MI – 308 & Mycology

Unit I : General

1.1 General characteristics and structural organization of 1.2 Cultivation of viruses A. Animal cultivation B. Cultivation in embryonated eggs C. In vitro culture: Cell line, primary and secondary cell lines, continuous cell lines, cytopathic effects D. Cultivation of 1.3 Enumeration of viruses: methods of enumeration of viruses 1.4 Classification of viruses: PCNV, ICNV and Cryptogram system of viral classification 1.5 Sub viral entities: viroids, virusoids, prions, introduction to persistent, latent and slow viruses, oncogenic viruses

Early Development of Virology

 Virology has become a basic biological science around the middle of the century.  The subject matter of virology, the viruses cannot be defined by the common sense criteria applied to animals or plants.  Many definitions have been proposed: 1. Strictly intracellular and potentially pathogenic entities with an infectious phase and possessing only one type of , multiplying in the form of their genetic material, unable to grow and undergo binary fission, and devoid of a Lipmann system (ie. System of enzymes for energy production) – Lwoff (1957) 2. Elements of genetic material that can determine in the cells where they reproduce the biosynthesis of a specific apparatus for their own transfer into other cells – Luria (1959) 3. Virus are entities whose genomes are elements of nucleic acid that replicate inside living cells using the cellular synthetic machinery and causing the synthesis of specialized elements that can transfer the viral genome to other cells – Modified from Luria and Darnell (1967)  Viruses Latin word virus, poison or venom  Louis Pasteur used the term virus for any living infectious disease agent.  Diseases caused by viruses have been recognized for thousands of years. Diseases caused by viruses like , yellow fever, potato leaf roll and tulip break have been known for centuries.  Mayer (1886) demonstrated the transmissibility of mosaic disease of tobacco by mechanical inoculation with sap of infected plants.  Dimitri Iwanowsky (1852) reported the transmission of tobacco mosaic by sap filtered through proof filter.

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 Beijerinck (1899) succeeded in proving the serial transmission of tobacco mosaic by bacteria free filter in which no microscopic organism could be detected. He described this causative agent as “contagiumvivumfluidum”  Walter Reed (1900) showed yellow fever diseases virus transmitted by mosquito  Beginning of 20th century viruses are different from bacteria, plant and humans.  VilhelmEllermann and Oluf Bang reported leukemia could be transmitted between chickens by cell free filtrate and was probably caused by virus.  Peyton Rous (1911) reported virus now known as Rous Sarcoma virus was responsible for malignant muscle tumor in chicken.  (1915) reported that bacteria also could be attacked by viruses.  Felix d’ Herelle (1917) established decisively the existence of bacterial viruses.  Schelsinger (1933) was the first to determine the composition of a virus. He showed that bacteriophage consists of only protein and DNA.  Wendell Stanley (1935) crystallized the tobacco mosaic virus (TMV) and found to be largely or completely protein.  Later on Frederick Bawden and managed to separate the TMV virus particles into protein and nucleic acid. Thus by the late 1930s it was becoming clear that viruses are complexes of nucleic acids and proteins able to reproduce only in living cells. General Characteristics of Viruses  Viruses are a unique group of infectious agents whose distinctiveness resides in their simple, acellular organization and patternof reproduction.  A complete virus particle or virionconsists of one or more molecules of DNA or RNA enclosed in a coat of protein, and sometimes also in other layers.  These additional layers may be very complex and contain carbohydrates, lipids, and additional proteins.

 Viruses can exist in two phases: extracellular and intracellular.  Virions, in the extracellular phase, possess few if any enzymes and cannot reproduce independent of living cells.  In the intracellular phase, viruses exist primarily as replicating nucleic acids that induce metabolism to synthesize virion components; eventually complete virus particles or virions are released.  In summary, viruses differ from living cells in at least threeways:

