 % NaCl (inhibits growth of most organisms but not Staph genus), and mannitol
(fermented mainly by Staph aureus, not other organisms).
Biochemical tests
4. Colonies can be tested for coagulase and catalase
For food poisoning and TSS, diagnosis is made based on clinical history.
Treatment
Infections acquired outside hospitals can usually be treated with penicillinase-resistant ß-lactams.
Hospital acquired infection is often caused by antibiotic resistant strains and can only be treated with
vancomycin.
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LECTURE 21: STREPTOCOCCAL INFECTIONS
• Describe the general features of Strep and differentiate between the three Streptococcal
species (S. pyogenis, S. agalactaiae, and S. pneumoniae) based on biochemical properties
and clinically
• List the virulence factors for GAS (capsule, M protein, sAg and pyogenic exotoxin)
• Describe Group A Streptococcal infections including strep throat, scarlet fever, rheumatic
fever, skin infections, and systemic invasive infections
• Describe the basis of GAS rapid antigen test
• Describe the disease mechanism of GBS and Describe the CAMP test
• Identify diseases caused by and the virulence factors for S. pneumoniae
• Describe the vaccines for S. pneumonaeie
Streptococcus
General features:
• G (+), cocci in chains or pairs
• Catalase negative
• Air-tolerant
• Three species will be discussed
(right)
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Streptococcus pyogenes (Group A Strep)
Virulence Factors
• Streptokinase lyses fibrin

• Hyaluronidase hydrolyses hyaluronic acid, part of the ground substance in host tissues
• C5a peptidase: C5a enhances chemotaxis of phagocytes.
• Capsule: hyaluronic acid is produced which inhibits phagocytosis.
• M protein is a fibrillar surface protein. Its distal end bares a negative charge that interferes
with phagocytosis. It also blocks complement deposition on the cell surface. Mutations during
the course of infection alter the structure of M proteins, rendering some antibodies ineffective.
Strains that persist in carriers frequently exhibit altered M proteins.
• Leukocidins, including streptolysin S and streptolysin O, are proteins secreted by the
streptococci to kill phagocytes (probably to release nutrients for bacterial growth).
• Antigenic variation. Antibody against M protein (antigen) is the only effective protective
antibody, but there are more than 50 different M types, and subsequent infections may occur
with a different M serotype.
• Antigenic disguise and tolerance provided by hyaluronic acid capsule
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Production of toxins and other systemic effects

Streptococcal pyrogenic exotoxin (SPE) is an exotoxin that acts as a superantigen. Without
being processed it binds directly to MHC II and links the MHC expressing APC to the T cell receptor
of many (up to 20% of) T cells. Exaggerated production of cytokines causes the signs of shock: fever,
rash, low blood pressure. This is similar to TSST-1 toxin effects.
Pathogenesis:
Suppurative Infections (active infections associated with pus) occur in the throat, skin, and
systemically.
Respiratory infections:
Pharyngitis is the most common bacterial cause of Strep throat (esp. in kids 5-15 yrs of age). The
pathogen is spread by large respiratory droplet and the symptoms are hard to distinguish from viral
pharyngitis unless bacteriological and serological tests are done. The lymph nodes in the neck
become swollen, tonsils, uvula and soft palate
become red, swollen and covered with yellow-
white exudate. Two complications are noted:
glomerulonephritis and rheumatic fever.
Scarlet fever - 1-3% of people with pharyngitis
will develop scarlet fever.
Some strains of Strep have been lysogenized by
bacteriophage that stimulates the production of
erythrogenic, pyrogenic exotoxin which
causes a rash on the chest, and face but not
around the mouth. A bright red tongue is also
characteristic of scarlet fever. Later in the
infection, skin peeling may be seen.
Skin infections: (see Staph section for pictures)

Impetigo: Impetigo involves the infection of epidermal layers of skin. It begins with localization of skin
by S. pyogenes which can then be introduced into subcutaneous tissue through cuts in the skin. Pus
filled vesicles develop which can rupture and crust. Pre-pubertal children are the most susceptible.
Systemic infections are unlikely.
Erysipelas- Acute infection of the dermal layer of the skin. Usually on face and legs, and may be
preceded by infection of the respiratory tract by Group A strep. Is not associated with systemic
disease but about 5% of patients may develop disseminated disease.
Cellulitis- Cellulitis occurs when the infection spreads subcutaneous tissues.
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Necrotizing fasciitis (systemic) is an

infection that spreads along fascial planes
and is characterized by severe destruction of
muscle and fat. This is often referred to by the
media as “flesh-eating bacteria”; the organism
is introduced into tissue though cuts, burns or
surgery causing systemic symptoms (fever,
shock, and multi-organ failure). Unlike
erysipelas which can be treated with
antibiotics, this disease must be treated with
aggressively with surgical removal of infected
tissue. The strain usually carries additional
toxins. This disease is more common with
Strep than with Staph.
Toxic shock syndrome (systemic): certain very invasive
strains of Gr. A Strep can cause systemic disease
characterized by inflammation at the initial site of infection,
then fever, chills, vomiting, diarrhea and multi-organ failure.
Whereas Staph-induced (TSS) is mainly due to the toxin
entering the blood stream, Strep-induced TSS is associated
with bacterial presence in the blood and is often linked to
necrotizing fasciitis.
Non-suppurative sequelae:
Some of the antibodies produced during the above infections cross-react with certain host tissues.
These can indirectly damage host tissues, even after the organisms have been cleared, and cause
non-suppurative complications.
Rheumatic fever:
The most feared complication of post -
Streptococcal pharyngitis (Strep throat)
disease – which is characterized by changes
in the heart and joints leading to myocarditis
and aortic valve stenosis. The mechanism for
this is molecular mimicry, where heart muscle
tissue cross react with antibodies formed to
Streptocococcus. Major symptoms of aortic
stenosis are chest pain (angina), fainting
(syncope), and shortness of breath.
In about one third of all patients
diagnosed with aortic valve stenosis, chest
pain is usually the first sign. Because the aortic valve is narrowed, this increases pressure in the
heart. It also increases the oxygen demand for the blood flowing into the heart because there is less
blood being pumped by the heart due to the narrowing of the aortic valve. This can also attribute to
shortness of breath.
Joint are also affected which is how the disease got its name.
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Aortic
stenosis
Glomerulonephritis:
Post-infectious acute inflammation of

the renal glomeruli can occur. This is
accompanied by edema, hypertension and
hemato and proteinuria. The mechanism of
damage is similar to either Type II or Type III
hypersensitivities.
In Type III reaction, immune complexes
with Strep antigen and anti-Strep antibodies
are deposited in the kidney basement
membrane (see Figure on the right).
In Type II reaction, antibody against
Strep cross-reacts with host cells in the
basement membrane of the kidney and
deposit there. In both cases this results in
inflammation of the kidney with dysregulation
of kidney function. Only a few M-types are
nephrogenic.
Diagnosis:
Stain: Gram staining of samples from affected tissue can provide INITIAL diagnosis. Strep. is often
part of the normal flora of the oropharynx and sometimes skin.
Culture: Specimen obtained from the oropharynx, skin or blood should be cultured on blood agar in
the presence or absence of oxygen and observed for beta-hemolysis.
Specific tests: Bacterial colonies can then be further analyzed for group-specific Lancefield antigens
using immunofluorescence (IF). Another test used in labs is bacitracin sensitivity; unlike other Strep
species, Gr. A Strep growth is inhibited by this antimicrobial agent.
Serology: usually used for diagnosis of post-strep sequel such as rheumatic fever. These test for
anti-streptolysin O (ASO) Ab.
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Rapid Ag test:
Many newer tests have been developed that test reactivity of lab anti-GAS antibodies with
group-specific carbohydrates in the cell wall. This can be done DIRECTLY on the throat swab and
takes ~20 min. However, due to the novelty of these tests, negative results should be confirmed by
culture.
Procedure of Rapid Ag test:
o The patient specimen is mixed

with beads coated with rabbit
anti-GAS Ab.
o This mixture is then applied to
a membrane and diffuses
down to the lane that contains
additional immobilized rabbit anti-GAS antibodies.
o If the beads carry GAS antigen they become trapped in first lane.
o Excess beads that do not carry GAS antigen get trapped in the last lane. This lane contains
goat anti-rabbit antibodies against bead-bound antibodies. There are always an excess of
beads, so even in infected patients there should be a reaction in the last lane. This lane serves
as a positive control.
o If both the middle and the last lane appear red (due to the aggregation of the beads) then the
patient is GAS positive, if only the last lane appears red, then the patient is negative.
Sreptococcus agalactiae (Group B Strep)

Pathogenesis:
GBS is the main cause of meningitis in the first few days of
infant's life. The organism can gain access to amniotic fluid or
colonize the infant during pregnancy and delivery. Group B strains
contain different polysaccharides in their capsules, which determines
their ability to colonize and cause disease. GBS also expresses
hydrolytic enzymes that facilitate tissue destruction and spread of the
pathogen. Up to 30% of women are vaginal carriers of this bacteria
and are thus under the greatest risk of passing this organism to the
baby.
Disease:
Neonatal meningitis/Early-onset disease occurs when GBS is
acquired in-utero; the disease develops during the first few weeks of
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life and is characterized by bacteremia, meningitis, failure to thrive and pneumonia.

• Many infants who survive the disease have permanent problems including blindness, deafness,
and retardation.
• Prenatal care includes screening for GBS in the third trimester.
• As a result of rapid diagnosis and better supportive care, the mortality rate has decreased to 5-
20%.
• Not all infants born to Group B strep positive moms will have the disease (about 50% will be
infected with bacteria but not progress to disease). A high titer of maternal anti-Group B strep
antibodies usually cross the placenta and protect the infant.
Diagnosis/Screen:
• Culturing of blood or CSF of affected neonates, or vaginal specimen of pregnant women in
blood-enriched agar would produce large colonies that may or may not be beta-hemolytic.
• Usually a selective medium is also used for culture. This media contains antibiotic that
suppress the growth of other organisms.
• PCR can be done on the cultures
Prevention:
In colonized pregnant women penicillin is administered during delivery to minimize contact of
bacteria with the neonate.
Streptococcus pneumoniae
Epidemiology:
• The bacterium generally resides in the nasopharynx and is carried asymptomatically in

approximately 50% of healthy individuals
• Leading cause of community-acquired pneumonia which is a class of pneumonias in someone
who has not recently been in a hospital
• Major cause of pneumonia in alcoholics, HIV patients, the elderly, asplenic patients and children
under 2.
• Attaches to human nasopharyngeal epithelial cells via bacterial surface adhesins
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• Under certain situations can spread to nasal sinuses, lungs and middle ear which are normally
sterile sites
• It can also be transported to the blood and the brain
• The normal action of mucus and ciliated cells usually prevent bacterial entry into the lungs, but
when these barriers are damaged, bacteria gains access to lungs, and subsequently to blood
• Stimulates local inflammatory response, but evades phagocytic killing
• Transient colonization of throat and nasopharynx occurs by a succession of different
serotypes
• Often preceded by respiratory viral infection (e.g., influenza, rhinovirus)
• Several conditions interfering with normal clearance of bacteria predispose host to pneumococcal
infections:
• Chronic pulmonary disease
• Alcoholism
• Neutrophil dysfunction
• Congestive heart failure
• Diabetes mellitus
• Chronic kidney disease
• Splenic dysfunction or splenectomy: Absence of spleen decrease clearance of opsonized
bacteria from blood and also causes a defect in antibody production
Morphology and Physiology:
• Encapsulated gram-positive diplococci or short chains

• The capsular antigen in virulent strains is made of
complex polysaccharide (there are more than 90
different types of capsule)
• Produce alpha hemolysis when grown aerobically
(which is the usual way to culture) and beta hemolysis
when grown anaerobically
• Anaerobic growth may show beta-hemolysis due to the
production of pneumolysin by bacteria which is an
oxygen labile pore-forming toxin
• Fastidious growth requirements
o Enriched media with 5% defibrinated blood
o This media provides catalase for destruction of H2O2
• S. pneumo ferment carbohydrates; major end-product is lactic acid
• Accumulation of hydrogen peroxide inhibits growth
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Virulence Factors:
1. Adhesion proteins
allow bacteria to bind to
oropharyngeal
epithelium and enable
initial colonization
2. Secretory IgA
protease assists
spreading by
enzymatically disrupting
secretory IgA (sIgA)
clearance of bacteria.
3. Pneumolysin functions as multifactorial cytotoxin. It assists spreading by destroying ciliated
epithelial cells though host cell membrane cholesterol binding. It also stimulated complement-
mediated inflammation.
4. Capsule-mediated phagocytic survival allows bacteria to escape from neutrophils
Pathogenesis:
Pneumonia
Pneumonia is characterized by severe shaking chills, fever, productive cough with blood-tainted
sputum and chest pain. Due to the lower oxygen tension preferred by bacteria, it is usually seen in
the lower lobes of the lungs. Tachycardia and tachypnea are often seen. Recovery is rapid in the
presence of antibiotics. Without antibiotics, the recovery is associated with high levels of opsonizing
antibody.
Facts about pneumonia:

• When endogenous oral
pneumococci are aspired into
lung alveoli it causes aspiration
pneumonia.
• Local inflammation causes
pulmonary capillaries to leak
fluid into the interstitium and
bacteria further multiply in
nutrient-rich edema.
235
• Capillaries may get damaged and erythrocytes leak from capillaries causing bloody sputum.
• Phagocytic cells (neutrophils, followed by alveolar macrophages) migrate and phagocytose and
destroy pneumococci.
• Children and elderly may have diffuse bronchopneumonia.
• Pneumococcal lesion can be observed by X-ray.
• Radiological resolution (clean chest X-ray) within 2-3 weeks with antimicrobial therapy
Otitis media and sinus infections (sinusitis)

