Introduction to the Fungi

In this part of the course, we will be studying the organisms that are referred to as fungi (sing.=fungus). Although you have now studied various groups of plants and algae, as well as other eukaryotic organisms, in other courses, you will find that the fungi are probably the least understood among the eukaryotes. Looking back at my undergraduate career, prior to taking my first mycology class, I had a very negative concept of the fungi. My impression of fungi was that they were disease-causing organisms that were found in unsanitary conditions. Although this impression was not entirely wrong, fungi are so much more than that. They are also very beneficial organisms. We have derived a number of useful antibiotics from them, including the "wonder drug" penicillin. Without fungi, we would not have leavened bread, Roquefort and Camembert cheeses, beer, wine and other alcoholic beverages and some mushrooms, morels and truffles are considered to be delicacies among gourmands. While these aspects of fungi are of interest, they will not be the emphasis on our discussions of fungi. If you are interested in learning about these aspects of fungi, you may go to the Botany 135 home page. The emphasis here, instead, will be to study the relationships of the various groups of fungi and attempt to make sense of their phylogeny.

Classification of Fungi

Once upon a time biologist only recognized two kingdoms: Plant and Animal (this was how organisms were classified when I was an undergraduate). Fungi, as well as bacteria and algae were classified in the plant kingdom under this system and that is the reason that these organisms are traditionally studied in botany. In the case of fungi, MYCOLOGY is that part of botany that studies fungi. Although fungi are no longer classified as plants, there is still good reason to study them in botany. Fungi are most often associated with plants, commonly as decomposers, and pathogens, and as their benefactors, e.g. mycorrhiza, but "What is a fungus?" Based on what your studies on plants, in this course, you know that plants are known to be derived from a single algal ancestor from the algal division: Chlorophyta, i.e. they are monophyletic. Once upon a time, the fungi were also believed to be monophyletic and to be derived from an algal ancestor that lost its ability to photosynthesize. However, over time, with the discovery of new techniques in determining relationships between organisms, it was discovered that the fungi are made up of a polyphyletic group of organisms that, in some cases, are very distantly related to one another. Thus, organisms that we call fungi are not grouped together because they are closely related, but rather because they share a combination of characteristics that we will now go over:

Characteristics of "fung" in the broad sense

Achlorophyllous: Fungi cannot make their own food like plants. They are heterotrophs and depend upon other organism for their carbon source. Heterotrophs can further be divided into the following categories: Parasites: Organisms that derives their nutrition from the protoplasm of another organism (=host). Saprobes: Organisms that obtains their carbon source (=food) from the by-products of organisms or dead organisms. However, if the opportunity arises, some saprobes may become parasitic. Such organisms are said to be facultative parasites. Symbiosis: In the strict sense, this term refers to the habitual "living together" of different species. As such, there are a number of different categories of relationships that may fit under this term. However, we will define it in its most common usage: "The intimate association of two dissimilar organisms in a mutually beneficial relationship, e.g. lichens and mycorrhizae." This type of symbiosis is specifically referred to as a mutualistic symbiosis. Eukaryotic: Fungi have membrane bound organelles, i.e. nucleus, mitochondrion, E.R., etc. Once upon a time filamentous bacteria called Actinomycetes were classified with fungi, but this is no longer the case. The the body or assimilative part of the fungus (=thallus) usually takes the following forms: Yeast: Unicellular fungi that reproduce, asexually, by budding or fission (terms to be defined later). Mycelium: The collective, filamentous strands that make up the fungal thallus. Strands of mycelium is referred to as hyphae (sing.=hypha). Mycelium may be of two types: Septate: Mycelium that is divided into discreet cells by cell walls that are laid down at regular intervals along the length of the mycelium. These cell walls are called septa (sing.= septum). Coenocytic: Mycelium that is not divided up by septa and forms a continuous tubular network. Septa, however, are present occasionally, especially where reproductive structures occur and where the cell wall of the mycelium has been compromised. Some species may have have thalli that are mycelium and yeast. Such fungi are said to be dimorphic (=two forms). The assimilative stage of the fungal body, i.e. mycelium or yeast, has a cell wall. In the strict sense organisms classified as fungi have cell walls composed primarily of chitin. However, we will be also be covering "fungi" that do not have chitin in their cell walls. Fungi have a common nutritional mode: Absorption: The transport of food from their substrate into their cell walls. The following events occur in this mode of nutrition: If the available food that the fungus is using is soluble, i.e. a simple organic compound, such as simple sugars and amino acids, the mycelium or yeast cells can transport the food directly through their cell wall.

If the available food is insoluble, i.e. a large, complex, organic compound, such as lignin, cellulose and pectin, then production the food must first be digested. Digestion is carried out by the production of various enzymes that are substrate specific and will break down insoluble food material to soluble compounds that can be transported through the cell wall. Although this appears to be very different from the way in which we (animals) digest food, it differs only in the sequence of events that takes place. Where we ingest food and then digest it, fungi first digest their food before ingestion. Either sexual or asexual reproduction or both may occur by spores. Spores and/or gametes can be motile or not. However, in the strict sense as fungi are currently defined, only those organisms that produce nonmotile spores and gametes are classified as fungi. Nevertheless, we will be going over organisms that have motile spores, called zoospores, and motile gametes.