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1. Their simple, acellular organization; 2. The presence of eitherDNA or RNA, but not both, in almost all virions (human cytomegalovirus has a DNA genome and four mRNAs) 3. Theirinability to reproduce independent of cells and carry out cell divisionas procaryotes and eucaryotes do.  Although bacteria such as Chlamydia and rickettsia are obligatory intracellularparasites like viruses, they do not meet the first two criteria. The Structure of Viruses  Virus morphology has been intensely studied over the pastdecades because of the importance of viruses and the realizationthat virus structure was simple enough to be understood. Progresshas come from the use of several different techniques: electron microscopy,X-ray diffraction, biochemical analysis, and immunology.  Although our knowledge is incomplete due to the large numberof different viruses, the general of virus structure isbecoming clear. Virion Size  Virions range in size from about 10 to 300 or 400 nm in diameter.  The smallest viruses are a little larger than ribosomes,whereas the poxviruses, like , are about the samesize as the smallest bacteria and can be seen in the light microscope.  Most viruses, however, are too small to be visible in thelight microscope and must be viewed with the scanning and transmissionelectron microscopes. General Structural Properties  All virions, even if they possess other constituents, are constructedaround a nucleocapsidcore (indeed, some viruses consistonly of a nucleocapsid).  The nucleocapsid is composed of anucleic acid, either DNA or RNA, held within a protein coatcalled the , which protects viral genetic material and aidsin its transfer between host cells.  There are four general morphological types of andvirion structure. . Some capsids are icosahedral in shape. An icosahedron is aregular polyhedron with 20 equilateral triangular faces and12 vertices. These capsids appear sphericalwhen viewed at low power in the electron microscope. . Other capsids are helical and shaped like hollow proteincylinders, which may be either rigid or flexible. . Complex viruses have capsid symmetry that is neither purelyicosahedral nor helical. They maypossess tails and other structures (e.g., many )or have complex, multilayered walls surrounding the nucleicacid (e.g., poxviruses such as vaccinia).

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 Both helical and icosahedral capsids are large macromolecularstructures constructed from many copies of one or a fewtypes of protein subunits orprotomers.  Probably the most importantadvantage of this design strategy is that the informationstored in viral genetic material is used with maximum efficiency. Many viruses have an envelope, an outer membranous layer surrounding the nucleocapsid. Enveloped viruses have a roughly spherical but somewhat variable shape even though their nucleocapsid can be either icosahedral or helical. 1. Helical Capsids  Helical capsids are shaped much like hollow tubes with proteinwalls.  The tobacco mosaic virus provides a well-studied exampleof helical capsid structure.  A single type of protomerassociates together in a helical or spiral arrangement to produce along, rigid tube, 15 to 18 nm in diameter by 300 nm long.  The RNAgenetic material is wound in a spiral and positioned toward the insideof the capsid where it lies within a groove formed by the proteinsubunits.  Not all helical capsids are as rigid as the TMV capsid.  virus are enclosed in thin, flexible helicalcapsids folded within an envelope.  The size of a helical capsid is influenced by both its protomersand the nucleic acid enclosed within the capsid.  The diameterof the capsid is a function of the size, shape, and interactionsof the protomers.  The nucleic acid determines helical capsidlength because the capsid does not seem to extend much beyondthe end of the DNA or RNA. 2. Icosahedral Capsids  The icosahedron is one of nature’s favorite shapes (the helix isprobably most popular).  Viruses employ the icosahedral shapebecause it is the most efficient way to enclose a space.  A fewgenes, sometimes only one, can code for proteins that self-assembleto form the capsid.  In this way a small number of lineargenes can specify a large three-dimensional structure.  Certain requirementsmust be met to construct an icosahedron. Hexagonspack together in planes and cannot enclose a space, and thereforepentagons must also be used.  When icosahedral viruses are negatively stained and viewedin the transmission electron microscope, a complex icosahedralcapsid structure is revealed.