These infections usually follow a viral infection after which the inflammatory cells obstruct the sinuses
and ear canal, creating a moist, warm environment allowing bacteria to multiply.
• Ear infections are usually in kids, sinus infections could occur in patients of all ages.
• These infection are often preceded by upper respiratory tract viral infection with PMN caused
obstruction of sinuses and ear canal,
preventing air flow and clearing.
Meningitis
• Bacteria can spread to CNS after a sinus or ear
infection. Alternatively it can enter CNS following
bacteremia that can accompany pneumonia.
• This organism is now the leading cause of bacterial
meningitis in older children and adults.
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• Mortality and severe neurological complications are 4-20 times more likely than with meningitis
caused by other organisms
Bacteremia
• Common in people with pneumonia or meningitis.
• Not common in people with ear or sinus infections.
Prevention:
• Immunization via polyvalent vaccine as prophylaxis.
• Streptococcus pneumoniae vaccine is given as either:
1) Prevnar 13; PCV13 is a 13-valent conjugated vaccine for kids under 5 years old. PCV13
was licensed in February 2010, supplanting PCV7. PCV13 is identical in formulation for the
previous vaccine and contains seven common serotypes in PCV7, with six additional antigens.
2) PPSV23 is a 23-valent polysaccharide vaccine for anyone over 65 years of age and adults
at high risk. For the 23-valent vaccine, purified capsular material from most common
serotypes (23 different polysaccharides covering 94% of clinically relevant serotypes) is
administered.
237
Diagnosis:
Gram stain:
Streptococcus pneumoniae
• Specimen - CSF, aspirate from ear, sinus, or lung, sputum
The disk contains optochin
• Gram stain more useful for CSF analysis than other specimens
and is surrounded by a zone
Culture:
of inhibition.
• Specimen in blood agar with antibiotics that suppresses normal flora
• Alpha hemolysis observed
Biochemistry:
• Test colonies for bile solubility – bacteria dissolve in bile
Specific tests:
• Urine, CSF, pleural fluid tested using rapid immunochromatographic test
NAAT:
• PCR for amplifying S. pneumo specific genes Bile solubility test
Practice questions:
1. Which of the following is the main mechanism behind rheumatic fever
caused by Streptococcus pyogenes?
a. IgE mediated mast-cell activation which leads to heart damage

b. CD8 T cell invasion into the myocardium with the subsequent lysis of the
heart cells
c. IgA recruitment of the inflammatory molecules and complement
d. IgG binding to the heart tissue causes subsequent inflammation
e. Group A streptococcus invades the heart and secretes super-antigen exotoxins
f. Immune complexes containing Streptococcal antigen and anti-Streptococcal antibody get
deposited in the heart valves causing damage
2. A 15 year old boy comes in to clinic with pain in his right ear. He is on his school swim team and
spends about 2 hours per day in the pool. Along with the pain in his ear, he has watery drainage
coming from the ear canal. On exam his vital signs are normal, and he appears healthy, but his
left external ear looks quite red and there is copious watery, slightly green drainage coming from
the ear. The organism mostly likely causing this is:
a. Staph aureus
b. Adenovirus
c. Streptococcus pneumoniae
d. Streptococcus pyogenes
e. Rhinovirus
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3. Which of the following statements about the 23-valent pneumococcal vaccine is not correct?
a. It is a protein-conjugated, polysaccharide vaccine
b. It is poorly immunogenic in young children and immunocompromised hosts
c. It is routinely recommended for immune competent adults and children >2 yrs. of age at risk for
serious pneumococcal disease
d. It protects against the major serotypes of pneumococci causing infection
e. An adult with asplenia would be a candidate for this vaccine
4. All of the following statements about the M-protein of Group A Streptococci are correct EXCEPT
a. There are over 80 distinct serotypes.

b. Antibodies to M proteins exhibit crossreactivity with heart tissue
c. Antibodies to M protein confer type-specific immunity.
d. M protein is the major virulence factor of Group A streptococci.
e. M protein is the major constituent of the capsule of Group A streptococci.
5. A 12 year old boy presents with acute onset of sore throat, fever to 38.9 degrees C and painful
anterior cervical lymphadenopathy. On exam the pharynx is red and swollen and the tonsils are
covered with yellow-white exudate. The child also has halitosis. Which of the following non-
suppurative complications are of concern?
a. Sinusitis
b. Acute rheumatic fever alone
c. Acute glomerulonephritis alone
d. Acute rheumatic fever and acute glomerulonephritis
e. Scarlet fever alone
6. Which of the following Streptococcal infections is commonly prevented by a vaccine?
a. Strep throat
b. Group B strep meningitis
c. Strep. pneumoneae meningitis
d. Flesh-eating bacteria disease
e. Skin infections
7. A 2 –day old infant born to a mother who has not received any prenatal care is brought to the
emergency room. The child has a fever, is febrile, and will not feed well. His CSF analysis shows
low glucose level, but elevated protein and white blood cell levels. Gram-positive cocci are
isolated from the CSF. Which of the following would have prevented this disease in the baby?
a. Intravenous antibiotic administration to the mother during labor

b. Vaccination of the mother
c. Treatment of neonate’s eyes with the antibiotic erythromycin
d. Vaccination of the baby at birth
e. Avoidance of sushi and other raw products by the mother
239
8. Which of the following diseases caused by Staphylococcus aureus is not mediated by a toxin, but
rather by bacterial replication?
a. Food poisoning
b. Toxic shock syndrome
c. Scalded skin syndrome
d. Impetigo
e. Scarlet fever
240
LECTURES 22 AND 23: FOOD INFECTIONS AND FOOD POISONING
• Describe major structural and metabolic properties of Bacilli

• Describe the clinical symptoms and virulence factors for anthrax
• Describe the clinical symptoms and virulence factors for B. cereus food poisoning
• Describe major biochemical differences between B. cereus from B. anthraces
Bacilli – Gram-positive rods, facultative anaerobic or strictly

aerobic, form highly resistant spores, catalase positive
Bacillus anthracis
Background:
• Spores of B. anthracis gain access to the soil from the

carcasses of large animals such as sheep, goats,
horses, and cattle that die of disseminated disease.
• The organism can be harbored in the gastrointestinal
tract of animals that do not develop symptoms and also in animals that die of disease.
General features:
• B. anthracis grow singly, in pairs, or in long chains and

are encapsulated.
• The spores resist heating at 100oC (i.e., boiling water
and steam) for up to 30 min; the spores may be killed
after heating to 121°C for 15 min or with steam-under-
pressure in an autoclave.
• The organisms are facultative anaerobes that grow best aerobically on blood agar at 37oC and
are non-hemolytic.
• Colonies appear rough with an irregular edge that gives a "Medusa head" appearance.
241
Virulence factors:
Exotoxin:
Diagram of the actions of the secreted anthrax toxins.
LT (lethal toxin) has a devastating effect on immunity, impairing

the immunogenic response by destroying macrophages, which
release the bacteria into lymph, and induce further spread of B.
anthracis and septicemia. Most importantly, the cellular effects of
LT on the heart result in decreased stroke volume and cardiac
output, which are further diminished by the effects of
hypovolumia, resulting in hypotension and death.
• The extracellular product associated with the pathogenicity of the organism is an exotoxin.
• The exotoxin is a heat-labile protein composed of 3 components referred to as:
o "protective antigen" (PA)
o "edema factor" (EF)
o "lethal or toxic factor" (LF), which is responsible for most of the toxicity.
• Combined these can activate signal machinery inside the cell (such as activation of adenylate
cyclase) which results in edema and leads to cell death. Maximal biological activity occurs only
when all components are present together with the capsule.
Structure:
• The capsule is composed of a polypeptide made up exclusively of D- glutamic acid.
• The capsule exhibits anti-phagocytic activity and does not stimulate protective antibody.
242
Transmission of anthrax:
Transmission appears to be exclusively by the Transmission of anthrax

exogenous route through contact with spores.
1. Cutaneous anthrax is the result of infection

with spores that gain access to humans
through small abrasions or scratches in the
skin while handling diseased animals or
animal products such as meats, skin, brushes
made from infected horse hair and drums
constructed of infected goat skins.
2. Inhalation anthrax or woolsorters' disease usually results from inhalation of spores from infected
animals or animal products.
Pathogenesis:
Example of cutaneous anthrax
1. Cutaneous anthrax
• Cutaneous anthrax is both a toxemic and
invasive disease.
• Spores are introduced into the skin via abrasions
or cuts and germinate.
• The vegetative cells multiply locally and the host
responds with an acute inflammatory response
characterized by an influx of polymorphonuclear
leucocytes.
• Phagocytosis by the polymorphonuclear
leucocytes is inhibited by the capsule and the
organisms continue to survive and multiply.
• The organisms release exotoxin locally and begin to invade adjacent tissue rapidly producing
extensive damage.
• The primary skin lesion is usually a nondescript, painless, pruritic papule that appears three to
five days after the introduction of endospores.
243
• In 24 to 36 hours, the lesion forms an ulcer that undergoes central necrosis and drying, leaving
a characteristic black eschar surrounded by edema and a number of purplish vesicles with
serosanguinous fluid.
• Rapid dissemination may occur via the lymphatics to the circulation resulting in septicemia and
tissue invasion, including the lungs.
• Profound toxemia and necrosis may result in death despite therapy.
2. Inhalation anthrax or Woolsorters' disease

• This syndrome is also both toxemic and invasive.
• It is acquired either by inhalation of vegetative cells in droplet nuclei from a patient with
respiratory disease or more commonly by the inhalation of spores from infected animals or
animal products.
• When spores are introduced, they germinate to vegetative cells in the trachea, bronchi and/or
lungs.
• Vegetative cells undergo multiplication in these organs with the host response, mechanism of
survival, and exotoxin release as described for the cutaneous form of the disease.
• Within 24 hours, pulmonary necrosis, septicemia, and meningitis occur which is most often
rapidly fatal despite therapy.
Inhalation anthrax
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Diagnosis:
1. Rapid presumptive diagnosis based upon history and clinical manifestations is essential
2. Specimens and stain

• Specimens obtained depend upon the disease process and
include material from lesions, blood for culture, spinal fluid, and
sputum.
• Gram-stain of the specimen will reveal characteristic gram-
positive rods with square cut ends and often in pairs or long
chains.
• Spores appear as an oval unstained space in the center of the
organism stained by Gram stain, and may be confirmed by the
use of a special spore stain called malachite green.
3. Cultures and biochemical or direct identification

• Cultivation on blood agar
• Overnight incubation under aerobic conditions at 37oC is optimal
for isolation of the organism, which is identified as a non-hemolytic, gram-positive, non-motile,
spore forming rod growing singly, in pairs, or in long chains
• Colonies have “medusa head” appearance.
• A direct immunofluorescence assay on the isolated organism is
available where antibody from the laboratory is directed against
the capsular polypeptide.
• PCR tests are also available.
Bacillus cereus
Morphology:
B. cereus is similar to B. anthracis, however it is:
• Beta-hemolytic on blood agar
• Non-encapsulated
• Does not express the edema causing exotoxin.
Virulence factors:
• Some strains express a heat-stable emetic toxin (mechanism unknown) and others heat-
labile diarrheal toxin which stimulates adenylate cyclase in intestinal epithelial cells
leading to diarrhea.
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Pathogenesis:
Bacillus cereus exists in water and soil. The organism may cause a self-limiting type of food
poisoning usually resulting from the ingestion of contaminated rice or meat dishes containing
enterotoxins. The incubation period and clinical manifestations resemble those seen in
staphylococcal food poisoning.
Gastroenteritis/ Emetic food poisoning

• Emetic food poisoning results from consumption of
contaminated rice. The steps in the development of
gastroenteritis:
1. Bacteria in rice are killed from cooking the rice but heat-
resistant spores survive if the rice is inadequately boiled.
2. If rice is not refrigerated, these spores can then germinate at
R.T, releasing live bacteria which multiply and secrete the
toxin.
3. This toxin may not be destroyed during reheating of the rice.
4. After ingestion of the food with the toxin, a person gets ill with
the symptoms that include: vomit, nausea, and abdominal
cramps.
• Diarrheal form results from contaminated meats, sauces and

veggies. This disease has a longer incubation period and lasts
longer.
Diagnosis:
• For emetic gastroenteritis, suspected food should be cultured.