In summary then, the organisms that we call fungi represent a heterogenous group, i.e., they are polyphyletic, that are not closely related as you will soon see.

When I was an undergraduate, organisms that were defined as fungi were heterotrophs, with cell walls, that have filamentous or yeast thalli. Today, fungi that are classified in the Kingdom Mycetae (=true fungi), have a more restrictive set of characteristics: Eukaryotes with cell wall material composed primarily of chitin and derive their nutrition by absorption. Why the change? As with any science discipline, knowledge in mycology is dynamic and we have accumulated a great deal of knowledge about the fungi since I first took mycology 30 years ago. The additional knowledge has led us to change our concepts as to the relationship of those organisms that were classified as fungi. Much of the knowledge that led to these changes began in the early 1960s, when a great deal of research was carried out in fungal ultrastructure. This was later followed by comparative studies on cell wall biochemistry (BarnickiGarcia, 1970) and more recently molecular approaches, utilized in studying relationships of organisms, has led to further changes in our concepts as to how we define fungi.

Another change that occurred during this period of time that affected not only fungi, but also "plants" and "animals" as well. When I was an undergraduate, the classification for plants and animals were very broadly defined. As I mentioned, above, organisms in those days were classified as either plants or animals. Fungi, as well as bacteria and algae were classified in the plant kingdom, based mainly on the presence of a cell wall and the lack of ingestion of food material. However, *Whittaker (1969) erected the five-kingdom system, which is currently still the accepted system of classification of organisms. As a result, the fungi, algae and bacteria were placed in different kingdoms. While, the concepts of the five kingdoms have changed since Whittaker (1969), the classification of organisms into five kingdoms have persisted.

Although, our definition of a fungus has changed a great deal, by tradition, mycology classes have continued to study the same organisms that have been studied since the 1960s and earlier. While mycologists have learned a great deal about the fungi in these last 30-35 years, there is still not agreement as to how best to classify the fungi, nor will there likely be any agreement at a later time. Some examples of the more popular classification schemes are reproduced below:Ainsworth and Bisby (1971) Bessey (1950) Alexopoulos (1962) Kingdom Fungi Kingdom Plantae

Division Mycota Myxomycota Acrasiomycetes Hydromyxomycetes Myxomycetes Plasmodiophoromycetes

Mycetozoa Myxomycetes

Myxomycotina

Eumycotina Plasmodiophormycetes Eumycota Mastigiomycotina Chytridiomycetes Hyphochytridiomycetes Oomycetes Phycomyceteae Chytridiomycetes

Hyphochytridiomycetes Oomycetes

Zygomycotina Zygomycetes Trichomycetes Zygomycetes Trichomycetes Carpomycetae Ascomycotina Hemiascomycetes Plectomycetes Pyrenomycetes Discomycetes Laboulbenomycetes Loculoascomycetes Ascomyceteae

Pyrenomycetes Ascomycetes Hemiascomycetidae Euascomycetidae Plectomycetes Pyrenomycetes Discomycetes Laboulbeniomycetes Loculoascomycetidae Basidiomycotina

Teliomycetes Hymenomycetes Phragmobasidiomycetidae Holobaasidiomycetidae Gastromycetes Class: Basidiomyceteae

Teliosporeae

Heterobasidiae

Hymenomyceteae

Gasteromyceteae

Basidiomycetes

Heterobasidiomycetidae

Homobasidiomycetidae

Gasteromycetes Deuteromycotina Blastomycetes Hyphomycetes Coelomycetes The Imperfect Fungi Moniliales Sphaeropsidales

Melanconiales Deteromycetes

Over the last ten years, there have a great deal of changes in the concepts of the relationships of the various groups of fungi. The classification below represents one of the more recent systems and is based, in part, on molecular research that has been carried out in recent years. Because of time constraints, not all of the different taxa of "fungi" will be listed below or covered in this course.

Kingdom: Protista

Division: Myxomycota (Currently classified with protozoans)

Flagellated Fungi Division: Hyphochytridiomycota Division: Oomycota

The above two divisions have also been placed in a recently erected Kingdom: Stramenopila. This kingdom includes the divisions Phaeophyta and Chrysophyta, which you have already studied in the algae portion of this course

Kingdom: Myceteae (=Fungi)

Division: Chytridiomycota

Division: Zygomycota

Division: Ascomycota Class: Ascomycetes Order: Saccharomycetales and Schizosaccharomycetales (Yeast) Filamentous Ascomycetes Order: Eurotiales (Fruiting body a cleistothecium) Order: Sordariales and Xylariales (Fruiting body a perithecium) Order: Pezizales (Fruiting body an apothecium) Order: Dothideales (Fruiting body an ascostroma)

Division: Basidiomycota Class: Basidiomycetes Order: Agaricales (Mushrooms) Order: Lycoperdales, Phallales and Nidulariales (Puffballs) Order: Aphyllophorales (Polypores) Order: Tremellales, Dacrymycetales and Auriculariales (Jelly Fungi) Class: Teliomycetes (Rust) Class: Ustomycetes (Smuts)

Division: "Deuteromycota" (Asexual Fungi)

As we study the various groups of fungi, I will attempt to point out the problems in using a classification scheme in a course such as this one where the goal is to study the phylogeny and relationship of the various organisms that we are calling fungi.

