Overview of Viruses and Virus Infection

The Nature of Viruses

viruses are sub-cellular, infectious agents that are obligate intracellular parasites

Sub-Cellular
viruses are very small viruses range in size but are smaller than the cells they infect

Overview of Viruses and Virus Infection

The Nature of Viruses -Infectious Agents
a simple test to see if an agent is a virus or a toxic compound is to serially dilute it
if it retains ability to cause damage it must be replicating therefore an infectious agent

practical importance: viruses are the cause of many human, animal and plant diseases
smallpox, AIDS, influenza, rindepest, foot and mouth disease

Overview of Viruses and Virus Infection

The Nature of Viruses
viruses are obligate intracellular parasites
only the simplest RNA viruses can have their genetic material replicated in a cell-free system must replicate in a living, usually replicating host cell

viruses are acellular

Overview of Viruses and Virus Infection

The Nature of Viruses
viruses consist of a genome and a protein covering or shell
genome is either RNA or DNA but only one naked genetic material without a capsid is called a viroid a virus can have a much more complicated structure including membranes (envelopes), multiple layers of protein, spikes, legs, and more

Overview of Viruses and Virus Infection

The Nature of Viruses
can broadly classify viruses into 3 main groups by the genetic material and method of replication DNA genomes RNA genomes retroviruses

Overview of Viruses and Virus Infection

The Nature of Viruses
DNA genomes either ds or ss
the viral DNA genome replicated by direct DNA to DNA copying viral DNA is transcribed to mRNA for translation into proteins

Overview of Viruses and Virus Infection

The Nature of Viruses
RNA genomes
the viral RNA genome replicated by direct RNA to RNA copying single-stranded RNA viral genomes can be positive (+) sense and act as mRNA single-stranded RNA viral genomes can be negative (-) sense and cannot directly act as mRNA some ss-RNA viruses are both some RNA viruses are double stranded

Overview of Viruses and Virus Infection

The Nature of Viruses
RNA genomes which replicate through an obligate DNA intermediate
retroviruses contain or encode the enzyme reverse transcriptase infectious particle contains RNA

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
before the 1950s there was no cell culture presumptive viruses were passed through live host animals
ethics selection of mutants contamination with other viruses and other microorganisms

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
embryonated chicken eggs many viruses can replicate in eggs multiple sites of infection
chorioallantoic membrane amniotic cavity or yolk sac or allantoic cavity

for some viruses you can get high titres still used as a general purpose viral screening medium

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
cell culture now the most important technique primary cell culture continuous cell culture

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
primary cell culture
cells are derived from tissue taken directly from an animal cells divide 5 to 10 times, 100 times in a diploid cell line derived from a homogenous cell type useful when virus cannot grow in a continuous cell line, there is a need for differentiated cells or for some vaccine applications

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
continuous cell culture
cells are transformed to immortality, they will reproduce forever usually derived from a single cell or cell type may be derived from a tumor, or from normal cells treated with mutagenic chemicals, radiation or a tumor virus may be frozen and restored, provide a uniform cell population, often well characterized and predictable

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
continuous cell culture
however may have lost characteristics of the normal cell dedifferentiated abnormal chromosomes drift over time and multiple serial passages may not support the replication of a given virus or the wt of that virus may be tumorigenic in animals (including humans)

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
cell culture
most cells are contact dependent they will grow until they touch other cells they form a monolayer in tissue culture flasks can be removed and converted into a suspension by treating with trypsin (trypsinization) suspension can be counted and used to inoculate another flask (serial passage, splitting)

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
viral cytopathic effects (CPE)
viral infection and replication inside a cell can have visible (microscopic) effects rounding up cells lose their normal morphology and often become more spherical detachment cells lose their contact with the substrate and float in the growth medium (also do this when they are dead)

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
viral cytopathic effects (CPE)
nuclear shrinking nuclear membrane proliferation vacuoles in the cytoplasm syncytia inclusion bodies in the nucleus or cytoplasm organelle fragmentation blebbing

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
plaque assay
animal virus equivalent to a bacteriophage assay or a colony count in bacteriology add viruses to cells (MO << 1) inoculate a flask with the cells, allow to grow to confluency or immobilize the cells in agar count the plaques viruses replicate in the original infected cell then spread to adjacent cells

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
plaque assay
infected cell die and lyse or demonstrate visible CPE the areas of infected cells are localized and appear as dots or plaques cells can be stained to make plaques more visible variation with transforming viruses, infected cells lose contact inhibition and pile up on top of each other, count the little piles or foci

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
endpoint dilution assay
used when viruses do not produce CPE dilute a stock of viruses (multiple serial10 fold dilutions) add a volume of each dilution to test animals observe effects (disease symptoms, death) endpoint is the dilution which caused 50% infection of the test organisms measures infectious dose (ID) rather than the number of viruses, ID50