• Stool can be used to identify the organism in patients with diarrheal form of disease; however,
fecal colonization of healthy individuals is common. Isolation of organism is usually done for
epidemiologically related studies.
246
B cereus
247
Clostridia:
General features:
• The clostridia are relatively large, gram-positive, rod-shaped bacteria.
• All species form endospores and
have a strictly fermentative
(anaerobic) mode of metabolism.
• Most clostridia will not grow under
aerobic conditions and vegetative
cells are killed by exposure to
atmospheric O2, but their spores are able to survive long periods of exposure to air.
• Their fermentation of organic
compounds, such as sugars,
produces large amounts of CO2
and H2 as well as volatile organic
compounds like acetic and butyric
acid, acetone, and butanol.
Metabolism of substrates like
amino acids and fatty acids results
in foul-smelling degradation
products.
• Clostridium tetani and Clostridium
botulinum produce the most
potent biological toxins known to affect humans.
o The virulence of C. tetani and C. botulinum is entirely due to their toxigenicity.
o C. botulinum is usually encountered in improperly sterilized (canned) foods in which
endospores have germinated.
o C. tetani is acquired through exposure to the spores of the bacterium Clostridium tetani
which are universally present in the soil.
• Clostridium perfringens produces a huge array of invasins and exotoxins and causes wound and
surgical infections that lead to gas gangrene, in addition to severe uterine infections.
o Clostridial hemolysins and extracellular enzymes such as proteases, lipases, collagenase
and hyaluronidase all contribute to the invasive process.
• Clostridium perfringens also produces an enterotoxin and is an important cause of food poisoning.
248
C. perfringens
Virulence factors:
• A single strain could produce up to 12 toxins, however the alpha toxin is the most important of
these toxins. This toxin is a lecithinase (phospholipase C) which breaks lecithin (also known as
phosphatidylcholine) in the cell membrane.
• The effect of the toxin is to cause massive hemolysis, tissue destruction, myocardial destruction
and hepatic failure.
• Beta-toxin makes necrotic lesions in the mucosa.
• Other toxins increase vascular permeability and have necrotic activity.
• Certain C. perfringens strains produce a superantigen-like entertoxin which is made during the
spore formation and is released together with the spore.
• Five main strains are identified based on toxin production
Pathogenesis:
C. perfringens is part of the normal flora in the human intestinal tract. However they can cause a
range of disease from mild disease to very severe. Infections result from: 1) contamination of an open
wound with spores or 2) due to ingestion of a large number of C. perfringens or toxin in inadequately
stored food
Soft-tissue infection/Gas gangrene

Wounds are colonized by the organism usually through the spores from the environment which
germinate and produce vegetative bacteria in the tissue. C. perfringens can cause cellulites, fasciitis
and gas gangrene.
249
• In the wound the bacterial alpha toxin splits lecithin into diacylglycerol and IP3 which in turn
activate the protein kinase C second messenger system.
• The toxin also causes hemolysis and bleeding by increased vascular permeability and
destruction of platelets
• Toxin can spread and cause hepatic toxicity, and myocardial dysfunction (bradycardia,
hypotension).
• Fermentation in anerobic conditions causes gas production.
• Gas gangrene is caused accumulation of hydrogen and CO2
gas
• The toxin causes accumulation of fluid, the pressure of gas
causes muscle pain.
• Muscle necrosis, shock, renal failure, and death can occur
within days.
Gastroenteritis/Food infection/Food poisoning
• Short incubation period (8-24 hours)

• Characterized by:
• Abdominal cramps
• Watery diarrhea with or without vomiting
• No fever
• It lasts about 2 days
In food poisoning, the symptom are usually due to ingestion of spore-contaminated meat
products where the spores did not get killed during the original cooking process and germinated in
the food. If the food is not refrigerated, the bacteria divided and secreted enterotoxin into the food.
• Since the toxin is heat-labile, reheating the food will result in destruction of the toxin. However
eating the food without reheating can result in an intoxication.
In food infection (C. perfringens can also cause a food infection rather than food poisoning) the
spores germinated in the spore-contaminated food. The bacteria proliferated and this was
followed by the ingestion of a large number of organisms. Once in the person’s gut, the bacteria
sporulate due to unfavorable conditions and this causes toxin production. When the spore is
released, the toxin is released with it and the person feels the symptoms of gastroenteritis.
250
Necrotizing enteritis:
• This is a rare, acute necrotizing process by

which the jejunum is destroyed.
• It is characterized by vomit, bloody diarrhea,
abdominal pain, ulceration of small intestine,
and peritonitis (inflammation of a two-
layered serous membrane covering both the
surfaces of the organs that lie in the
abdominal cavity and the inner surface of
the abdominal cavity itself) and results in
50% mortality.
• Beta-toxin of C. perfringnens Type C strain
is responsible for this disease. The beta-
toxin can be inactivated by pepsin and
trypsin proteases which are found in people
on protein rich diet: These enzymes are low
in small children and malnutritioned people
so they are more susceptible.
Diagnosis:
• Clinical diagnosis is of primary importance.

• For soft-tissue disease, microscopic detection of these bacteria in specimen in the absence of
leukocytes (which differentiates it from other soft tissue infections) is enough to initiate
immediate therapy. Since gas gangrene is a toxin-mediated disease, there is little leukocytes
infiltration.
251
• For food poisoning, the recovery of 105 organisms per gram of food or a quantifiable number of
bacteria from feces is indicative of infection.
• Bacteria grow quickly anaerobically. On blood agar, they display a double layer of hemolysis:
inner beta-hemolysis and outer alpha-hemolysis. The beta-hemolysis is by theta toxin and
alpha-hemolysis is by alpha toxin (see figures below).
C. tetani
General features:
• The spores of the organism

are found in soil, especially
heavily-manured soils, and in
the intestinal tracts and feces
of various animals.
• The spores are capable of
surviving in oxygen.
252
Virulence factors:
Exotoxin:
• Tetanus is a highly fatal disease of humans.

• The disease stems not from invasive
infection but from a potent neurotoxin
called tetanus neurotoxin or
tetanospasmin produced when spores
germinate in anaerobic conditions of the
woundand vegetative cells grow. The
toxin is also made during sporulation.
• The toxin is heat-labile. It is destroyed at
56 degrees C in 5 minutes.
• Upon entering the blood stream the toxin
is initially carried to and binds to
peripheral nerve terminals.
• The toxin then migrates along neural paths from a local wound to sites of action in the central
nervous system.
• It is transported within the axon and across synaptic junctions until it reaches the central
nervous system. There, it becomes rapidly fixed to gangliosides at the presynaptic
inhibitory motor nerve endings of the Renshaw cells, and is taken up into the axon by
endocytosis.
o Within these cells it cleaves and blocks the release of the inhibitory
neurotransmitters glycine and gamma-amino butyric acid across the synaptic cleft.
o Presynaptic blockade is specific to the synapse between these inhibitory Renshaw cells
in the CNS and the alpha motor neurons. The Renshaw cells that handle acetylcholine
transmission.
o Tetanospasmin appears to act by selective cleavage of a protein component of synaptic
vesicles, synaptobrevin II. This prevents synaptobrevin II within the membrane of the
vesicle from fusing with syntaxin within the plasma membrane. This blocks the release
of GABA and glycine by the Renshaw cells.
• If nervous impulses cannot be checked by normal inhibitory mechanisms, they produce the
generalized muscular spasms characteristic of tetanus.
253
• Because of the widespread use of the tetanus toxoid for prophylactic immunization, fewer
than 150 cases occur annually in the U.S.
Pathogenesis:
Generalized tetanus/Adult tetanus:
• Tetanus is the only vaccine-preventable disease that is not communicable but acquired through
environmental exposure to the spores of Clostridium tetani.
• Most cases of tetanus result from small puncture wounds or lacerations which become
contaminated with C. tetani spores which germinate and produce tetanospasmin.
• The bacteria remain localized with only minimal inflammatory damage but the toxin spreads in the
blood. The clinical pattern of generalized tetanus consists of spastic paralysis, severe painful
spasms and rigidity of the voluntary muscles and irritability. The characteristic symptom of
"lockjaw" involves spasms of the masseter muscle accompanied by grotesque, grinning
expression.
• Spasms of the pharyngeal muscles cause difficulty in swallowing.
• Death usually results from interference with the mechanics of respiration.
Neonatal tetanus:
• Accounts for about half of the tetanus deaths in developing countries
• Neonatal tetanus follows infection of the umbilical stump in infants born to non-immune mothers
(therefore, the infant has not acquired passive immunity). The main symptom of neonatal tetanus
is failure to thrive.
• Unlike other diseases, such as diphtheria, recovery from the natural disease usually does not
confer immunity, since even a lethal dose of tetanospasmin is insufficient to provoke an immune
response.
Prevention:
• Prophylactic immunization is accomplished with tetanus toxoid, as part of the DTaP vaccine.
“T” denotes the tetanus toxoid portion of the vaccine.
• Three injections are given in the first year of life, and a booster is given about a year later, and
again on the entrance into elementary school.
Diagnosis:
Clinical presentation is most indicative. Neither tetanus toxin nor antibodies to the toxin are
detectable in the patient because it is rapidly absorbed by muscle motor neurons and internalized.
254
Microscopic examination and culture can be done but are not useful because they are not sensitive
enough.
C. botulinum
C. botulinum is a large anaerobic bacillus with endospores. It is widely distributed in soil,

sediments of lakes and ponds, and decaying vegetation. Hence, the intestinal tracts of birds,
mammals and fish may occasionally contain the organism as a transient flora.
Virulence factors:
Exotoxin:
Botulin toxin
• Botulinum neurotoxins
predominantly affect the
peripheral nervous system
with a preference of the toxin for
stimulatory alpha-motor
neurons at a neuromuscular
junction.
• The toxins bind to the neuron
and is internalized.
o The heavy chain of the
toxin mediates binding to
presynaptic receptors.
The A fragment of the
toxin enters the cell by
receptor mediated endocytosis.
o The toxin blocks the release ACH across the synaptic cleft by cleaving synaptobrevin
within the alpha motor neuron and thus producing the paralysis of the skeletal muscles.
o The recovery of function requires sprouting of a new presynaptic axon and the
subsequent formation of a new synapse.
• With a defect in acetylcholine release, the primary symptom is weakness or flaccid paralysis.
• Seven toxigenic types of the organism exist, each producing an immunologically distinct form
of botulinum toxin.
• Botulinum toxins are very similar in structure and function to the tetanus toxin, but differ
dramatically in their clinical effects because they target different proteins in the nervous
system.
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• Botulin toxins are designated A, B, C, D, E, F, and G.

• In the U.S. type A is the most significant cause of botulism, involved in 62% of the cases.
Pathogenesis:
Food-borne Botulism
• C. botulinum spores are relatively heat resistant and may
survive the sterilizing process of improper canning
procedures.
• The steps during exposure to botulin toxin are as follows:
• The spores from foods that are grown in soil
contaminate a can during canning process
• In the anaerobic conditions of the can, the spores germinate and bacteria begin to
divide.
• The germination of spores occurs only in the right conditions, i.e. if the canned food is
too acidic, too sugary, or kept refrigerated, the spores will not germinate.
• If spores do germinate, then bacteria divide and release botulin toxin into the food.
• The toxin is heat labile, so reheating the food before ingestion will inactivate it, however,
because it is a very potent neurotoxin, even small amounts will cause morbidity.
• The botulinum toxin is ingested with food. When the food is ingested, the toxin is
absorbed by the upper part of the GI tract in the duodenum and jejunum, and passes
into the blood stream by which it reaches the peripheral neuromuscular synapses.
• The toxin then binds to the presynaptic stimulatory terminals and blocks the release of
the neurotransmitter acetylcholine which is required for a nerve to simulate the muscle.
• Food-borne botulism is not an infection but an intoxication since it results from the ingestion
of foods that contain the preformed clostridial neurotoxin.
• Clinical symptoms of botulism begin 18-36 hours after toxin. The symptoms include:
Somatic motor:
o Descending weakness of skeletal muscles (Flaccid paralysis)
o Inability to swallow (block in CN IX)
o Blurred vision/Double vision (block CN III, CN IV, CN VI)
o Difficulty in speech (block in CN IX - XII)
o Extreme cases - respiratory paralysis
Autonomic motor:
o Dilated pupils (block in CN III)

o Dizziness Dry mouth (block in CN VII, and IX)
o Vomiting
256
o Lack of sweating (Block ACh release)

o In botulism, CNS involvement is rare
Neurologic features of blurred vision (diplopia), inability to swallow (dysphagia) and difficulty
in speech (dysphonia) are often called the “3D symptoms”
Infant Botulism:
• Infant botulism is due to the infection caused by C. botulinum.
• The disease occurs in infants 5 - 20 weeks of age who have been
exposed to solid foods, presumably the source of contamination with
spores.
• Production of toxin by bacteria in the GI tract induces symptoms.
• In adults, neither the organism nor the spores survive the low pH of the GI tract, whereas in the
infant (1-6 months) the higher pH and the lack of normal flora allow spores to germinate and the
organism to flourish.
• The symptoms in babies are usually “failure to thrive”. A child diagnosed with infant botulism is
unable to move or open his eyes. His cry is barely audible. His breathing becomes shallow and
this frequently leads to death.
• C. botulinum organism as well as the toxin can be found in the feces of infected infants.
Immunity and Prevention:
• The toxins that cause botulism are each specifically neutralized by an antitoxin.
• As with tetanus, immunity to botulism does not develop, even with severe disease, because the
amount of toxin necessary to induce an immune response is deadly.
• The most important aspect of botulism prevention is proper food handling and preparation.
• Because the toxin is heat-labile boiling or intense heating of contaminated food will inactivate the
toxin.
• A multivalent toxoid evokes good protective antibody response but its use is unjustified due to the
infrequency of the disease.
Diagnosis:
• Electrodiagnostic by repetitive nerve stimulation
• Testing of multiple muscles may be needed to see increments
• Muscle biopsy: scattered angular, small muscle fibers
• Analysis of serum, feces & implicated food
C. difficile
• Pseudomembranous colitis in humans is caused by overgrowth of Clostridium difficile in the colon,
usually after the normal flora has been disturbed by antimicrobial chemotherapy.
257
• C. difficile produces two toxins: Toxin A is referred to as an enterotoxin because it causes fluid
accumulation in the bowel. Toxin B is an extremely lethal (cytopathic) toxin.
ENTEROBACTERIACEAE
• Describe the biochemical features of Enterobacteriacece

• Describe the use of McConkey and Hektoen agars
• Describe the diseases caused E. coli, Salmonella, Shigella
• Describe the biochemical features of Vibrio cholera
• Describe the manifestation and treatment of cholera
• Understand the mechanisms of toxins when applicable
General features:
• Gram-negative rods, facultative anaerobes
• Catalase positive, Oxidase negative (lack complex IV)
• Ferment glucose to produce acid and gas
• Can undergo “anaerobic” respiration and reduce nitrogen rather than oxygen
• In the presence of oxygen, they reduce oxygen, but with an enzyme other than cytochrome C
oxidase which is the reason behind the oxidase negative test result
• Selective/differential media is used to isolate different bacteria in this group
Main sites of infection
258
Media used for isolation:
MacConkey Agar:
• Selective and differential