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 The capsids are constructedfrom ring- or knob-shaped units called capsomers, eachusually made of five or six protomers. Pentamers (pentons) havebfive subunits; hexamers (hexons) possess six.  Pentamers are atthe vertices of the icosahedron, whereas hexamers form its edgesand triangular faces. 3. Viruses with Capsids of Complex Symmetry  Although most viruses have either icosahedral or helical capsids,many viruses do not fit into either category.  The poxviruses and large bacteriophages are two important examples.  The poxviruses are the largest of the animal viruses and can even be seen with a phasecontrast microscope or in stained preparations.  They possess an exceptionally complex internal structure with an ovoid- to brickshaped exterior. The double-stranded DNA is associated with proteins and contained in the nucleoid, a central structure shaped likea biconcave disk and surrounded by a membrane.  Two elliptical or lateral bodies lie between the nucleoid and itsouter envelope, a membrane and a thick layer covered by an arrayof tubules or fibers.  Some large bacteriophages are even more elaborate than thepoxviruses.  The T2, T4, and T6 phages that infect E. coli have beenintensely studied.  Their head resembles an icosahedron elongated byone or two rows of hexamers in the middle and containsthe DNA genome.  The tail is composed of a collar joining it tothe head, a central hollow tube, a sheath surrounding the tube, and acomplex baseplate.  The sheath is made of 144 copies of the gp18 proteinarranged in 24 rings, each containing six copies.  In T-evenphages, the baseplate is hexagonal and has a pin and a jointed tailfiber at each corner. The tail fibers are responsible for virus attachmentto the proper site on the bacterial surface.  There is considerable variation in structure among the largebacteriophages, even those infecting a single host.  In contrastwith the T-even phages, many coliphages have true icosahedralheads. T1, T5, and lambda phages have sheathless tails that lacka baseplate and terminate in rudimentary tail fibers.  ColiphagesT3 and T7 have short, noncontractile tails without tail fibers.  Clearly these viruses can complete their reproductive cycles usinga variety of tail structures.  Complex bacterial viruses with both heads and tails are saidto have binal symmetry because they possess a combination oficosahedral (the head) and helical (the tail) symmetry.

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Tobacco mosaic virus Helical Capsid (Influenza Virus)

Nucleic Acids  Viruses are exceptionally flexible with respect to the nature of theirgenetic material. They employ all four possible nucleic acid types: 1. single-stranded DNA 2. double-stranded DNA 3. single-stranded RNA 4. double-stranded RNA  All four types are found in animal viruses.  Plant viruses most often have single-stranded RNA genomes.  Althoughphages may have single-stranded DNA or single-strandedRNA, bacterial viruses usually contain double-stranded DNA.

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 Most RNA viruses employ single-stranded RNA (ssRNA) as their genetic material.  The RNA base sequence may be identical with that of viral mRNA, in which case the RNA strand is called theplus strand or positive strand.  However, the viral RNA genome may instead becomplementary to viral mRNA, and then it is called a minus ornegative strand.  Polio, tobacco mosaic, brome mosaic, and Roussarcoma viruses are all positive strand RNA viruses; rabies,, measles, and influenza viruses are examples of negativestrand RNA viruses.  Many of these RNA genomes are segmentedgenomes—that is, they are divided into separate parts.  It is believedthat each fragment or segment codes for one protein. Usuallyall segments are probably enclosed in the same capsid eventhough some virus genomes may be composed of as many as 10to 12 segments.  Plus strand viral RNA often resembles mRNA in more thanthe equivalence of its nucleotide sequence.  In fact, plusstrand RNAs can direct protein synthesis immediately after enteringthe cell.  A few viruses have double-stranded RNA (dsRNA) genomes.  All appear to be segmented; some, such as the reoviruses, have 10to 12 segments. These dsRNA viruses are known to infect animals,plants, fungi, and even one bacterial species.

Nucleic Acid Type Nucleic Acid Structure Virus Examples DNA Single-Stranded Linear single strand Parvoviruses Circular single strand øX174, M13, fd phages Double-Stranded Linear double strand Herpesviruses (herpes simplex cytomegalovirus, Epstein-Barr virus), adenoviruses, T coli phages Linear double strand with T5 coliphage single chain breaks Double strand with cross- Vaccinia, smallpox linked ends Closed circular double Polyomaviruses (SV-40), papillomaviruses, PM2 strand phage,cauliflower mosaic RNA Single-Stranded Linear, single stranded, Picornaviruses (polio, rhinoviruses), togaviruses, positive strand RNA bacteriophages, TMV, and most plant viruses Linear, single stranded, Rhabdoviruses (rabies), paramyxoviruses

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negative strand (mumps, measles) Linear, single stranded, Brome mosaic virus (individual segments segmented, positive strand inseparate virions) Linear, single stranded, (Rous sarcoma virus, segmented, diploid(two humanimmunodeficiency virus) identical single strands), positive strand Linear, single stranded, Paramyxoviruses, orthomyxoviruses (influenza) segmented, negative strand Double-Stranded Linear, double stranded, Reoviruses, wound-tumor virus of plants, segmented cytoplasmic polyhedrosis virus of insects, phage