During the final lab in this section of the course, I will bring in several dated textbooks, such as Alexoupolous (1962) and Bessey (1950), which were considered outstanding textbooks for their time. At that time, I want you to compare the concepts of fungi in these books with our present concept.

Whenever I look back at these textbooks, it never ceases to amaze me as to how much we have progressed in our knowledge of the fungi. Perhaps, this will also be something that you will appreciate by the end of this section of the course.

*Whittaker, R.H. 1969. New concepts of kingdoms of organisms. Science 163: 150-161.

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GENERAL PROPERTIES OF VIRUSES

MM 526-541

Table of Contents Educational Objectives General Properties of Viruses Replication Cycle Summary

EducationalObjectives General 1. To familiarize you with the structural components of the virus, which can act as antigens during the infection process.

2. To emphasize the unique nature of viral nucleic acid and its role in the infection process.

3. To familiarize you with the morphological types of virus in order that this information can be used in making a diagnosis.

4. To develop an understanding of the virus replication cycle in order to appreciate how the physician can interrupt this cycle. Specific educational objectives (terms and concepts upon which you will be tested) Capsid Plus strand Capsomere Protomere Complex viruses Replicative form Cubic symmetry Replicative intermediate Eclipse Ribonucleoprotein (RNP) Envelope Spikes Gaps Viral morphology & structure Helical symmetry Viral nucleic acid types Icosahedral symmetry Viral replication cycle Inclusion body Viroid

Negative strand Viropexis Nucleoprotein (NP) General Properties of Viruses Structure

1. Nucleic acid -contains 3-400 genes Deoxyribonucleic Acid (DNA) -unique features Single and/or double stranded Glycosylated and/or methylated Gaps present in double stranded molecule Circular or linear Bound protein molecules Unique purine and/or pyrimidine bases present Ribonucleotides present Ribonucleic Acid (RNA) - Unique features Single or double stranded Segmented or unsegmented Bound protein molecules Unique purine and/or pyrimidine bases present Folding pattern 2. Capsid -The capsid accounts for most of the virion mass. It is the protein coat of the virus. It is a complex and highly organized entity which gives form to the virus. Subunits called protomeres aggregate to form capsomeres which in turn aggregate to form the capsid.

3. Envelope -this is an amorphous structure composed of lipid, protein and carbohydrate which lies to the outside of the capsid. It contains a mosaic of antigens from the host and the virus. A naked virus is one without an envelope.

4. Spikes. These are glycoprotein projections which have enzymatic and/or adsorption and/or hemagglutinating activity. They arise from the envelope and are highly antigenic.

Morphology (Symmetry) 1. Icosahedral -The protomeres aggregate in groups of five or six to form the capsomere. In electron micrographs, capsomeres are recognized as regularly spaced rings with a central hole. The shape and dimensions of the icosahedron depends on characteristics of its protomeres. All icosahedral capsids have 12 corners each occupied by a penton capsomere and 20 triangular faces, each containing the same number of hexon capsomeres. Icosahedral symmetry is identical to cubic symmetry.

2. Helical -The protomeres are not grouped in capsomeres, but are bound to each other so as to form a ribbon-like structure. This structure folds into a helix because the protomeres are thicker at one end than at the other. The diameter of the helical capsid is determined by characteristics of its protomeres, while its length is determined by the length of the nucleic acid it encloses.

3. Complex -e.g., that exhibited by poxvirus and rhabdovirus. This group comprises all those viruses which do not fit into either of the above two groups.

Replication Cycle 1. Adsorption -Viruses can enter cells via phagocytosis, viropexis or adsorption. Adsorption is the most common process and the most highly specific process. It requires the interaction of a unique protein on the surface of the virus with a highly specific receptor site on the surface of the cell.

2. Penetration -This occurs by one or more processes. Enveloped viruses fuse their envelope with the membrane of the host cell. This involves local digestion of the viral and cellular membranes, fusion of the membranes and concomitant release of the nucleocapsid into the cytoplasm. Naked viruses bind to receptor sites on the cellular membrane, digest the membrane and enter into the cytoplasm intact. Both naked and enveloped viruses can be ingested by phagocytic cells. However, in this process they enter the cytoplasm enclosed in a cytoplasmic membrane derived from the phagocytic cell. 3. Uncoating -During this stage cellular proteolytic enzymes digest the capsid away from the nucleic acid. This always occurs in the cytoplasm of the host cell. The period of the replication cycle between the end of the uncoating stage and maturation of new viral particles is termed the eclipse. Thus during the eclipse stage, no complete viral particles can be viewed within the

cell.

4. Replication of nucleic acid. Replication of viral nucleic acid is a complex and variable process. The specific process depends on the nucleic acid type.

NOTE: Symmetrical transcription of DNA gives rise to double-stranded RNA.