• Selective: Contains bile salts and the dye crystal violet, which inhibit the growth of gram-
positive bacteria and selects for gram-negative bacteria
• Differential: Has lactose, which allows differentiation of gram-negative bacteria based on their
ability to ferment lactose
o Organisms which ferment lactose produce acid end-products which react with the
pH indicator neutral red, and produce a pink color. The medium itself is light
pink/yellow in color.
Hektoen Enteric agar: selective and differential agar used to culture infected blood or stool
• Selective: Bile salts

and the dyes
bromthymol blue
and acid fuchsin
inhibit the growth of
most gram-positive
organisms and
serve as acid base
indicators
• Differential: designed for the differentiation between Salmonella and Shigella
259
• Differentiates between bacteria that ferment lactose and those that don't as well as
between bacteria that can produce H2S and those that can’t
• Acid produced from fermenting lactose imparts a yellow-orange color to the medium
due to the presence of a pH indicator (ex. E. coli in top figure, previous page)
• Non-lactose fermenters do not significantly change the color of the medium (ex. Shigella
in top figure)
• Production of hydrogen sulfide gas, turns parts of the medium black
• Sodium thiosulfate in the media provides a source of sulfur
• Ferric ammonium citrate in the media provides a source of iron
to allow visualization of black precipitate (figure right)
• Ex. Salmonella does not ferment lactose and appears light H2S- H2S+
gray but it also produces H2S which is seen as a black zone in
the middle of colony
Various routes of acquiring

Enterobacteriaceae
260
E. coli
Pathogenesis of E. coli
Specific infections:
Septicemia: The E. coli infections that cause septicemia originate in the urinary tract or GI tract. They
cause the highest number of systemic diseases within the Enterobacteriaceae group.
Neonatal meningitis:
• Bacterial strains with K1 capsular antigens are commonly present in the GI tract of pregnant
women and newborns. Along with Group B Strep., E. coli K1 is a major causes of CNS
infections in infants younger than 1 month.
• The capsule is a made of polysialic acid polysaccharide.
• These E. coli strains invade the blood stream of infants from the nasopharynx where the
infants first come in contact with bacteria during delivery and the bacteria are then carried to
the meninges.
261
Urinary tract infections:
• These infections are usually due to

contamination of urethra and the bladder by
bacteria originating in colon.
• Certain bacterial serotypes produce adhesive
pili that attach to the cells lining the bladder
and the urinary tract are prevent the bacteria
from being eliminated in the voided urine.
• The bacteria causing UTI could also affect
kidney and prostate.
Gastroenteritis:
• Virulent strains causing GI infections differ
from nonvirulent E coli in the GI only in
possessing genetic elements for virulence
factors. For example, strains producing
enterotoxins are called enterotoxigenic E coli
(ex. ETEC) and can cause disease in humans. These strains are not part of the normal flora.
• Depending on the virulence factors, virulent Escherichia coli strains can cause either
non-inflammatory diarrhea (watery diarrhea) or inflammatory diarrhea (stools containing
any or all of the three: blood, mucus, leukocytes).
Pathogenesis:
• Transmission is by the fecal-oral route.
• Pathogenesis involves adhesins and/or exotoxins.
• Pili (fimbriae) allow the bacteria to colonize and adhere to the ileal mucosa.
Epidemiology:
• Infections are common where sanitation is poor; both infants and susceptible travelers to
developing countries are particularly at risk. The disease is most serious in infants.
Diagnosis:
• The diagnosis is suggested by the clinical picture and confirmed by stool culture. Serotyping
and tests for virulence factors are occasionally performed for outbreaks.
262
Control:
• Prevention depends on sanitary measures to prevent fecal-oral transmission, hand-washing
and proper preparation of food, chlorination of water supplies, sewage treatment and disposal.
• Parenteral or oral fluid and electrolyte replacement is used to prevent dehydration.
• Broad-spectrum antibiotics are used in chronic or life-threatening cases.
Summary of the characteristics of various gastroenteritis-causing E. coli strains:
EPEC (Enteropathogenic)
• The most important
virulence factor is
attachment
• Bundle-forming pilus-
mediated attachment
• Non-fimbrial (non-pilus)-
mediated adhesion via
intimin
• Causes infantile diarrhea
ETEC (Enterotoxigenic)
• The most important
virulence factor is
exotoxin
• Bacteria is non-invasive to
the cell and stays
extracellular
• Exotoxins can penetrate cells
• Watery diarrhea in infants and travelers
The typical symptoms of traveler's diarrhea caused by either ETEC and EPEC include:
• Abrupt onset of diarrhea
• Nausea and vomiting
• Bloating
• Urgent need to have a bowel movement
• Malaise (weakness or discomfort)
• Loss of appetite
• Cramps
263
EIEC
• The most important virulence factors are adhesins and ability to move laterally within cells
• Non-fimbrial adhesins
• This strain is invasive, i.e., they penetrate and multiply within epithelial cells of the colon similar
to Shigella but EIEC strains do not produce Shiga toxin
• Symptoms include: inflammatory, dysentery-like diarrhea with mucous, white blood cells and
erythrocytes in the stool
• Fever
EHEC
• Found in cattle GI track and fecal matter of cattle
• Acquired by eating undercooked meat, unpasteurized milk or
contaminated apple cider, lettuce and other uncooked food
• Require low inoculation dose (fewer than 100 bacteria can cause
disease)
• The most important virulence factors are adhesins and Stx-1 (shiga-like) toxin
o Shiga-like toxin
 Shiga-like (Stx-1) disrupts ribosome structure and
inhibits protein synthesis (Figure right)
 Shiga-like toxin targets red blood cells and endothelial
cells:
• vasculature of colonic mucosa
• kidney vasculature, kidney tubules
• cerebral vessels
 Toxin has enteric, cytolytic and neurological effects
• Diseases: Hemorrhagic colitis (copious bloody discharge), pediatric diarrhea
• Complication: hemolytic uremic syndrome (HUS, about 10%) which is characterized by
anemia, thrombotic thrombocytopenic purpura (TTP) and kidney failure
• Mild or no fever
• Main cause- strain O157:H7
264
• Receptor for toxin is more common in kids

• Invasive due to the toxin rather than bacteria, so leukocytes may be absent in stool
Various attachment means of E. coli:
EPEC (e.g. E. coli O127:H6) and EHEC

Intimin Pilus/Fimbria (e.g. E. coli O157:H7) have intimin for
attachment. It belongs to a class of
proteins called attaching and effacing
(A/E) proteins. They and other
virulence factors are responsible
for diarrhea.
ETEC have pilus which adheres to the

microvilli of small intestinal epithelial
cells and delivers enterotoxins
Salmonella
General features:
• Causes gastroenteritis and typhus
• Do not ferment lactose or sucrose

• Produce abundant H2S
• Genus contains 3 species identified on basis of biochemical activity, metabolic end product
difference, and antigenic difference.
a. S. typhi
b. S. cholerae-suis
c. S. enteritidis (1700 + serotypes)
• For Salmonella, animals are main reservoir of human disease except for bacteria responsible
for typhoid fever
• At risk: anyone consuming foods contaminated with large numbers of Salmonella, particularly
children younger than 1 year old, elderly, patients with reduced gastric acids, and patients with
AIDS, for S. typhi foreign travelers or individuals exposed to carriers
• Transmission is through the ingestion of contaminated food products (especially poultry, eggs,
dairy products) or direct human to human fecal-oral spread for typhus
265
Pathogenesis:
Transmission of Salmonella
Gastroenteritis:
• Many infections are due to ingestion of
contaminated food, especially poultry (see figure
on the right)
• Organism can grow while food is stored outside
the fridge
• Can also be transmitted to humans from reptiles
• Onset at about 48 hours after ingestion of
contaminated food
• Characterized by vomiting, diarrhea, colicky abdominal cramps, headache and fever (see
figure on bottom right).
• Bacteria invade the GI epithelium
through M cells, invade the Peyer's
patches and migrate to lamina propria of
the small intestine. An acute
inflammatory reaction is then initiated.
• Some strains appear to produce an
enterotoxin that stimulates adenylate
cyclase activity (cholera-like).
• Usually of short duration
• No septicemia
266
Spread of Salmonella
267
S. typhi
Enteric fever (typhoid fever):
Transmission of typhoid fever in the body
268
• A strictly human disease with the incubation period of about 10 days

• The pathogenesis of enteric fever is more complex than that of watery diarrhea or dysentery
• See figure below:
o Bacteria invade mucosa of the distal small bowel through M-cells of Peyer's patches in
the small intestine (1)
o They pass into mesenteric lymph nodes where they multiply (2)
o Enter the blood stream via the thoracic duct (3)
o Subsequently spread outside the bowel to the liver, gall bladder, biliary tract, mesentery
and spleen may occur (4).
o Multiplication in the gall bladder (5) and spill over into the biliary tract leads to seeding
the intestine with large numbers of bacteria (6).
• Little or no diarrhea is present upon initial infection, but diarrhea may be present later in the
course of the illness
• The most prominent features are fever, headache and abdominal pain, which develop
gradually over a
4
few days. 1 2
• Bacteria multiply in
macrophages and
monocytes and in
3
the organs that
house them. 6
• Characterized by 4
prolonged febrile
illness with 5
splenomegaly,
leukopenia,
abdominal pain,
and positive blood,
urine and stool
cultures.
269
• Rose spots on the abdomen reflex systemic involvement

Rose spots
• Infection of intestinal lymphoid tissue may lead to necrosis
and ulceration.
• Bacteremia is common, occasionally causing a chronic
infection in the gall bladder. This infection may or may not
be asymptomatic.
• Although it is usually self-limiting, enteric fever carries a significant risk of serious disease and
significant mortality.
Shigella – Can cause Shigellosis or Bacillary dysentery
General features:
• Non-motile - no H (flagella) antigens
• Closely related to E. coli
• Four species that are pathogenic for humans:
o S. dysenteriae- 10 Serotypes (Group A)
o S. flexneri- 6 Serotypes (Group B)
o S. boydii- 15 Serotypes (Group C)
o S. sonnei- 1 Serotype (Group D)
 S. sonnei most common in U.S
• Some strains produce a protein synthesis-

inhibiting toxin
Epidemiology:
• Humans and higher primates are the only
reservoirs
• Transmitted by oral-fecal route
• Very low infectious dose: ~ 200 bacteria.
• So sanitation can be fairly good and transmission will still occur.
• High incidence of unapparent infection makes control difficult
270
Pathogenesis:
• Invasion of the large bowel

via M cells
• Infection of macrophages in Peyer’s
patches
• Infection of basolateral side of the
epithelium
• Cell-to-cell spread
• Similar to how EIEC spreads
Virulence factors:
• Invasion of colon epithelium

• Secretion of a potent Shiga exotoxin
• This toxin has enterotoxic, cytotoxic, and neurotoxic effects
Diseases
Shigellosis- Mild acute diarrheal disease (ADD)

• Fever
• Abdominal pain
• Vomiting
• Watery stools may progress to dysentery (exudate with blood, pus, etc)
• Last 1 to 7 days without treatment
• No bacteremia
Dysentery - more severe diarrhea due to S. dysenteriae

• Begins with the rapid onset of frequent intestinal evacuations
• Stools are of smaller volume than in watery diarrhea and contain blood and pus (including
WBCs).
• High fever, abdominal pain, and tenesmus are frequent complaints.
• Vomiting occurs less often.
• In dysentery, the focus of pathology is the colon (although small intestine is also affected).
• S. dysenteriae is invasive to the cell, but bacteria do not penetrate the bloodstream
• Exotoxin is secreted, can penetrate beyond the mucosa and has the following
properties:
• It inhibits protein synthesis
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• Toxin cleaves off an adenine molecule from 28S ribosomal RNA causing a
structural alteration in 60S ribosomal subunit.
• Cytotoxic effect:
o Vascular endothelial cell damage to the intestine causing hemorrhage (blood and fecal
leukocytes in stool)
o Usually the toxin only effects intestinal epithelium, BUT in rare cases can be
disseminated in blood and cause renal failure - HUS: hemolytic uremic syndrome or
neurological problems.
• Neurotoxic effect: Abdominal cramping, damage to smaller cerebral blood vessels
• Enterotoxin effect: Blocks absorption of electrolytes, glucose, and amino acids from the
intestinal lumen by adhering to small intestine receptors.
• This damage produces the pus and blood seen in the stools, but does not result in substantial
fluid loss because the absorptive and secretory capacity of the colon is much less than that of
the small bowel.
• Dysenteric infections generally last longer than the common watery diarrheas, but most cases
still resolve spontaneously in 2 to 7 days.
• Without treatment dysentery leads to dehydration-related shock and has a mortality rate of
20%.
Diagnosis:
Direct IF of stool sample, culture stool on Hektoen media, biochemical tests on the colonies. Slide
agglutination tests using anti-O Ag antibodies can also be done on the stool isolates.
Cholera (not an Enterobacterieae)
General features:
• Gram-negative, facultative anaerobic
• Comma-shaped bacillus
• Oxidase positive
• Motile
• Catalase positive
• Optimal growth: 18-37° C; pH - 7.0, tolerates up to
pH 9
• V. cholerae can grow with or without salt, but
other Vibrios prefer salt environment
• There are 139 serogroups of V. cholerae based on
O-somatic antigen (O-polysaccharide side chain of lipopolysaccharide)
• V. cholerae endemic in regions of Southern and Southeastern Asia and is responsible for
pandemic cholera
• Do not have a capsule
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• Limited to the intestinal tract
Vibrio cholera
Transmission:
• Vibrio species survive in fecal-contaminated water and grow well in increased salt environment
Unlike other Vibrio species cholera do not require salt to grow.
• Shellfish often carry this organism. So the spread is usually by ingestion of fecal- contaminated
food or water.
• Since a very large number of bacteria are needed to cause disease (108), direct person to
person transmission is rare.
• Found in area of poor sanitation
Virulence factors:
• Pili – helps attach to small

intestine
•
• Cholera toxin - encoded by
a bacteriophage and
functions in activating
adenylate cyclase which, in
turn, converts ATP to cAMP
increasing its levels. cAMP
activates Protein Kinase A. This results in the opening of CFTR chloride channel:
• When the CFTR channel is forced to open, it is sends chloride ion to the lumen, sodium
will follow chloride and water will follow both electrolytes. Bicarbonate from pancreas
and potassium from diet are not reabsorbed. This results in secretory isotonic diarrhea.
• Hypersecretion of water follows the concentration gradient which ultimately results in the
induced secretion and reduced absorption of electrolytes from the lumen.
• In the absence of cholera toxin, a less violent disease similar to gastroenteritis is produced
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• Main sites of toxin function