Viral Envelopes and Enzymes  Many animal viruses, some plant viruses, and at least one bacterialvirus are bounded by an outer membranous layer called an envelope.  envelopes usually arise fromhost cell nuclear or plasma membranes; their lipids and carbohydratesare normal host constituents.  In contrast, envelope proteinsare coded for by virus genes and may even project from the envelopesurface as spikes or peplomers.  Thesespikes may be involved in virus attachment to the host cell surface.Since they differ among viruses, they also can be used toidentify some viruses.  Because the envelope is a flexible, membranousstructure, enveloped viruses frequently have a somewhatvariable shape and are called pleomorphic.  However, the envelopesof viruses like the bullet-shaped rabies virus are firmly attachedto the underlying nucleocapsid and endow the virion witha constant, characteristic shape.  In some viruses the envelope is disrupted by solvents like ether to such an extentthat lipid-mediated activities are blocked or envelope proteins aredenatured and rendered inactive.  The virus is then said to be“ether sensitive.”  Influenza virus is a well-studied exampleof an enveloped virus.  Spikes project about 10 nm from the surfaceat 7 to 8 nm intervals.  Some spikes possess the enzyme neuraminidase,which may aid the virus in penetrating mucous layersof the respiratory epithelium to reach host cells.  Other spikeshave proteins, so named because they can bind thevirions to membranes and cause .  participate in virion attachmentto host cells. Proteins, like the spike proteins that are exposed on the outer envelope surface, are generally glycoproteins— that is, the proteins have carbohydrate attached to them.

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 A nonglycosylated protein, the M or matrix protein, is found on the inner surface of the envelope and helps stabilize it.  Although it was originally thought that virions had only structural capsid proteins and lacked enzymes, this has proven not to be the case.  In some instances, enzymes are associated with the envelope or capsid (e.g., influenza neuraminidase).  Most viral enzymes are probably located within the capsid. Many of these are involved in nucleic acid replication.  For example, the influenza virus uses RNA as its genetic material and carries an RNA dependent RNA polymerase that acts both as a replicase and as an RNA transcriptase that synthesizes mRNA under the direction of its RNA genome.  The polymerase is associated with ribonucleoprotein. Although viruses lack true metabolism and cannot reproduce independently of living cells, they may carry one or more enzymes essential to the completion of their life cycles. 1.2 Cultivation of Viruses  Viruses are unable to reproduce independent of living cells,viruses cannot be cultured in the same way as bacteria and eukaryotic microorganisms. Animals, plants, human, bacteria, fungi, protozoa and algae are the natural host of viruses.  Cultivation of animal viruses can be done as per following methods. 1. Animal cultivation 2. Cultivation in embryonated egg 3. In vitro cell culture 1. ANIMAL CULTIVATION  Some viruses cannot be cultivated in cell culture or in embryonated chicken eggs and must be propagated in living animals.  Mice, guinea pigs, monkeys, rabbits and primates are used for this purpose.  Virus to be grown is inoculated in the animal through nasal instillation or intracerebral inoculation or intraperitoneal inoculation or subcutaneous inoculation.  The inoculation method is depending on the type of virus and its target site of infection. Animal should be kept in hygienic condition in laboratory.  Animal inoculation is also good diagnostic tool because the animal can show typical disease symptoms and histological (tissue) sections of infected tissue can be examined microscopically. 2. Embryonated Chicken Eggs  One of the most economical and convenient methods for cultivating a wide variety of animal viruses is the chick embryo technique. The discovery that viruses could be cultivated by the simple technique was made in 1931.

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 For many years researchers have cultivated animalviruses by inoculating suitable host animals or embryonatedeggs—fertilized chicken eggs incubated about 6 to 8 days after laying.  To prepare the egg for virus cultivation, the shellsurface is first disinfected with iodine andpenetrated with a smallsterile drill.  After inoculation, the drill hole is sealed with gelatin or paraffin wax andthe egg incubated.  Viruses may be able to reproduce only in certainparts of the embryo; consequently they must be injected into theproper region.  For example, the myxoma virus grows well on thechorioallantoic membrane, whereas the mumps virus prefers the allantoiccavity.  The infection may produce a local tissue lesion knownas a pock, whose appearance often is characteristic of the virus.