DNA virus replication -with the exception of the poxviruses, all DNA viruses replicate in the nucleus. In some cases one of the DNA strands is transcribed (in others both strands of a small part of the DNA may be transcribed) (step 4) into specific mRNA, which in turn is translated (step 5) to synthesize virusspecific proteins such as tumor antigen and enzymes necessary for biosynthesis of virus DNA. This period encompasses the early virus functions. Host cell DNA synthesis is temporarily elevated and is then suppressed as the cell shifts over to the manufacture of viral DNA (step 6). As the viral DNA continues to be transcribed, late virus functions become apparent. Messenger RNA transcribed during the later phase of infection (step 6) migrates to the cytoplasm and is translated (step 7). Proteins for virus capsids are synthesized and are transported to the nucleus to be incorporated into the complete virion (step 8). Assembly of the protein subunits around the viral DNA results in the formation of complete virions (step 9), which are released after cell lysis.

The single-stranded DNA viruses first form a double stranded DNA, utilizing a host DNA-dependent DNA polymerase. They then undergo a typical replication cycle.

RNA virus replication -with the exception of the orthomyxoviruses and retroviruses, all RNA viruses replicate in the cytoplasm of the host cell. The exact process varies with the species of virus. The singlestranded RNA that is released after uncoating will act as either: (a) the mRNA to synthesize viral-coded proteins; or (b) a template to synthesize mRNA; or (c) a template to synthesize double stranded RNA, which is then used as a template to synthesize mRNA; or (d) a template to synthesize double-stranded DNA, which is then utilized as a template to synthesize mRNA. This latter process occurs only with the retroviruses (oncornaviruses).

The replication of poliovirus, which contains a single-stranded RNA as its genome, provides a useful example. All of the steps are independent of host DNA and occur in the cell cytoplasm. Polioviruses absorb to cells at specific cell receptor sites (step 1), losing in the process one virus polypeptide. The sites are specific for virus coat-cell interactions. After attachment, the virus particles are taken into the cell by viropexis (similar to pinocytosis) (step 2), and the viral RNA is uncoated (step 3). The singlestranded RNA then serves as its own messenger RNA. This messenger RNA is translated (step 4), resulting in the formation of an RNA-dependent RNA polymerase that catalyzes the production of a replication intermediate (RI), a partially double-stranded molecule consisting of a complete RNA strand and numerous partially completed strands (step 5). At the same time, inhibitors of cellular RNA and protein synthesis are produced. Synthesis of (+) and (-) strands of RNA occurs by similar mechanisms. The RI consists of one complete (-) strand and many small pieces of newly synthesized (+) strand RNA (step 6). The replicative form (RF) consists of two complete RNA strands, one (+) and one (-).

The single (+) strand RNA is made in large amounts and may perform any one of three functions: (a) serve as messenger RNA for synthesis of structural proteins; b) serve as template for continued RNA replication; or (c) become encapsulated, resulting in mature progeny virions. The synthesis of viral capsid proteins (step 7) is initiated at about the same time as RNA synthesis.

The entire poliovirus genome acts as its own mRNA, forming a polysome of approximately 350S, and is translated to form a single large polypeptide that is subsequently cleaved to produce the various viral capsid polypeptides. Thus, the poliovirus genome serves as a polycistronic messenger molecule. Poliovirus contains four polypeptides. 5. Maturation and Release Naked viruses -Maturation consists of two main processes: the assembly of the capsid, and its association with the nucleic acid. Maturation occurs at the site of nucleic acid replication. After they are assembled into mature viruses, naked virions may become concentrated in large numbers at the site of maturation, forming inclusion bodies. Naked virions are released in different ways, which depend on the virus and the cell type. Generally, RNA-containing naked viruses are released rapidly after maturation and there is little intracellular accumulation; therefore, these viruses do not form predominant inclusion bodies. On the other hand, DNA-containing naked icosahedral viruses that mature in the nucleus do not reach the cell surface as rapidly, and are released when the cells undergo autolysis or in some cases are extruded without lysis. In either case they tend to accumulate within the infected cells over a long period of time. Thus, they generally produce highly visible inclusion bodies. Enveloped viruses -In the maturation of enveloped viruses, a capsid must first be assembled around the nucleic acid to form the nucleocapsid, which is then surrounded by the envelope. During the assembly of the nucleocapsid, virus-coded envelope proteins are also synthesized. These migrate to the plasma membrane (if assembly occurs in the cytoplasm) or to the nuclear membrane (if assembly occurs in the

nucleus) and become incorporated into that membrane. Envelopes are formed around the nucleocapsids by budding of cellular membranes. NOTE: Enveloped viruses will have an antigenic mosaicism characteristic of the virus and the host cell. Viruses are slowly and continuously released by the budding process with the results that: (a) the cell is not lysed; and (b) little intracellular accumulation of virus occurs; and (c) inclusion bodies are not as evident as with naked viruses. Complex viruses -These viruses, of which the poxvirus is a good example, begin the maturation process by forming multilayered membranes around the DNA. These layers differentiate into two membranes: The inner one contains the characteristic nucleoid, while the external one acquires the characteristic pattern of the surface of the virion.

These form very characteristic cytoplasmic inclusion bodies. The viruses are generally released from the cell via cell lysis.