are the crypt cells of small
intestine
which induces
hypersecretion.
• Absorption is effected less,
and that’s the reason why
rehydration therapy can work
in cholera.
• Fluid loss can approach 15 to
20 liters/day
Pathogenesis:
Gastroenteritis or Cholera:
• Infections range from mild disease to severe fatal diarrhea, and depends on whether the strain
carries cholera toxin.
• V. cholera can attach to the epithelial tract by toxin co-regulated pili and prevent the flushing
out of the organism during fluid loss.
• Rapid onset of watery, non-bloody diarrhea, and colorless, odorless stool, speckled with
mucus
• Vomiting
• The name “rice-water” stools refer to cholera disease where mucus specks are found in the
stool.
• Fever, and presence of leukocytes or blood in the stool are uncommon.
• Severe electrolyte loss due to CTFR channel disruption can lead to dehydration, metabolic
acidosis (HCO3 loss), hypovolemic shock, hypokalemia (potassium loss), and renal failure.
• Kidney failure occurs when the kidneys shut down from increased constriction as a result of
sympathetic activity. They then lose their filtering ability leading to build up of excess amounts
of fluids and wastes in the body
• Prompt replacement of electrolytes reduced the mortality rate from 60% to 1%.
• In the absence of cholera toxin, a less violent disease similar to gastroenteritis is produced.
• Cholera serotypes O1 and O139 are the most severe.
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• Cholera can resolve spontaneously.
Diagnosis:
• Stain: Bacteria is very thin and curved and are difficult to visualize by gram stain.
o Darkfield microscopy is specialized illumination technique that capitalizes on oblique
illumination to enhance contrast in specimens that are not imaged. A bright image of the
specimen is superimposed onto a dark background
• Culture: Vibrios grow on most media including blood agar and
MacConkey agar. Therefore a selective medium for cholera is
used.
o The most typical medium is called thiosulfate citrate bile salts
sucrose (TCBS) agar. It is selective and differential
Selective:
• The high concentrations of thiosulfate and citrate and the
strong alkalinity of this medium largely inhibit the growth of Enterobacteriaceae.
• Alkaline pH of 8.0-9.0 selects Vibrio and suppresses other bacteria
• Ox bile and cholate (bile acid) suppress primarily enterococci.
Differential:
• Sucrose is added with the pH indicator blue-bromothymol which changes its color to
yellow, when acid is formed (even in this strongly alkaline medium).
• Fermenters such as Vibrio form distinctive yellow-orange colonies.
• Cholera is a lactose non-fermenter and sucrose fermenter.
• Biochemical tests: can then be done on the bacterial colonies to confirm Cholera.
Treatment, Prevention & Control:
• Rehydration & support - Oral or IV

• Tetracycline
• Sanitary and sewage treatment
• Water purification
• Vaccines are not yet totally efficacious
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Comparison of E. coli, Shigella and Salmonella
Summary of lecture
276
Practice questions:
1. Within a few hours of eating a delicious blinchik, a group of picnickers get violently ill with vomiting,
nausea and abdominal cramps. Symptoms last less than a day. A gram positive, aerobic bacillus
growing in chains is isolated from the food. What else is likely true about this organism?
a. It forms spores
b. It produces LOS
c. It has a poly-D glutamic acid capsule
d. It encodes a potent endotoxin
e. It is salt tolerant
2. All Enterobacteriaceae share all of the following characteristics EXCEPT:

a. Ferment glucose
b. Reduce nitrates to nitrites
c. Gram negative
d. Oxidase positive
e. Rod-shaped (bacilli)
3. Which of the following statements regarding enterotoxigenic E. coli are CORRECT?
a. They are important causes of traveler's diarrhea.

b. Transmission occurs from ingestion of contaminated food and water.
c. Disease is caused by production of one or both of two types of enterotoxins.
d. None of the above are correct.
e. All of the above are correct.
4. Two sisters presented to the emergency department with abdominal cramping, fever, and frequent
blood-tinged stools. They indicated that hamburgers that they had eaten 3 days earlier at a
restaurant were pink and undercooked. A methylene blue-stained smear of stool noted very few
leukocytes. What was the most likely cause of their illness?
a. Heat-stable enterotoxin produced by enterotoxigenic (ETEC) E. coli

b. Shiga-like toxin produced by EHEC E. coli 0157:H7
c. Toxin A and toxin B produced by Clostridium difficile
d. Bundle forming pili produced by enteropathogenic (EPEC) E. coli
e. An AB toxin secreted by Vibrio cholera
5. Which of the following is the most likely source of Salmonella typhi infection?
a. Contact via infected person

b. Steamed rice
c. Undercooked chicken
d. Undercooked hamburger
e. Contaminated marine water
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6. Which of the following best describes the structure and growth of Cholera?
Ability to ferment Encodes a Tolerates Anaerobic

lactose neurotoxic exotoxin high salt
medium
a. yes no yes no
b. no no yes no
c. no yes yes no
d. yes no no yes
e. no yes no yes
7. Which of the following best described the clinical symptoms of Cholera?
Fever Invasive to the cell Penetrates the Blood in the

mucosa stool
a. no yes yes no
b. yes no yes no
c. no no no no
d. yes yes no yes
e. yes yes no yes
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Campylobacter:
Characteristics:
• Spiral, gram-negative rods
• No fermentation or oxidation of carbohydrates
• Microaerophilic growth,
• Growth at 42oC (C. jejuni)
• Motile, catalase +, oxidase +
• The major carbon and nitrogen sources are amino
acids, and it possesses several enzymes for the
amino acid deamination. Serine catabolism is
especially significant.
Pathology:
Gastroenteritis:
• Infectious dose - few hundred bacteria
• However bacteria can be killed by gastric acid, so larger inoculation dose or conditions that
neutralize the gastric acid favor disease. Infections are zoonotic and it is usually the
consumption of contaminated milk, food or water that causes the infection.
• No person-to-person spread
• Diarrhea, fever, malaise, abdominal pain. More than 10-20 stools a day
• Stools contain blood
• Septicemia is uncommon but happens often with other species of Campylobacter such as C.
fetus.
• Histological damage to mucosal surface of the jejunum, ilium and colon
• See ulcerated, fluid filled mucosal surfaces, infiltration of neutrophils, eosonophils and
mononucler cells into lamina propria (a thin vascular layer of connective tissue beneath the
epithelium of an organ's mucous membrane)
• The important factors for pathogenesis of this organism are adhesins and flagella
• A very rare sequel to gastroenteritis has been associated with Guillain-Barre syndrome (a
neurological disorder in which auto-immune response reacts to peripheral nervous system
causing weakness), recovery may take months.
• The mechanism of Guillain-Barre is due to cross-reactivity between antigens of C. jejuni and
glycosphingolipids in the surface of neural tissues. Arthritis has also been observed as a
complication for this disease.
Diagnosis:
Stain: Stool specimen can be analyzed by gram stain or dark-field microscopy (it is hard to see the
organism, because it is too thin and very small).
Culture: the organism grows in reduced oxygen (5-7%), and increased CO2 (5-10%). It grows better
at 42oC than 37oC. Selective media containing blood and antibiotics are usually used. Additives that
remove toxic oxygen radicals can also be added. A media used to grow C. jejuni is called Campy
agar contains 10% sheep blood, sodium bisulfite, and three antibiotics. The sodium bisulfite reduces
some of the oxygen in the medium which enhances recovery of Campylobacter. The antibiotics
prevent other gram-negative bacilli and yeast from growing.
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Listeria
General features:
• All Listeria species are small, Gram-positive rods, which are
sometimes arranged in short chains.
• In direct smears they may be coccoid, so they can be mistaken
for streptococci.
• Flagella are produced at room temperature rather than at 37°C.
• Tumbling motility in fluid cultures is due to flagella being
produced at 250C (not at 370C)
• Catalase-positive, beta-hemolytic
• Can grow at 1oC
• Hemolysin production is an important marker for L monocytogenes
• Strains of Listeria are usually serotyped to characterize surface
antigens, such as O antigens (teichoic acids) and H antigens
(proteins)
Virulence factors:
An invasion factor secreted by the pathogenic bacteria enables
them to penetrate host cells of the epithelial lining. Bacteria enters
through the GI tract epithelial cells and lyses the acidic vesicles with
listeriolysin O. Listeriolsin O is a cytolytic, thiol-activated, pore-forming
toxin protein that is activated by reducing agents and inhibited by
oxidizing agents. It works best at pH of 5.5. After lysis of the vesicle,
bacteria multiplies in the cytoplasm of the cell, then propels itself
using bacterial protein ActA and host actin into the next cell, where
the process of vesicle lyses and multiplication begins again.
Pathogenesis:
Listeria monocytogenes is ingested with raw, contaminated food. Listeriosis has recently been
recognized as an important
public health problem in the
United States in neonates,
elderly, pregnant women and
patients with defective
cellular immunity. Post-
pasteurization contamination
is often the cause of an
outbreak. Normally, the
immune system eliminates
the infection before it
spreads. Indeed, most adults
who have no history of
listeriosis have T
lymphocytes primed
specifically by Listeria
antigens. If the immune system is compromised, however, systemic disease may develop. Listeria
monocytogenes multiplies not only extracellularly but also intracellularly within macrophages after
phagocytosis and even within parenchymal cells which are entered by induced phagocytosis. It
therefore belongs to the large group of facultatively intracellular pathogens.
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Listeriosis also may be transmitted congenitally across the placenta. The immunocompetent
mother suffers at worst a brief, flu-like febrile illness, but the fetus, whose defense system is still
immature, becomes seriously ill. Depending on the stage of gestation, the fetus is either stillborn or
born with signs of congenital infection. Typically, multiple pyogenic foci are found in several organs
(granulomatosis infantiseptica). The onset of listeriosis is delayed (i.e., a few days after birth) when
infection is acquired during labor by bacteria colonizing the genital tract of the mother.
Diagnosis:
Gram stain of CSF usually shows no organism (it is present below detection level) which is in
contrast to most other bacteria in the CNS. If it is isolated from CSF, it must be differentiated from a
similar-looking G(+) rod (Corynebacterium) which is often a contaminant of specimen from other parts
of the body.
Bacteria can be cultured from blood and CSF on most lab media, and selected by cold enrichment.
Beta-hemolysis is hard to detect on blood agar. Listeria also shows motility in semi-solid agar and
liquid culture.
Biochemical tests and very specific genetic markers for pathogenic Listeria strains are used for
definitive diagnosis.
Control:
Hygienic food processing and storage may reduce the risk of listeriosis. Individuals in high-risk
groups (i.e., immunocompromised individuals and pregnant women) should avoid uncooked food or
should at least marinate salads for a long time in a vinegar-based dressing to kill adherent bacteria.
Since a cell-mediated immune response (the most potent weapon against L monocytogenes) is
induced only by injection of living antigen, vaccination is difficult. Antimicrobial agents are the
mainstay of treatment.
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LECTURE 24: BACTERIAL MENINGITIS AND RESPIRATORY INFECTIONS
1. Describe the structure and clinical symptoms of respiratory pathogens: Corynebacterium

diphtheria, Bordetella pertussis, Haemophilus influenza
• Describe clinical features of diphtheria

• Describe the function of diphtheria toxin and its role in myocarditis
• Describe the Elek test and the media used for C. diphtheria
•
• Describe the disease mechanism of whooping cough
•
• Describe the difference between H. Influenzae type b and nontypable H. Influenzae
• Describe the clinical features of epiglottitis
• Describe the media used to grow Haemophilus
• Understand the composition of the vaccines for these pathogens
2. Describe clinical presentation of meningitis caused by Neisseria and the symptoms of

meningococcemia
• Describe the composition of N. meningitides vaccine

• Differentiate biochemically between N. meningitides and N. gonorrhoeae
3. Describe the clinical presentation of gonorrhea in men and women
• List the features of gonococcemia and ophthalmia neonatorum

• Describe ways to diagnose gonorrhea (media used, biochemical tests, gram-stain, etc.)
4. Describe and differentiate structurally/biochemically the common causes of non-neonatal

meningitis (Streptococcus pneumoniae, Neisseria meningitides, Haemophilus influenzae).
Describe the pathogens that cause neonatal versus non-neonatal meningitis.
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Corynebacterium diphtheriae
General features:
Anatomy and histology of upper respiratory tract

The nasal cavity, the upper part of the pharynx, the trachea, and the bronchi are lined with pseudostratified ciliated
columnar epithelium
• Diphtheria are small, nonmotile, irregularly staining pleomorphic Gram-positive rods with club-
shaped swelled ends
• Arrangement of cells in short chains of "V" or "Y"
configurations or in clumps
• May contain phosphate rich inclusion bodies, known
as metachromatic granules
• Facultative anaerobes – produce acid but not gas
from carbohydrates
• Disease causing strains carry a phage-encoded
exotoxin
• Widely distributed in nature; worldwide in occurrence
• Humans are the only natural hosts
• Carried asymptomatically in the oropharynx of immune individuals
283
Virulence factors:
• A bacteriophage-encoded toxin is
responsible for the pathogenicity of
this organism.
• Diphtheria A-B exotoxin interrupts
peptide formation at the ribosomal
level
• The B component has receptor-binding
region and a translocation region,
whereas the A subunit is the catalytic
subunit.
• The A subunit terminates host cell protein elongation by adding ADP-ribose to the EF-2.
• The B subunit of the toxin has a receptor on a variety of cells including heart and nerve cells.
• The toxin is distributed to tissues distal of the respiratory tract by the circulation.
• Another enzyme, called phospholipase D, increases vascular permeability, thus allowing C.
diphtheriae to spread through tissues of the naso-pharyngeal area
Pathogenesis:
Corynebacterium diptheriae: toxigenic strains with a phage-encoded
toxin cause diphtheria in humans
Diphtheria - respiratory disease:
• Local multiplication in the in the pharynx and adjacent areas
causes initial damage
• This is followed by low grade fever, sore throat, swollen neck,
cough and exudative pharyngitis with a thick
pseudomembrane (right) as a result of fluid from the dying
cells that coagulates
• The pseudomembrane is composed of:
• Bacteria
• Necrotic epithelium
• Macrophages
• Fibrin
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• The pseudomembrane covers tongue, uvula, and can be extended into the nasopharynx and
the larynx
• The membrane firmly adheres to tissue and is difficult to dislodge without causing bleeding
• Spreading of the membrane down the bronchial tree can occur, causing respiratory tract
obstruction and dyspnea
• If the patient recovers, the membrane eventually dislodges
• Toxin may contribute to the initial adhesion of bacteria
• Bacteria itself is not invasive to the bloodstream
Complications
• Result from systemic bacterial spread of the exotoxin to other target organs in the body
• Myocarditis (about 20% of cases)
• Peripheral neuropathy (about 10% of cases)
• Most mortality results from systemic toxin-mediated heart failure
Cutaneous diphtheria (extra-respiratory disease)
• Acquired by skin contact; organism enters through break in subcutaneous tissue