3. In vitro culture  Cell cultures are today the method of choice for the propagation of viruses for many reasons. As this method is convenient, economy of maintenance compared to animals, observable cytopathic effects and choice of cells for their susceptibility to particular viruses.  Animal tissues cultures established from individual cells are called cell lines.  On the basis of their origin and characteristics cell cultures are of three types, primary culture, secondary and continuous cell lines. 1. Primary cell culture: Primary cell culture are derived from normal tissue of an animal (such as mouse, hamster, chicken, or monkey tissue) or a human (gingival

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tissue). When cells from these tissues are processed and cultured, the first monolayer is referred to as primary culture.  A monolayer is a confluent layer of cells covering the surface of a culture vessel.  Processing of Primary cell culture:  Primary cell cultures are prepared from fresh tissue, which is usually minced with sharp sterile razor and dissociated with the aid of proteolytic enzymes (such as trypsin) into a cell suspension.  The cells are washed with physiological buffer (to remove the proteolytic enzymes used) and then suspended in a special containing a balanced salt solution, a buffer, necessary nutrients (vitamins, coenzymes, amino acids, glucose) and serum.  Antibiotics may be added to inhibit bacterial growth.  The cell suspension in the growth medium is placed in a tissue culture vessel and incubated.  The cells settle on the surface of the vessel and grow into a monolayer. 2. Secondary cell culture:  The cell sub cultured from primary cell culture.  Cell cultures prepared from fresh tissue resemble more closely the cells in the whole animal than do the cells in continuous cell lines.  Unfortunately, cells derived in this manner can be sub cultured only a limited number of times before dying.  For some types of cells only a few divisions are possible. For 50 to 100 divisions occur.  Cell cultures derived from embryonic tissue are generally capable of a greater number of divisions in vitro than those derived from adult tissue.  Diploid cell strains are derived from primary cell cultures established from a particular type of tissue, such as lung or kidney, which is of embryonic origin.  They are of a single cell type and can undergo 50 to 100 divisions before dying. They possess the normal diploid karyotype. Such diploid cell strains are host of choice for many viral studies, especially in the production of human virus.  prepared from tissue cultures have an advantage over those prepared from embryonated chicken egg in minimizing the possibility of a patient developing hypersensitivity or allergy to egg albumen.  The Salk poliomyelitis vaccine, which is produced in tissue culture, was developed after basic research had shown that the poliovirus would grow satisfactorily on monkey kidney cell cultures. 3. Continuous cell line:

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 Continuous cell lines appear to be capable of an infinite number of doublings. Such cell lines may arise with the mutation of a cell strain, or more commonly from the establishment of cell cultures from malignant tissue.  The karyotype of these cells is aneuploidy (a variable multiple of the haploid chromosome number) and not diploid.  These cells are also different morphologically from the cells of origin.  They are usually less fastidious in their nutritional requirements. They don’t attach as strongly as other cell cultures to the surface of the culture vessel, so under certain circumstances they can grow in suspension.  They also have a tendency to grow on the top of each other in multilayer on culture- vessel surfaces.  Even though cells from continuous cell lines are very different from normal cells in both genotype and phenotype, they are very useful in studies where large numbers of cells are required.  Furthermore, they are easy to propagate serially, but because of their derivation from malignant tissue or their possession of malignant characteristics, such cells obviously are not used in virus production for human vaccines.  Nevertheless, continuous cell lines have been extremely useful in cultivating many viruses previously difficult or impossible to grow. Detection of virus growth:  Growth of viruses in tissue culture can be detected by the following effects 1. Cytopathic effect (CPE): The tissue structure deteriorates as the virus multiplies. Generally morphological changes produced by viruses. 2. Hemadsorption: If hemagglutinating viruses are multiplying in the cell culture, the erythrocytes will adsorb on to the surface of cells. Patches of red areas (blood clots) appear on the monolayer 3. Interference: The growth of first virus will inhibit second virus infection due to some inhibitory effect. This property of cell culture is called interference. It is useful to detect the growth of non-cytopathic viruses in cell cultures. It is useful to detect the growth of non-cytopathic viruses in cell cultures. 4. Transformation: If oncogenic viruses are inoculated into cell cultures, the infected cells grow fast and produce microtumors in the culture. This is called transformation. It indicates the presence of oncogenic viruses in the culture. 5. Immunofluorescence test: some cells from the cell culture are stained with fluorescent dye conjugated and viewed under UV microscope. Viral present on the cell surface binds with the antiserum. from the cell is the positive indication for presence of virus in the cells.  More recently animal viruses have been grown in tissue (cell)culture on monolayers of animal cells.