Summary 1. Viruses contain either DNA or RNA as their genetic material, but not both. This nucleic acid usually has unique chemical and/or physical features which makes it distinguishable from human nucleic acid.

2. Viral nucleic acid is enclosed in a capsid made up of protein subunits called protomeres.

3. Some species of viruses have a membrane, the envelope, surrounding the capsid; other species do not have an envelope, i.e., they are naked. Enveloped viruses have glyco-protein spikes arising from their envelope. These spikes have enzymatic,

absorptive, hemagglutinating and/or antigenic activity.

4. The morphology of a virus is determined by the arrangement of the protomeres. When protomeres aggregate into units of five or six (capsomeres) and then condense to form a geometric figure having 20 equal triangular faces and 12 apices, the virus is said to have icosahedral (cubic) morphology. When protomeres aggregate to form a capped tube, they are said to have helical morphology. Any other arrangement of the protomeres results in a complex morphology.

5. All viruses undergo a replication cycle in their human host cell consisting of adsorption, penetration, uncoating, nucleic acid replication, maturation and release stages.

6. During the viral replication cycle, an accumulation of mature viruses, incomplete viruses and viral parts occurs within the cell. This accumulation is the inclusion body. The size, shape, location and chemical properties of the inclusion body are used by the pathologist to diagnose viral infectious disease.

7. A virally-infected cell generally presents three signals that it is infected. The first is the production of double-stranded RNA, which induces interferon; the second is the expression of viral protein on the surface of the plasma membrane, thus causing activation of cytotoxic T-cells, natural killer cells and sometimes induction of antibody synthesis. The third is the formation of an inclusion body either within the cytoplasm or the nucleus or very rarely within both the cytoplasm and nucleus.

8. In general, all DNA-containing viruses replicate in the host cell nucleus. The exceptions to the rule are the poxviruses.

9. In general, all RNA-containing viruses replicate in the host cell cytoplasm. The exceptions to the rule are the retroviruses and the orthomyxoviruses. -----------------------------------------------------------------------------------------------------------------------------------------Previous Lecture | Syllabus | Next Lecture

INTERFERENCE WITH VIRAL REPLICATION

MM 526-541 and 145-152

Table of Contents Educational Objectives Discussion of Interference Summary

EducationalObjectives General 1. To provide an awareness of the mode of action and potential clinical use of interferon.

2. To provide insight into the uses and misuses of antiviral vaccines.

3. To develop the concept of viral-mediated interference with viral replication.

4. To relate the lysogenic state to diseases thought to be caused by "dormant," "latent" or "slow" viruses.

5. To define the rational use of antiviral agents.

6. To gain familiarity with the structure and function of antiviral drugs. Specific educational objectives (terms and concepts upon which you will be tested) Antiviral agents Lysogeny Attenuated virus vaccine Phenotypic mixing Inactivated virus vaccine Refractory period Interferon Target organ theory Interferon specificity Translation inhibitory protein Latency Viral interference

Discussion of Interference The physician's job is to interfere with viral replication in order to prevent or ameliorate the disease process. This can be done by manipulating the biological system of the patient or by utilizing antiviral antibiotics. In general, the more complex a system is (such as the viral replication cycle), the more easily that system is disrupted. However, in viral replication, the virus is utilizing mostly host cell proteinsynthesizing systems. Disruption of the viral replication cycle can also affect cellular metabolism in not only the infected cell but also in the noninfected cell. However, there are ways to interfere with viral replication with minimal effect on the host cell. Interferon Interferon is a class of glycoproteins which interferes with virus replication. There are numerous types of interferon and each is produced by an animal or by cultured animal cells. Interferon synthesis is induced by viruses or by certain biochemicals and can be of three types:

IFN- a = a - Interferon (20 subtypes) - from many different cell types IFN - b = b - Interferon (2 subtypes) - from fibroblasts, macrophages IFN -g = g - Interferon (3 subtypes) - from T-lymphocytes Characteristics a. Interferons are cell specific in both their production and their effects.

b. Interferons are virus non-specific.

c. Interferons are induced by viruses, chemicals, some species of bacteria and some extracts of fungi.

d. Although all animal cells appear able to produce one or more types of interferon, cells of the bone marrow, spleen, and macrophages, appear to play a special role, i.e., they produce a larger volume of interferon and a more potent interferon. Induction of interferon The nature of the stimulus to interferon production has been clarified by the discovery that doublestranded RNAs, such as reovirus RNA and certain synthetic polynucleotides, can induce a large production of interferon in many animals and in tissue cultures.

For most RNA viruses, double-stranded RNA segments produced during replication mediate the induction of interferon. Production of interferon In virus-infected cells, the synthesis of interferon begins after viral maturation is initiated and then continues for many hours.

Interferons are synthesized on membrane-bound polysomes. As they are formed, they are segregated into vesicles and are glycosylated; from the vesicles they are excreted outside the cells. Therefore until they are excreted, interferons do not act on the cell that produces them.

Mechanism of interferon action

Suggested mechanisms for the antiviral action of interferon.

Interferons cause antiviral resistance not directly, but by activating cellular genes for antiviral proteins; they are ineffective in enucleated cytoplasts or in the presence of actinomycin D.