• Chronic non-healing ulcer results
• Most strains causing skin disease are non-toxigenic unless associated with an outbreak of
pharyngeal diphtheria.
Prevention/ Vaccine:
• Diphtheria toxoid vaccine is given as a part of DTaP (diphtheria toxoid, tetanus toxoid,
acellular pertussis) vaccine at ages 2, 4, 5, 15 months and 5-6 years
Diagnosis:
• Clinical diagnosis is used to give initial treatment
• Direct examination: of clinical material is unreliable as several normal
flora inhabitants resemble this organism
• Culture: of the nasopharynx or throat specimen is done on special
medium:
Cysteine-tellurite blood agar (right) is selective and differential:
• Selective: Tellurite inhibits growth of most upper
respiratory tract bacteria and gram-negative rods, and is reduced by C. diphtheriae
to tellurium to produce a gray color.
285
• Differential: H2S production from cysteine and its interaction with tellurite salt
produces a brown halo around the colonies.
• Toxigenicity test called Elek test is done on bacterial cultures (see figure below).
• Anti-toxin filter is placed
on an agar plate
containing C. diphtheriae
• Isolates of C. diphtheriae
are streaked across the
plate at an angle of 90°
to the antitoxin strip
• Toxigenic C. diphtheriae
is detected because
secreted toxin diffuses
from the area of growth
and reacts with antitoxin
to form lines of precipitin
Control of infection:
• Antitoxin is used for neutralizing exotoxin
o Effective in conjunction with antibiotic therapy
• Antibiotics
Penicillin G
Erythromycin
Bordetella pertussis
General features:
• Small, gram-negative coccobacilli
• Obligate/strict aerobes
• Oxidase positive
• Highly communicable and infectious
• Person-to-person spread via inhalation of infectious aerosols
• Man is the only natural host
• Multiply among cilia of epithelial cells
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Virulence factors (table right):
• Bacteria attach and destroy ciliated

epithelial cells
• Hemagglutinin and special adhesive pili
are used for attachment
They promote binding to the sialic acid on
ciliated respiratory cells
• Massive proliferation of bacteria
• Production of localized tissue damage by
several toxins
• Bacterial toxins stimulate histamine and causes systemic
toxicity
Pathogenesis: Whooping cough (right table)
This pathogen causes a strictly human disease.

Disease is divided in stages:
1st stage- catarrhal stage- common cold-like.
Most bacteria is produced at this stage and
therefore a person at this stage is very
contagious.
2nd stage- paroxysmal stage –ciliated epithelial
cells are extruded from the respiratory tract
and the clearance of mucus is impaired. At
this stage whooping cough develops (series
of repetitive coughs followed by a deep noise inspiration – taking a breath). Mucus production can
287
restrict airways. Often vomiting and exhaustion follows these coughs. In adults the infection can
cause a chronic cough which can be contagious to children.
3rd stage: convalescent stage- paroxysms diminish in number and severity. However secondary
complications may occur. In vaccinated people, organism can colonize but not cause disease -
a “bad cold” is often due to B. pertussis outgrowth in immunized people (possibly due to the less
efficient nature of the acellular vaccine). Unvaccinated infants have a very high mortality rate.
Components of the mucociliary system (below): the

cilia (hair type projections of the epithelia cells), the
periciliary fluid layer and the mucus. The mucus is
secreted from the goblet cells and the submucosal
glands. Mucociliary clearance is the primary mechanism
of clearance of mucus.
Prevention/Vaccine:
• Acellular B. pertussis, diphtheria and tetanus toxoids are prepared in formalin for inclusion in
DTaP vaccine.
• The acellular pertussis contains pertussis toxoid, hemagglutinin, and fimbriae.
Diagnosis:
• Organism is very sensitive to drying. Therefore the nasopharyngeal aspirate must be collected
on special non-cotton fiber swabs that are low in fatty acids.
• Specimen should either be directly cultured or transported in a special medium such as
Regan-Lowe (moist, charcoal-horse blood medium).
o Aspirated specimen can be directly observed with fluorescin-labeled anti-pertussis Ab.
288
Culture:
• Regan-Lowe media supplemented with charcoal and starch (to absorb toxic substances in lab
media), glycerol, peptones
• Media should also be supplemented with NAD (Factor V) an incubated at 35oC in humidified
incubator.
• Bacteria are slow growing.
Specific assays:
Antigen-Antibody tests and PCR tests:
• Specific latex agglutination assay (using anti-pertussis specific Ab) and IF tests can be done
directly on the specimen
• PCR test can also be done
Left: Cases of pertussis

declined rapidly in the
1940s. The all-time low
was in 1976, with only 75
cases reported in the
United States. Since the
early 1980s, there has
been an increase in
reported cases of
whooping cough.
Haemophilus influenzae
General features:
• Gram-negative rods
• Facultative anaerobe
• Require hemin (factor X) and NAD (factor V) for
growth
• Catalase positive, oxidase positive
289
• Non encapsulated organisms from sputum are pleomorphic and often exhibit long threads and
filaments
• H. influenzae does not produce any demonstrable exotoxins
Virulence Factors:
• Encapsulated strains:
• Polysaccharide capsule
((polyribitol phopshate
(PRP) is found in type b
strain and was the main
cause of invasive bacteria before the vaccine.
o Encapsulated H. influenza are not often found normally in the upper respiratory tract
but can cause several diseases in unvaccinated kids.
o Encapsulated bacteria can cross epithelial and endothelial cells and enter blood
causing high grade bacteremia.
o The capsule material is antiphagocytic, and it is ineffective in inducing the alternative
complement
o After the vaccine for Hib, encapsulated H. influenzae type c and f and non-
encapsulated strains are responsible for most invasive diseases.
• Non-encapsulated strains (NTHi – non-typeable H. influenzae):

• H. influenzae colonize upper respiratory tract early in life, and can spread locally to cause
otitis media, sinusitis, and bronchitis, but disseminated disease is relatively uncommon.
• For both encapsulated and non-encapsulated strains, fimbiae and adhesins impair the
function of the ciliated cells in the oropharynx leading to damage of the respiratory epithelia.
• All virulent strains produce neuraminidase and an IgA protease.
290
Pathogenesis:
• Encapsulated organisms can penetrate the epithelium of the nasopharynx and invade the blood
capillaries directly. Their capsule allows them to resist phagocytosis and complement-mediated
lysis in the non-immune host.
• Non-typable (non-encapsulated) strains are less invasive, but they are able to induce an
inflammatory response that causes disease.
• The direct role of endotoxin in meningitis or bacteremia is unclear, although the Gram-negative
bacterium's outer membrane lipooligosaccharide is thought to play a role in inflammation
associated with otitis media.
• Vaccination with type b polysaccharide (in the form of Hib conjugate vaccines) is effective in
preventing infection, and several vaccines are now available for routine use.
• Naturally-acquired disease caused by H. influenzae seems to occur in humans only.
Meningitis (Hib)
• Between 40 and 60% of all serious Hib-related diseases present as meningitis

• Before the vaccine, H. Infuenzae type b was the most common cause of pediatric meningitis
• Mainly in infants and young children under 5 years of age
• Often following an upper respiratory infection or pneumonia caused by another pathogen
• Infections can also be caused by patient’s bacterial flora (endogenous)
• Person-to-person spread in common
• Up to 10% mortality rate in patients that receive treatment
Initial manifestations of meningitis, seen in most patients with Hib meningitis are:
• Altered cry
• Lethargy
• Nausea or vomiting
• Fever
• Headache
• Photophobia
• Irritability
• Anorexia
• Seizures
• An altered cry in small children appears as high pitch cry, lethargic moaning or an attempt to cry
without a noise.
• Vomiting is reported as an early manifestation in nearly 50% of Hib meningitis cases. If vomiting
occurs, it generally does so within hours to days after the onset of fever.
291
• This meningitis form has a high level of morbidity although not as high as pneumococcal or
meningococcal disease.
Epiglottitis (Hib)
Anatomy and histology of epiglottis
• Epiglottis is a flap of tissue that sits at the base of the tongue that keeps food from going into
the trachea, or windpipe, during swallowing.
• When it gets infected and inflamed, it can obstruct, or close off, the windpipe, which may be
fatal unless promptly treated.
Symptoms:
• Patients with
epiglottitis usually
have an upper
respiratory infection
• Epiglottis swells,
turns bright red and
protrudes into the
airway
• Fever
• Stridor (high pitched sound heard on inspiration that is indicative of airway obstruction)
• Patients can’t swallow saliva- and present with drooling, head held forward with tongue
protruding, insisting on sitting up in bed, “hot-potato cough”
292
• Since the development of a vaccine, there are fewer cases since epiglottitis was mainly
caused by Hib.
Cellulitis ((Hib) – (right image):
• Cellulitis is an inflammation of the connective tissue

underlying the skin, usually caused by a bacterial infection.
• Hib cellulitis usually involves the face, head, or neck. Most
cases occur in children aged 2 years or younger.
• In Hib cellulitis, patients often present with reddish-blue
patches on cheeks and periorbital (of the tissues
surrounding the eye) areas.
o Hib used to be the most common cause of
periorbital cellulitis but post vaccine,
streptococcal organisms are now the most common cause.
• In other areas affected by cellulitis the local inflammatory reaction stems from granulocytic
infiltrations, hyperemia, and capillary leakage.
o This is the basis for the skin disruption inherent in cellulitis. The patient may guard the
tender area. If the cellulitic area overlies a mobile area such as a joint, the patient may
display resistance or anxiety with limb movement, either passive or active.
Additional images of cellulitis
Otitis media, sinusitis, lower respiratory tract disease (NTHi)
• Acute otitis media usually follows a cold after which the ear becomes involved
• Causes severe pain
• The pain will usually settle within a day or two, but can last over a week.
• Sometimes the ear will discharge pus, but this is rare
293
• The tissues surrounding the Eustachian tube swell due to inflammation within. It remains
blocked for a period of time.
• The air present in the middle ear is slowly absorbed into the surrounding tissues creating
negative pressure and pain. This pain can be severe because the sensory nerve endings in
the eardrum respond to increased pressure with pain.
Pneumonia (NTHi)
• Indistinguishable from other pneumonias
Vaccine:
The use of protein-conjugated PRP, has vastly reduced the frequency of infection due to type b H.
influenzae.
o The PRP vaccine consists of the type b capsular polysaccharide and either a diphtheria
toxoid or tetanospasmin (also called tetanus toxoid).
Diagnosis:
Stain:
• For meningitis- take blood or CSF specimen, for localized infection

take specimen by needle aspiration from affected areas.
o Blood specimen can also be collected for cellulatitis and
epiglottitis.
o Detecting of H. influenza (gram-negative rods that range
in size) is useful in preliminary diagnosis.
294
Culture:
• Chocolate agar (heat blood agar to release V and X factors).

• Bacteria can also be detected as small satellite colonies in regular blood culture inoculated with S.
auerus, because Staph. can lyse red blood cells allowing the release of factor X and V.
• However, because the blood is not previously inactivated, there are inhibitors of factor V present,
and therefore the colonies of H. influenza are very small (satellite).
Antigen detection –latex agglutination test:
• PRP Antigen can be detected in CSF and urine where some of the antigen is eliminated intact
• This test only works for Type b which is rare in US.
• PRP is detected with particle agglutination which can detect less than 1 ng/ml of PRP in a clinical
specimen
295
Control of infection:
Treatment:
• Virtually all patients treated early in the course
of H. influenzae meningitis are cured.
• Ampicillin has been the drug of choice, but
presently over 20 percent of all strains of H.
influenzae are resistant to ampicillin because
of plasmid-mediated ß-lactamase production.
• The recommended treatment for H. influenzae
meningitis is ampicillin for strains of the
bacterium that do not make ß-lactamase, and
a third-generation cephalosporin or chloramphenicol for strains that do.
296
Neisseria
General features:
• Gram-negative cocci, usually seen in pairs with the adjacent
sides flattened.
• Oxidase positive, catalase positive
• Both Neisseria gonorrhoeae and meningitides are
considered to be an obligate aerobe; they have been shown,
however, to grow in the absence of oxygen by anaerobic
respiration by using nitrite as a terminal electron acceptor.
• Can make acid byproducts from glucose and/or maltose during glucose oxidation rather than
fermentation.
Oxidase test (Figure below)
• Detects the presence of the enzyme cytochrome c oxidase

using a dye, tetramethyl-p-phenylenediamine
dihydrochoride.
• The dye is colorless in the reduced state but purple in the

oxidized state (when it is oxidized by cytochrome c
oxidase).
• Have lipooligosaccharide (LOS) composed of Lipid A +

core polysaccharide in addition to the typical LPS (Lipid A +
core polysaccharide + O antigen).
Some feature that differentiate Neisseria gonorrhoeae from Neisseria meningitides are listed in the ta
297
Neisseria meningitidis
Virulence factors: (Table bottom right)
• N. meningitidis is grouped on the basis of capsular

polysaccharides,
into 12 serogroups,
some of which are
subdivided
according to the
presence of outer
membrane protein and lipopolysaccharide antigens.
Pathogenesis:
• Asymptomatic carriage of meningococci in the nasopharynx in 5 - 20% of population provides a
reservoir for infection but also enhances host immunity.
• Meningococcal meningitis occurs both sporadically (mainly groups B and C meningococci) and in
epidemics (mainly group A meningococci), with the highest incidence during late winter and early
spring.
• Infection is by aspiration of respiratory particles from an infected person.
• Bacteria which attach to epithelial cells of the nasopharyngeal and oropharyngeal mucosa, cross
the mucosal barrier, and enter the bloodstream.
• If not cleared, blood-borne bacteria may enter the central nervous system and cause meningitis.
• Meningococci establish systemic infections only in individuals who lack serum bacterial antibodies
directed against the capsular or noncapsular antigens of the invading strain, or in patients
deficient in the late-acting complement components.
Diseases:
Meningitis:
• Purulent inflammation of the meninges is mainly due to types A, B (sialic acid capsule), C, W-135
• Headache, fever, meningeal signs are common (typical age 18 – 25)
298
• Meningeal signs include:

o Kernig’s Sign – if positive then a supine patient experiences pain when his/her hips and
knees are flexed at 90% and a physician extends his/her knees further beyond 135
degrees.
o Brudzinski’s Sign – If positive then flexing the patient’s neck causes flexion of the
patient’s hips and knees.
• Arthritis and hearing loss can occur
• People with complement deficiency are more predisposed for this disease
• This is the most common cause of meningitis in infants (6-24 months old) and young adults
(age18-24).
• Sequel of the infection include arthritis and hearing loss. This has a close to 100% mortality rate if
untreated, and 10% in treated patients.
• Although the bacteria can be carried in the pharynx for reasons not fully known, it can sometimes
overwhelm the body’s defenses allowing the infection to spread through the bloodstream and to the
brain.
Meningococcemia: Meningiococcemia
• This is a life-threatening disseminated disease.