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
particle to plaque forming ratio
not all viral particles cause an infection need to independently measure the number of viral particles count the number of plaques formed (or other effect) by a known number of viral particles cannot be higher than 1, usually 10 10,000 or more for animal viruses

Overview of Viruses and Virus Infection

Techniques in Virology
how do you isolate an unknown virus in order to study it?
Serological techniques
most viruses induce immunological reactions in their hosts including specific antibodies Abs can be obtained in large quantities from animals (or humans) for use as analytical reagents can detect viruses can detect exposure to viruses can determine similarities or differences between viruses can monitor the course of infection

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Hemagglutination Inhibition
red blood cells (RBC) can absorb antigens such as proteins and carbohydrates as well as entire viruses a specific Ab will cause the RBC to agglutinate or stick together if they do so in a microtitre plate they will carpet the entire bottom of the well if they are prevented (inhibited) from agglutinating then they will roll down the well to form a button at the bottom of the well (negative reaction)

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Hemagglutination Inhibition

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Hemagglutination Inhibition
a fixed amount of treated RBC are added to each well in a microtitre plate a serial dilution of an antisera is made and added to each well if the antisera contains Ab to that specific virus coating the RBC then hemagglutination will occur the reciprocal of the highest dilution which causes hemagglutination is the titre of the antisera

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Hemagglutination Inhibition
repeat the first step but now adding to all wells the amount of antisera which just causes hemagglutination add to the wells samples which contain the virus free virus competes for Ab with the virus absorbed to the RBC, uses up the antibody and prevents (inhibits) hemagglutination from occurring by making a 2-fold dilution series of the test virus solution you can measure (titre) the virus can also be used to identify viruses if you have antisera (Ab) to known viruses

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Virus Neutralization
Ab to a particular virus are raised Ab has to be able to block infection of the host cell by the virus a specified virus preparation is incubated with dilutions of the specific Ab and then used to infect host (cell culture, animal) can be used to assay virus, Ab or to identify a particular virus
only a specific Ab to that virus will inhibit infection

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Complement fixation
the complement system is a collection of proteins which interact with Ab and eventually lyse cells RBS are used as the target cell since their lysis can be visually monitored a sample containing a virus is added to specific antiserum if the Ab are specific to the virus they will bind to the virus and use up or fix the complement system present RBC and an anti-RBC protein are added to the sample if all the complement is used up (fixed) there will be no hemolysis of the RBC

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Complement fixation
no hemolysis of the RBC (carpet of RBC in the microtitre plate well) = positive reaction hemolysis of the RBC = negative reaction not a very specific test but is still used to identify which group of viruses that you are dealing with still used as a screening tool only requires very small amounts of sample

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Enzyme-Linked ImmunoSorbent Assay (ELISA)
probably the most widespread and important diagnostic technique in virology, immunology and clinical microbiology many different formats for measuring either an antigen or a specific Ab

Overview of Viruses and Virus Infection

Enzyme-Linked ImmunoSorbent Assay
an antigen is absorbed onto a solid support
in this case the virus or a protein from the virus is absorbed onto polystyrene of a 96 well microtitre plate

a sample is added which contains the specific Ab to be detected the specific Ab binds to the antigen and the well is rinsed a secondary Ab is added which binds to the first Ab
ie anti-human Ab or anti-human, anti-IgG Ab the 2o Ab is conjugated to an enzyme

Overview of Viruses and Virus Infection

Enzyme-Linked ImmunoSorbent Assay
the enzyme catalyzes a reaction with a coloured product
horseradish peroxidase alkaline phosphatase

the enzyme substrate is added and the reaction proceeds for a fixed time the absorbance of the well is measured
absorbance is proportional to the amount of specific Ab present (captured) in the sample

Overview of Viruses and Virus Infection

Enzyme-Linked ImmunoSorbent Assay
the enzyme catalyzes a reaction with a coloured product
the coloured product can be soluble, more useful for measurement in a spectrophotometer the coloured product can be insoluble, this is more useful for immunoprecipitation or cell labeling studies some substrates are chemoluminescent, produce a photon of light as a product, very sensitive

Overview of Viruses and Virus Infection

Enzyme-Linked ImmunoSorbent Assay
there are many types of ELISAs
non- competitive
a capture reagent is bound to the substrate and a sample added, the analyte is captured by the reagent, has a fixed capacity

competitive
a fixed amount of enzyme-labeled Ab is added to the well along with the patient sample any Ab in the patient sample compete with the enzymelabeled Ab and prevent its capture the more patient Ab in the sample, the less enzymelabeled Ab bound and the less colour developed

Overview of Viruses and Virus Infection

Enzyme-Linked ImmunoSorbent Assay
there are many types of ELISAs
either the anigen (virus, viral protein) is bound to the well and patient Ab captured or: the specific Ab is bound to the well and antigen is captured

Overview of Viruses and Virus Infection

Techniques in Virology - Serological
Enzyme-Linked ImmunoSorbent Assay (ELISA)