Interferons induce the cell response by interacting with the cell surface. At the cell surface, interferons bind to receptors containing gangliosides (glycosylated phospholipids); transformed cells, which are deficient in gangliosides, are less interferon-sensitive than normal cells. In human cells, genes specifying the receptors are present on chromatosome 21; 21-trisomic (Down's syndrome) cells are especially sensitive to interferon. The molecular mechanisms of interferon-induced anti-viral resistance are multiple, and probably differ in different cell-virus systems. However, in vitro studies with extracts of interferon-treated cells show that the main target of interferon action is translation, which is blocked by two mechanisms, involving a protein kinase and a nuclease. In both, the block requires the presence of minute amounts of double-stranded RNA (dsRNA), which seems to signal to the cells the presence of a viral infection. (As in the induction of interferon, the dsRNA may be that of a viral replicative intermediate, or it may result from symmetric transcription of the viral DNA). Both translation blocks are therefore specific for virus-infected cells (containing dsRNA), although they do not distinguish cellular and viral messengers within such cells.

Interferons exhibit a wide variety of cell regulatory activities. They can be considered a family of hormones involved in regulation of cell growth and differentiation. The cell regulatory activity of IFN - g is much greater than that of IFN - a or IFN - b . Effects of interferon on the functions of the uninfected cells: a. Inhibition of cell replication;

b. Inhibition of activation of spleen lymphocytes;

c. Inhibition of liver regeneration;

d. Inhibition of production of platelets and leukocytes;

e. Enhancement of the expression of histocompatibility antigens on the lymphocyte surface;

f. Induction of liver degeneration;

g. Reduction of antiviral antibody production by inhibition of B-cell activity;

h. Enhancement of T-cell activity; and

i. Increase in the cytotoxic activity of natural killer cells against virus-infected cells. These effects denote a shift from humoral to cell-mediated immunity, which has a defensive role in many viral infections, but a pathogenic role in some. The combination of cell growth inhibition and enhancement of cell-mediated immunity accounts for the antitumor effect of interferon in experimental animals. Vaccination Viral vaccination refers to the administration of virus (inactivated or live), viral protein or antibody to a virus to a patient. Neither vaccination nor recovery from natural infection always results in total protection against a later infection with the same virus - REMEMBER THAT THE ANTIGENIC DETERMINANTS OF AN ENVELOPED VIRUS CAN VARY WITH THE TYPE OF CELL IN WHICH THE VIRUS GREW. Control is achieved by limiting the multiplication of virulent virus upon subsequent exposure and preventing its spread to target organs where the pathologic damage is done.

Principal vaccines used in prevention of viral diseases of humans

Immunization Recommended for General Public Disease Source of Vaccine Poliomyelitis Condition of Virus Route of Administration

Tissue culture (human diploid cell line, monkey kidney) Live attenuated

Killed

Oral

Subcutaneous

Measles

Tissue culture (chick embryo)

Live attenuated Subcutaneous

Mumps Tissue culture (chick embryo)

Live attenuated Subcutaneous

Rubella Tissue culture (duck embryo, rabbit, or human diploid ) Live attenuated Subcutaneous Hepatitis type B Purified HBsAg from "healthy" carriers; HBsAg from recombinant DNA in yeast Subunit Subcutaneous Chickenpox/Zoster Tissue culture Live attenuated Subcutaneous

Immunization Recommended Only Under Certain Conditions (Epidemics, Exposure, Travel, Military)Diseases Source of Vaccine of Administration Smallpox Lymph from calf or sheep (glycerolated, lyophilized) Live vaccinia Intradermal: multiple pressure, multiple Condition of Virus Route

Chorioallantois, tissue cultures (lyophilized) puncture Yellow Fever

Tissue cultures and eggs (17D strain) Subcutaneous

Live attenuated Subcutaneous or intradermal

Hepatitis type A Tissue culture Killed Influenza irradiated)

Highly purified or subunit forms of chick embryo allantoic fluid (formalinized or UVKilled Subcutaneous or intradermal Killed Subcutaneous

Rabies Duck embryo or human diploid cells Adenovirus Human diploid cell cultures

Live attenuated Oral, by enteric-coated capsule Killed Subcutaneous

Japanese B encephalitis Mouse brain (formalinized), tissue culture Venezuelan equine encephalomyelitis Guinea pig heart cell culture Eastern equine encephalomyelitis Western equine encephalomyelitis Russian spring-summer encephalitis Chick embryo cell culture Chick embryo cell culture Mouse brain (formalinized

Live attenuated Subcutaneous Killed Killed Killed Subcutaneous Subcutaneous Subcutaneous

Inactivated virus vaccines - Inactivated vaccines generally stimulate the development of circulating antibody against the capsid or envelope proteins of the virus, conferring some degree of resistance. However, there are disadvantages to this type of vaccine: a. Extreme care is required in their manufacture to make certain that no residual live virulent virus is present in the vaccine;

b. The immunity conferred is often brief and directed against only external antigens (capsid, envelope, spike);

c. Parenteral administration of inactivated vaccine sometimes gives limited protection because local resistance is not induced adequately at the natural port of entry or primary site of multiplication of the wild virus infection; and

d. Some inactivated virus vaccines induce hypersensitivity to subsequent infection or booster shots. Live attenuated virus vaccines. An attenuated virus is one which has lost its virulence or pathogenicity. This is usually accomplished by passing the virus through an animal host. A vaccine made with an attenuated virus has the advantage of acting like the natural infection with regard to its effect on immunity. The viruses multiply in the host and tend to stimulate longer-lasting antibody, antibody directed against all antigens of the virus (including internal antigens), and resistance at the portal of entry. The disadvantages of live attenuated vaccines include: a. The risk of reversion to greater virulence during multiplication within the host;

b. Unrecognized adventitious agents latently infecting the culture substrate may enter the vaccine stocks; and

c. Limited shelf life.