• Early recognition is critical to implement prompt antibiotic
therapy and supportive care and to prevent shock
• Meningococcal bacteria produce 100 to 1,000 times
greater amount of endotoxin than other gram negative
bacteria. As the bacteria multiply and move through the
bloodstream, it sheds concentrated amounts of LOS/LPS
toxin. Vessel wall damage
• The endotoxin directly affects the heart, reducing

Platelets activation Activation of coagulation
its ability to circulate blood.
• Thrombosis of small blood vessels, petechial rash Fibrin platelet End-Organ
thrombosis Damage
(pinpoint, nonraised, round, purplish red spots,
Coagulation Low platelet Fibrinolysin
caused by intradermal or submucous factor deficiency count activation
hemorrhaging) on trunk and extremities Fibrin degradation

Generalized Products generated
• The effects of LPS are shown on the right Bleeding
tendency
• Can form large hemorrhagic lesions
Effect of LPS
299
• As some blood vessels start to hemorrhage, major organs like the lungs and kidneys are
damaged.
• Disease may culminate in disseminated intravascular coagulation, shock and bilateral destruction
of the adrenal glands (endocrine glands above the kidney).
• Of the cases of meningococcal disease, 30-50% present with meningitis alone, 40% have
meningitis with septicemia, and 7-10% have only septicemic features.
• Treated with antibiotics
• Complication - Waterhouse-Friderichsen
syndrome: Bilateral destruction of the adrenal
glands due to hemorrhage of adrenal blood
vessels
• N. meningitides has a more frequent bloodstream
spread than gonorrhea.
Control/Prevention
• Penicillin is the drug of choice. Household contacts require chemoprophylaxis with rifampin.
• Menactra vaccine - a quadrivalent (protects against serogroups A, C, W, and Y)
meningococcal conjugate vaccine given at age 11 – 12 yr.
• Bexsero vaccine – as of 2014, this recombinant vaccine is available for Meningococcal
Serogroup B.
Diagnosis:
• Gram stain: of CSF or blood

• Culture: Specimen from sputum, CSF or blood is grown on enriched blood agar or
Thayer-Martin (TM) media. Colonies are oxidase positive.
Control:
300
Neisseria gonorrhoeae:
Virulence factors: (see table on the right)
Note on endotoxin:
• In N. gonorrhoeae the endotoxin is not

only released when bacteria dies but also
when it grows (unlike other gram-negative
in which endotoxin is released during
bacterial death).
• Levels of endotoxins are much larger in
Neisseria.
• Lipid A in the bloodstream binds to LPS
binding protein and then is carried to TLR-4 receptor on macrophages.
• Macrophages are activated and secrete IL-1, IL-6, IL-8.
Pathogenesis:
• Gonorrhea is a sexually transmitted disease of worldwide importance.
• The highest attack rate in both men and women occurs between 15 and 29 years of age.
• Host-related factors such as the number of sexual partners, contraceptive practices, sexual
preference, and population mobility contribute to the incidence of gonorrhea.
• Although gonorrhea is usually acquired by sexual contact, any mucous membrane can be infected
by this microorganism.
Gonorrhea
• Gonococci adhere to mucus secreting columnar epithelial cells, penetrate them, and multiply in
the basement membrane
• Bacteria can also reach sub-epithelial space by traveling between cells
• In sub-epithelial layer bacteria can adhere to polymorphonuclear leukocytes (PMNs) and get
phagocytized by them
301
• Neutrophils kill some bacteria, however some

PathogenesisPurulent
of gonorrhea
urethral discharge
bacteria survive and multiply inside PMNs
(see figure on the right)
• Gonococcal lipopolysaccharide stimulates
the production of tumor necrosis factor, which
causes cell damage.
• Adherence is facilitated through pili and opa

proteins.
• A typical host response is characterized by
invasion of neutrophils, followed by epithelial
sloughing, formation of submucosal
microabscesses, and purulent discharge.
Women
• Women are at 50 % risk of infection after exposure to an infected man
• In women cervix is the primary site of infection, specifically the infection of endocervical
columnar endothelial cells is most common
• 50% of infected women may be asymptomatic.
• Symptomatic women experience vaginal discharge, dysuria, and ascending infections
such as inflammation of fallopian tubes (salpingitis) and ovarian abscesses.
• In 15% of women pelvic inflammatory disease (PID= inflammation of the upper
reproductive tract) is observed. This infection can result in infertility due to tubal scarring
and/or ectopic pregnancy.
• Repeated infections in sexually active people with multiple partners are very common.
Men
• Men are at 20% risk of infection after exposure to infected woman.

• Most men are initially symptomatic
• Symptoms begin 2 – 7 days after infection
• Urethra is the primary site of infection.
• Purulent urethral discharge, and dysuria (pain when urinating)
are common when symptoms are seen. Also common are
unilateral testicular pain and swelling.
• Bacteria can attach to sperm and carry it to upper reproductive tract of women
302
• Although serum antibodies can be detected after the infection, it is not known whether these
antibodies are protective against future infection with gonorrhea.
Gonococcemia (Disseminated gonococcal infection DGI):
 Gonococci may disseminate via the bloodstream. Strains that cause

disseminated infections are usually resistant to serum and
complement. Individuals with genetic defects in late-acting
complement components are at an increased risk for disseminated
infections. Systemic infection stimulates systemic inflammation.
Protection, if it exists, may be strain specific.
• 1-3% of infected women experience disseminated infection due to untreated asymptomatic
infections.
• Manifestations include fever, arthritis in knees, ankles, wrists, and pustular rash. The rash
presents as small elevated area on the skin filled with pus and erythematous base. The rash
is usually seen over extremities but not head or trunk.
• Three fourths of the cases of DGI occur in women; susceptibility is increased if the primary
mucosal infection occurs during menstruation or pregnancy
Ophthalmia neonatorum:
• Purulent conjunctivitis in neonates infected during vaginal delivery.

• Untreated scarring of cornea, blindness
• The most common cause of this disease is Chlamydia
trachomatis.
• Whenever you treat a pregnant woman for gonorrhea you also
treat for Chlamydia not vice versa.
Diagnosis: Culture and non-culture methods

Gonorrhea cannot be diagnosed solely on clinical grounds.
Gram-stain: - Good for symptomatic urethral infections
o For men, a smear of urethral exudate showing intracellular Gram-negative diplococci is

diagnostic.
o For women, culturing on selective medium is often required.
303
Culture: In women and asymptomatic men, Thayer- Martin media is used.
• The specimen cultured is usually obtained from male urethra or female cervix. In case of arthritis it
is obtained from the fluid of joints affected by arthritis.
• Thayer-Martin is a complex, moist, selective media with heated blood (chocolate agar) as a base
and added cysteine, extra amino acids, purines, pyrimidines and starch to neutralize fatty acids. It
is enhanced (10%) CO2 and several antibiotics to inhibit normal flora.
Biochemical test: catalase and oxidase tests can be quickly performed on the cultures.
New tests: Not FDA-cleared for pharyngeal or rectal specimens but approved for urethral and cervical
specimen
o In many labs, culture has been replaced with sensitive nucleic acid tests done directly on
collected specimen
o These tests do not require viable organisms and are useful where maintenance of viability
during specimen transport is a problem; however, it is not as sensitive as culture
Example of such tests include:

1) Nucleic acid amplification tests (NAAT). NAATs detect and make many copies of the genetic
material (DNA) of gonorrhea bacteria. NAATs include polymerase chain reactions (PCRs) and
transcription mediated amplification (TMA). These tests are very accurate and can be done either on
a urine specimen or a sample of body fluid from the potentially infected area.
2) Nucleic acid hybridization test (DNA probe test, molecular probe test). Molecular probe
testing detects genetic material (DNA) of gonorrhea bacteria. This test is done on the body fluid
collected from the potentially infected area, most often the cervix or urethra. Often the molecular
probe test for gonorrhea also tests for chlamydia, another STI with symptoms similar to those caused
by gonorrhea. Serologic tests are not recommended for uncomplicated infections.
Control:
Recommended treatment for uncomplicated infections is a third-generation cephalosporin or a
fluoroquinolone plus an antibiotic (e.g., doxycycline) and is effective against possible coinfection with
Chlamydia trachomatis. Sex partner(s) should be referred and treated. No effective vaccine yet
exists. Condoms are effective in preventing gonorrhea.
304
Practice questions:
1. Which characteristic pertains to meningitis- causing oxidase negative bacteria that have a well-defined
capsule serotype and the infection with which is preventable by conjugating this capsule to a protein?
a. Bile soluble
b. Expresses high levels of LOS
c. Requires hemin and NAD for growth
d. Requires Thayer-Martin media to grow
e. Appears as gram-negative diplococcic
2. After performing a latex agglutination test on a respiratory specimen of a patient symptoms epiglottitis, you
notice that the beads form a clump on the test slide. Which of the following is the best conclusion?
a. The patient’s respiratory secretion contain antibodies to Haemophilus influenzeae
b. The patient’s serum contains antibodies to Haemophilus influenzae
c. The patient’s serum is negative for Haemophilus influenzae antigen
d. The patient’s respiratory secretion is positive for Haemophilus influenzae
e. This test is not specific for Haemophilus influenzae and needs to be repeated
3. A 3-month-old infant is brought to the pediatric ER in severe respiratory distress. The child appears
dehydrated, and there is prominent peripheral lymphocytosis. The chest radiograph reveals perihilar
infiltrates. The child’s grandmother who watched the infant now that the mother returned to work has had a
dry hacking cough. The most likely causative agent is:
a. Haemophilus influenzae type b

b. Bodetella pertussis
c. Streptococcus agalacatiae
d. Corynebacterium diphtheria
e. Streptococcus pyogenes
4. What is the best way to isolate Neisseria gonorrhea?
a. To culture a blood sample on Thayer-Martin agar

b. To culture blood sample on blood agar
c. To culture genital specimen on Thayer-Martin agar
d. To culture a genital specimen on blood agar
e. To culture a genital specimen on chocolate agar
5. Which of the following statements about Neisseria meningitides is true?
a. A polysaccharide vaccine conjugates can be administered

b. People that lack complement components are as susceptible as normal patients
c. It is the most common cause of neonatal meningitis
d. It can only grow on Thayer Martin medium
e. It only leads to a local inflammatory disease
6. Which of the following describes the morphology of the pathogen that causes gonorrhea?
a. Gram negative cocci in chains

b. Gram positive cocci in chains
c. Gram positive rods in pairs
d. Gram positive rods in chains
e. Gram negative cocci in pairs
305
LECTURE 25: MYCOBACTERIA
Describe the structure and pathogenesis of Mycobacteria tuberculosis, and detail how these bacteria
are structurally different from all the others discussed.
• Describe acid‐fast stain

• Describe reasons for the environmental resistance of TB
• Describe the various presentations and stages of TB (Asymptomatic TB, Acute/Primary TB,
Latent/Reactivated TB, Systemic/Military TB)
• Describe the histology and function of granulomas
• Describe what happens in caseating necrosis
• Detail the mechanism behind PPD test
• List the populations at risk
Mycobacterium tuberculosis
General features:
 Mycobacteria are classified as acid-fast bacilli

(AFB) because they retain the carbol-fuchsin red
dye after washing with acid, alcohol, or both.
 In humans they are obligate intracellular pathogens
preferentially utilizing mononuclear phagocytes as their
habitats and only rarely, if at all, inhabiting
nonprofessional phagocytes
 Obligate aerobes growing most successfully in tissues Structure of M. tb envelope
that have the highest partial pressure of oxygen, such

as lung apices
 Slow-growing with a generation time of 12 to 18 hours
 Lesions typically evolve over a chronic course
 60% of cell wall is made of long fatty acids called mycolic acids
 High lipid content resists environmental pressure
 Because the cells are hydrophobic and tend to clump together, they are impermeable to the usual
stains, ie, Gram stain.
 Mycobacterium tends to be quite resistant to most antibiotics such as Penicillin, but there are
some exceptions such as Streptomycin and Rifampin.
 No exotoxin produced
306
The Acid fast stain:
1. Add carbol fuchsin to slide

2. Heat slide
3. Wash slide with acid-alcohol
4. Counter stain with Methylene Blue
Virulence factors:
Active disease/
Primary Progressive
Reactivated disease/
Secondary Progressive
Symptoms of active TB
are abnormal
307
Pathogenesis:
• The transmission is via evaporated droplet called droplet nuclei that are coughed or
sneezed.
• They are 1 to 5 microns in diameter and can remain suspended in the air for several hours,
depending on the environment.
Once organisms have made their way into the lung, they have four potential fates:
1. The initial host response to primary infection can be completely effective and kill all
bacilli, such that the patient has no chance of developing tuberculosis at any time in the
future. These patients have resolved infection.
2. Bacilli may become dormant and never cause disease at all, such that the patient has
what is referred to as latent infection and will manifest only by a positive tuberculin skin
test.
3. The organisms can begin to multiply and grow immediately after infection, causing
clinical disease known as pulmonary tuberculosis or progressive (primary) TB.
4. The latent organisms can eventually begin to grow, with resultant clinical disease,
known as reactivation (secondary) tuberculosis.
• In most people primary lesions are controlled and go through fibrosis and calcification.
• However, M. tuberculosis can remain viable for a long time.
• Infections may never be apparent or may progress to disease in the lungs.
• Multiplication in the non-activated macrophage can also be followed by dissemination to lymph
nodes and bloodstream.
Details of progression:
Stage 1
 The larger aerosol particles are trapped in the mucosal

surfaces and are removed by ciliated cells in the respiratory
tract.
 Smaller particles with as little as 1-3 bacilli can reach alveolar
spaces and establish infection in susceptible host (inside a
macrophage).
 Infection correlates to the number of organisms in the sputum
of a person with active disease.
 Alveolar macrophages uptake bacteria.
Stage 2
2a. Organism prevents fusion of

phagosome and lysosome and evade
intravesicular killing by destroying any
oxidants that are formed.
2b. Bacterial replication eventually causes

macrophage to burst
308
2c. Surrounding blood DCs phagocytose M. tb and bring it to regional lymph nodes.
Stage 3 – Walling off
• Th1 cells can activate macrophages which can subsequently

control the bacterial growth.
• If the infectious load is small, granulomas /tubercles (localized
collection of activated macrophages, lymphocytes and fused
Langerhans cells) prevent bacterial spread.
• In patients with a higher bacterial load the center of the tubercle
is characterized by "caseation necrosis" meaning semi-solid
or "cheesy" consistency. These eventually calcify and wall-off
bacteria. M. tb cannot multiply within these tubercles because of
the low pH and anoxic environment.
• 90% of people infected with TB reach this
stage but feel no symptoms.
• Another 5% of people develop symptoms
because the Th1 cells have a hard time fighting
bacteria and become over-activated (see stage
4/5).
• Yet in additional 5% of people without the initial
symptoms, M. tb. can persist within the tubercles
for extended periods and be reactivated later in
in life when the immune response wanes.
Immune response weakening is the reason why
in many patients disease does not develop until
later in life
•
2c. Surrounding blood DCs phagocytose M. tb and bring it to regional lymph nodes.
Stage 3 – Walling off
• Th1 cells can activate macrophages which can subsequently control the bacterial growth.
• If the infectious load is small, granulomas /tubercles (localized collection of activated
macrophages, lymphocytes and fused Langerhans cells) prevent bacterial spread.
• In patients with a higher bacterial load the center of the tubercle is characterized by "caseation
necrosis" meaning semi-solid or "cheesy" consistency. These eventually calcify and wall-off
bacteria. M. tb cannot multiply within these tubercles because of the low pH and anoxic
environment.
• 90% of people infected with TB reach this stage but feel no symptoms.
• Another 5% of people develop symptoms because the Th1 cells have a hard time fighting bacteria
and become over-activated (see stage 4/5).
309
• Yet in additional 5% of people without the initial symptoms, M. tb. can persist within the tubercles
for extended periods and be reactivated later in in life when the immune response wanes. Immune
response weakening is the reason why in many patients disease does not develop until later in life
Stage 4/5 - Primary progressive or secondary progressive pulmonary TB
• In some patients, overactive Th1 response that for some reason can’t contain the bacteria and
causes the release of a high level of cytokines that cause systemic symptoms.
• Primary TB - In about 5% of people, within a year of infection, without containment, the caseating
centers of tubercle liquefy, the pathogen starts growing and spreads.
• From the bronchi TB can be disseminated to various other lung regions which then causes
pulmonary symptoms to develop. This damages the lung severely.
• Secondary (reactivated) TB- In 5% of patients disease does not develop until later in life when
immune response wanes.
• TB symptoms are attributed to infections with an overreaction or under-reaction of the immune
response.
Chest radiographs in pulmonary active tuberculosis

A: Infiltrates in left lung
B: Bilateral advanced pulmonary tuberculosis and
cavitation in apical area of right lung. Gohn’s focus
310
Extra-pulmonary (Miliary) TB
• The tubercle can also invade an artery.

• The hematogenous spread may result in extrapulmonary TB otherwise known as miliary
tuberculosis.
311
Predisposition to tuberculosis:
• HIV infection is the #1 predisposing factor for M. TB. infection.

• 10 percent of all HIV-positive individuals harbor M. TB.
• This is 400-times the rate associated with the general public.
• Having HIV increases risk of TB reactivation 300 times.
Other factors include:
• Close contact with large populations of people, i.e., schools, day cares, nursing homes,
dormitories, prisons, etc.
• Poor nutrition
• Homeless
• IV drug use
• Alcoholism
Difference between TB Infection and TB disease:
• TB infection means that M. tb is in

the body but the immune system is
keeping the bacteria under control.
The immune system does this by
producing macrophages that
surround the tubercle bacilli. The
cells form a hard shell that keeps
the bacilli contained and under
control.
• Most people with TB infection have
a positive reaction to the tuberculin
skin test. People who have TB
infection but not TB disease are NOT infectious, i.e., they cannot spread the infection to other
people. These people usually have a normal chest x-ray.
Summary:
Primary TB infection (usually asymptomatic)
• 75-90% of people activate good cellular response and in 3-6 weeks bacteria ceases to
replicate.
Active disease (Primary progressive):

• ~5% of patients, pulmonary macrophages are unable to contain the bacilli and are
overwhelmed, leading to a clinically apparent infection.
• Symptoms may include lower respiratory react infection with night sweats, cough, weight loss,
and bloody sputum.
• Usually both lung lobes are affected.
312
Reactivated disease (Secondary progressive, latent progressive)

 In 5-20% of patients who cannot completely eradicate bacteria during primary stage, disease
develops later in life due to a reactivation of a primary lesion that may have been quiescent for
years.
 Large necrotic areas in the lungs, the bronchus and small blood vessels can be noted.
Untreated, this will lead to disseminated disease and death.
Untreated tuberculosis has a mortality rate of 50%.
Diagnosis/Screening:
Screening:
TB test:
 An individual infected with TB will test positive in a PPD test 3-4 weeks after initial immune
conversion and will remain positive for life.
 In the test, induration will develop 24-72 hours after injection of PPD antigens.
 A positive result should be followed by a chest X–ray.
313
Quantiferon Gold test (Figure right):
 Interferon Gamma Release

Assays measure the cell
mediated response in infected
individuals through the levels of
interferon gamma released.
 During an infection, T cells from
the individual will be sensitized
(via MHC II proteins) to the
antigens presented by macrophages of the infected organism.
 Memory Th1 cells will then be releasing interferon-gamma upon recognizing a macrophage
presenting tuberculin antigen. Interferon gamma release assays take advantage of this natural
process in infected immunocompetent individuals.
 When tuberculin purified protein derivatives are applied to whole-blood samples from infected
individuals, Th1 cells previously sensitized to the antigens will be present in the blood, and will
bind to the MHCII/antigen complex.
 The T cells will then release Interferon-Gamma which can be detected by an ELISA type assay;
the presence of sensitized T-cells in infected individuals will result in far higher levels of IFN-G
release than among uninfected individuals.
Chest X-ray:
 In active pulmonary TB, infiltrates or consolidations and/or cavities are often seen in the upper
lung.
 However, lesions may appear anywhere in the lungs.
 Old healed tuberculosis usually presents as pulmonary
nodules in the hilar area or upper lobes, with or without
fibrotic scars and volume loss.
 Pleural scarring may be present.
 Nodules and fibrotic scars may contain slowly multiplying
Chest X-ray
tubercle bacilli with the potential for future progression to
active tuberculosis.
 Persons with these findings should be considered high-priority candidates for treatment of
latent infection.
314
 Conversely, calcified nodular lesions pose a very low risk for future progression to active
tuberculosis.
 Abnormalities on chest radiographs may be suggestive of, but are never diagnostic of,
TB.
To have a definitive diagnosis of TB infection, suspected patients can have the following tests:
Stain:
 Clinical sputum specimen can be directly analyzed by acid-fast stain,
and then particular mycobacterial specie is confirmed by PCR.
Culture:
 M. tb grows very slowly, so collected specimen is decontaminated with
Culture
2% NaOH to kill other contaminating, rapidly dividing bacteria and are
then plated on a Lowenstein-Jensen medium which is an egg based
medium. Increased CO2 (5-10%) also helps bacterial growth.
 Bacteria will appear in 2 weeks. These colonies can be tested for niacin
production which is unique for M. tb.
315
Practice questions:
1. Which of the following statements about the purified protein derivative (PPD) and tuberculin test is
most correct?
a. It is strongly recommended that the medical and other health science students have PPD test
every year.
b. Persons immunized with BCG rarely if ever convert to positive PPD skin test.
c. A positive tuberculin test result indicates that an individual has been infected with M. tuberculosis
in the past.
d. A positive PPD skin test results imply that a person is immune to active tuberculosis.
e. The first intradermal PPD test may result in the sensitization of a person’s immune system and
cause that individual to develop anti-tuberculin response.
2. A 10-year-old child has a primary pulmonary tuberculosis disease. Which of the following features
of tuberculosis is most correct?
a. In primary tuberculosis an active exudative lesion develops and spreads to the to the lymphatics
and regional lymph nodes
b. The exudative lesion of primary tuberculosis rarely heals spontaneously
c. If tuberculosis develops later, it usually is a result of exposure to a different strain of tuberculosis
d. A PPD test, which tests for the antibody titer again tuberculosis, should test positive in the child
e. A PPD test will be positive in the child only after secondary exposure to tuberculosis
3. Which of the following statements regarding interferon-gamma release assay is correct?
a. It is useful for evaluating immunocompromised patients for active tuberculosis

b. It detects antigens common to all Mycobacteria species
c. It is used to detect antibodies in a patient exposed to tuberculosis
d. It uses a molecular probe to detect the DNA of the organism
e. It is used as an alternative to the tuberculin skin test to evaluate for latent
316
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MERP Medical Immunology

Course: Microbiology and Immunology

Lectures 2 and 3: B cell differentiation

Lecture 4: Antigen Processing, MHC Presentation, and T cell Development

Lectures 5 and 6: Activation of Immune Response

Lecture 8: Hypersensitivity Reactions

Lectures 9 and 10: Introduction to Virology I

Lecture 11: DNA Viruses: Poxviridae and Parvoviridae

Lecture 12: DNA Viruses: Papillomaviridae, Polyomaviridae and Adenoviridae

Lecture 13: DNA Viruses: Herpesviridae

Lecture 14: DNA Viruses: Hepadnaviridae

Lecture 15: RNA Viruses: Picornaviridae and Orthomyxoviridae

Lecture 16: RNA Viruses: Paramyxoviridae, Togaviridae and Flaviviridae

Lecture 17: RNA Viruses: Rhabdoviridae and Retroviridae

Lecture 18: Introduction to Bacteriology

Lecture 19: Staphylococcus aureus

Lectures 20: Streptococci

Lectures 21: Bacilli and Clostridia

Lectures 22: E coli, Shigella, Salmonella, Vibrio

Lecture 24: Corynebacterium, Bordetella, Haemophilus. Neisseria

Lecture 25: Mycobacterium tuberculosis

LECTURE 1: INTRODUCTION TO IMMUNOLOGY

* List the cells of the immune system and their origin

Protection against Infectious Disease

Innate pathogen non-immune barriers are first defense:

Innate immune response are second defense:

Cytokines are released: Inflammation is part of the complex biological response of

chemotaxis. Chemokines and other cytokines are "locally" produced in tissues

Below is an example of inflammatory response mediated by cytokines

Complement and chemical agents participate in inflammation. Some chemical agents

1. is found only in vertebrates

Adaptive immune responses is mediated by lymphocytes and includes:

Remember that lymphocytes are specialized types of

1. They are antigen-specific leukocytes.

Differences between antibodies of B cells and T cell receptors of T cells

Similarities between antibodies and T cell receptors

Innate and adaptive immune responses collaborate to fight off infection.

Innate immune mechanisms including inflammation and

Figure (right): If an individual lacks adaptive immunity, the innate

Organs of the Immune System

An antigen is NOT required for the development of mature antigen-specific T and B

2. Secondary (peripheral) lymphoid organs:

Secondary lymphoid organs function

Mature naïve lymphocytes get

a. Lymph nodes which are

The trafficking of immune cells

1b. Naïve B cells and T cells leave the primary

1c. High endothelial venules are specialized

2. In the lymph node, antigen comes in contact

4. From circulation, activated lymphocytes go to

5. At any given time many leukocytes recirculate

Function of the adaptive immune system – response to an infection

Adaptive immunity can be acquired in two different ways:

1. The main advantage of passive immunization over active immunization is that:

2. A lymphoid progenitor cell differentiates into ______ .

3. The innate immune system is mainly mediated by _____.

a. T cells and B cells

4. Cytokine release _____ vascular permeability.

5. T cells finish development in the _____.

6. T cell receptors can bind to _____.

LECTURES 2 AND 3: GENERATION OF B CELL DIVERSITY AND B CELL DEVELOPMENT

• Understand the structure of antibody genes and proteins

• Understand the process of recombination

To reach the lymph nodes, B cells and T cells travel