Recombinant vaccines. These are live virus vaccines where the virus used has been altered via genetic engineering to produce an avirulent but immune organism. This is done in one of two ways: a. The virus normally causing a disease is attenuated by deleting one or more of the genes required for virulence. This deletion may partially inactivate the virus but it is still able to reproduce; and

b. A virus that is not pathogenic, e.g., the vaccinia virus, is altered by the insertion of a gene from a pathogenic virus. The inserted gene codes for protein (e.g., the capsid protein) which elicits an immune reaction and protects against the pathogenic virus. Purified protein vaccines. Viral genes are cloned into plasmids. That cloned DNA is then transformed into a bacterial cell where it can be expressed if appropriate genetic engineering techniques are used. As the bacterial cell grows and reproduces, it synthesizes large quantities of the cloned protein. The protein is purified and then used as a vaccine.

DNA vaccines (gene vaccines). Viral DNA is inserted into human cells where it codes for proteins that are expressed on the surface of the human cell, thus stimulating the immune system.

C. Lysogenization, latency, dormancy. Normally when a virus enters a cell, it reproduces at a rapid rate and produces large numbers of progeny virus. In rare cases, the virus enters the cell and persists in the cell with little or no detectable effect on the host cell. This is gusually called a latent or dormant infection. These latent infections are sometimes activated by systemic shock. The basis for this latency has been attributed to: 1. Lysogenization;

2. Lack of pathogenicity of the virus; and

3. Suppression of reproduction by the immune system (humoral, cell mediated, interferon). Antiviral agents. Because of the great variability among the reproductive cycles of viruses, it has not yet been possible to develop a broad spectrum anti-viral compound. However, there are a few narrow spectrum chemotherapeutic agents approved for the treatment of viral diseases.

1. Acycloguanosine (Acyclovir) (Zovirax) This drug is used to treat herpesvirus skin infections. It inhibits viral DNA-dependent DNA polymerase activity.

2. Valacyclovir (Valtrex)

This is the L-valyl ester of acyclovir. It has the same spectrum of activity as acyclovir.

3. Adenine arabinoside (Ara A) (Vidarabine) This drug is used to treat herpesvirus eye infections, encephalitis, neonatal herpes, and herpes skin infections. It inhibits DNA polymerase.

4. Cytosine arabinoside (Ara C) This drug is used to treat herpesvirus eye infections. It acts as an analog of deoxy-cytidine and thus inhibits DNA synthesis.

Licensed only as an anti-tumor drug.

5. Iododeoxyuridine (Idoxuridine) This is an analog of thymidine and works by inhibiting DNA replication and transcription. It is used topically against herpesvirus keratitis.

6. Trifluorothymidine (Trifluridine) This is a deoxyribonucleoside analog which inhibits viral DNA replication and transcription. It is used to treat herpes simplex keratitis.

7. Foscarnet Sodium (Phosphonoformic acid)

This drug is used to treat retinitis of cyto-megalovirus etiology.

8. Ganciclovir (Cytovene) This drug is used to treat all types of cytomegalovirus infections in AIDS patients. Because of bone marrow toxicity, its use is limited to AIDS patients. It inhibits viral DNA polymerase.

9. Fomivirsen (Vitravene) This drug was approved by the U.S. Food and Drug Administration in 1998 for the treatment of cytomegalovirus retinitis. It is unique in that it is the first "antisense" antiviral approved by the FDA. An "antisense" vaccine is one which is a single-stranded DNA complement to a mRNA. The DNA and mRNA bind to each other and prevent the translation process. It is administered directly into the eye. 10. Amantadine This drug is used to treat influenza A. It interferes with penetration and/or uncoating of the virus.

11. Rimantadine This is a structural analog of amantadine. It inhibits penetration, uncoating and release of the influenza A virus.

12. Isatin- b-Thiosemicarbazone (Methisazone) This drug is used to treat smallpox (a poxvirus disease) where it acts by inhibiting translation of late mRNA.

13. N-methyl-isatin-b -Thiosemicarbazone (Marburan) This is a methylated derivative of methisazone. It inhibits the translation of late messenger RNA of the smallpox virus. 14. Alpha interferon This compound inhibits viral protein synthesis (translation) via phosphorylation of elongation factor 2 and/or induction of a ribonuclease which degrades mRNA. It is used to treat condylomata acuminata (genital warts). 15. Ribavirin (Virazole) This drug is used to treat Lassa fever, respiratory syncytial disease and Hanta virus respiratory/renal disease (Muerto Canyon fever). It interferes with the synthesis of viral mRNA by inhibiting DNAdependent RNA polymerase, inosine monophosphate dehydrogenase and various viral GTP-dependent enzymes. It has the broadest spectrum of activity of any of the anti-viral compounds.

16. Azidothymidine (AZT, Zidovudine, Retrovir) This drug is used to treat AIDS. It is a thymidine analog. It interferes with viral RNA dependent DNA polymerase (reverse transcriptase).

A chain terminator activated via phosphorylation by thymidine kinase.

17. Dideoxyinosine (ddI) (Videx) This drug is used to treat AIDS. It is a nucleoside analog that is converted to dideoxyadenosine triphosphate to inhibit the reverse transcriptase of the human immunodeficiency virus.

Dideoxyinosine

18. Dideoxycytidine (ddC) This drug is used to treat AIDS. It is a nucleoside analog that is converted to dideoxycytidine triphosphate to inhibit the reverse transcriptase of the human immuno-deficiency virus.

19. Lamivudine - 3TC (Epivir) This drug is used to treat AIDS, but it can only be used in combination with AZT and a protease inhibitor in patients who have never been treated previously with AZT. It is a nucleoside analog that inhibits reverse transcriptase.

20. Stavudine, d4T This drug is used to treat AIDS, but it can only be used in combination with AZT and a protease inhibitor in patients who have never been treated previously with AZT. It is a nucleoside analog that inhibits reverse transcriptase.

21. Delavirdine This is a nonnucleoside inhibitor of reverse transcriptase.

22. Nevirapine

This is a nonnucleoside inhibitor of reverse transcriptase.

23. Saquinavir (Invirase) (Hoffman - LaRoche) This drug is used to treat AIDS, but it can only be used in combination with AZT and Lamivudine - 3TC in patients who have never been treated previously with AZT. It is a protease inhibitor. 24. Ritonavir (Norvir (Abbott) This drug is used to treat AIDS, but it can only be used in combination with AZT and Lamivudine - 3TC in patients who have never been treated previously with AZT. It is a protease inhibitor.

25. Indinavir (Crixivan) (Merck) This drug is used to treat AIDS, but it can only be used in combination with AZT and Lamivudine - 3TC in patients who have never been treated previously with AZT. It is a protease inhibitor.

26. Viracept (Agouron Pharmaceuticals) This drug is used to treat aids, but it can only be used on combination with AZT and Lamivudine - 3TC in patients who have never been previously treated with AZT. It is the only protease inhibitor approved for children as well as for adults.

Summary 1. When human cells become infected by a virus, they may produce interferon. Interferon is a class of glycoproteins which interfere with viral replication by inducing the synthesis of a translation inhibitory protein and a nuclease.

2. The induction of interferon is mediated by double-stranded RNA.

3. In uninfected cells interferons act like a family of hormones involved in regulation of cell growth and differentiation where they often effect a shift from humoral to cell-mediated immunity.

4. Effective vaccination for viral disease can be achieved using live viruses, inactivated viruses, viral antigens or antibody directed against viruses.

5. Pediatric patients are routinely immunized against the viral diseases of polio, measles (rubeola), mumps and rubella. Several other viral vaccines are available for special situations.

6. In general the live attenuated viral vaccines induce the broadest range of immunity (i.e., stimulate IgA, IgG and IgM production) and give the longest acting immunity.

7. Some viruses have the ability to be dormant or latent in a cell during which time they do not reproduce themselves. Most commonly they do this by integrating their DNA into the host cell DNA.

8. The antiviral agents currently available are narrow spectrum compounds, most of which are analogs of the purine or pyrimidine bases of DNA or RNA.

9. The antiviral drugs and the diseases they are effective against are: A. Herpesvirus infections:

Acycloguanosine - herpesvirus skin infections Adenine arabinoside - herpesvirus eye infections, skin infections, CNS infections, and neonatal herpes Cytosine arabinoside - herpesvirus eye infections Iododeoxyuridine - herpesvirus eye infections Trifluorothymidine - herpesvirus keratitis Valacyclovir - herpesvirus skin infections B. Cytomegalovirus infection

Foscarnet sodium - cytomegalovirus retinitis Ganciclovir - cytomegalovirus retinitis Fomivirsen - cytomegalovirus retinitis C. Influenza A virus infection

Amantadine Rimantadine D. Smallpox virus infection

Isatin- b-Thiosemicarbazone (Methisazone) N-methyl-isatin- b-Thiosemicarbazone (Marburan) E. Papilloma virus infection (genital warts)

Alpha interferon F. Lassa fever virus, respiratory syncytial disease virus, MuertoCanyon fever virus

Ribavirin G. Human immunodeficiency virus (HIV)

REVERSE TRANSCRIPTASE INHIBITORS PROTEASE INHIBITORS Azidothymidine (AZT) Saquinavir Dideoxyinosine (ddI) Ritonavir

Dideoxycytosine (ddC) Indinavir Lamivudine - 3TC Stavudine, d4T Delavirdine Nevirapine ----------------- ------------------------------------------------------------------------------------------------------------------------Viracept