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 This technique is made possibleby the development of growth media for animal cells and bythe advent of antibiotics that can prevent bacterial and fungal contamination.  A layer of animal cells in a specially prepared petridish is covered with a virus inoculum, and the viruses are allowedtime to settle and attach to the cells.  The cells are then coveredwith a thin layer of agar to limit virion spread so that only adjacentcells are infected by newly produced virions.  As a result localizedareas of cellular destruction and lysis called plaques often areformed and may be detected if stained with dyes,such as neutral red or trypan blue that can distinguish living fromdead cells.  Viral growth does not always result in the lysis of cellsto form a plaque. Animal viruses, in particular, can cause microscopicor macroscopic degenerative changes or abnormalities inhost cells and in tissues called cytopathic effects.  Cytopathic effects may be lethal, but plaque formation from celllysis does not always occur. 4. Cultivation of bacteriophages  Bacterial viruses or bacteriophages arecultivated in either broth or agar cultures of young, actively growingbacterial cells.  So many host cells are destroyed that turbidbacterial cultures may clear rapidly because of cell lysis.  Agarcultures are prepared by mixing the bacteriophage sample withcool, liquid agar and a suitable bacterial culture.  The mixture isquickly poured into a containing a bottom layer of sterileagar.  After hardening, bacteria in the layer of top agar growand reproduce, forming a continuous, opaque layer or “lawn.”  Wherever a virion comes to rest in the top agar, the virus infectsan adjacent cell and reproduces. Eventually, bacterial lysis generatesa plaque or clearing in the lawn.  As can be seenin figure, plaque appearance often is characteristic of thephage being cultivated. 5. Plant Viruses:  Plant viruses are cultivated in a variety of ways. Plant tissuecultures, cultures of separated cells, or cultures of protoplasts may be used.  Viruses also can be grown in wholeplants.  Leaves are mechanically inoculated when rubbed with amixture of viruses and an abrasive  Whenthe cell walls are broken by the abrasive, the viruses directly contactthe plasma membrane and infect the exposed host cells. (Therole of the abrasive is frequently filled by insects that suck orcrush plant leaves and thus transmit viruses.)

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 A localizednecrotic lesion often develops due to the rapid death of cells inthe infected area.  Even when lesions do not occur,the infected plant may show symptoms such as changes in pigmentationor leaf shape.  Some plant viruses can be transmittedonly if a diseased part is grafted onto a healthy plant. 1.3 ENUMERATION OF VIRUSES: METHODS OF ENUMERATION OF VIRUSES  Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration.  The quantity of viruses in a sample can be determined either bycounting particle numbers or by measurement of the infectiousunit concentration.  Although most normal virions are probablypotentially infective, many will not infect host cells because theydo not contact the proper surface site.  Thus the total particle countmay be from 2 to 1 million times the infectious unit number dependingon the nature of the virion and the experimental conditions. 1. Electron microscope enumeration  Despite this, both approaches are of value.Virus particles can be counted directly with the electron microscope.  In one procedure the virus sample is mixed with a knownconcentration of small latex beads and sprayed on a coated specimengrid.  The beads and virions are counted; the virus concentrationis calculated from these counts and from the bead concentration.  This technique often works well with concentratedpreparations of viruses of known morphology.  Viruses can be concentratedby centrifugation before counting if the preparation is toodilute.  However, if the beads and viruses are not evenly distributed(as sometimes happens), the final count will be inaccurate. 2. Hemagglutination assay  The most popular indirect method of counting virus particlesis the hemagglutination assay.  Many viruses can bind to the surfaceof red blood cells.  If the ratio of viruses tocells is large enough, virus particles will join the red blood cellstogether, forming a network that settles out of suspension or agglutinates.  In practice, red blood cells are mixed with a series ofvirus preparation dilutions and each mixture is examined.  Thehemagglutination titer is the highest dilution of virus (or the reciprocalof the dilution) that still causes hemagglutination.

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 Thisassay is an accurate, rapid method for determining the relativequantity of viruses such as the influenza virus.  If the actual numberof viruses needed to cause hemagglutination is determined by another technique, the assay can be used to ascertain the numberof virus particles present in a sample. 3. Plaque assay  A variety of assays analyze virus numbers in terms of infectivity,and many of these are based on the same techniques usedfor virus cultivation.  For example, in the plaque assay several dilutionsof bacterial or animal viruses are plated out with appropriatehost cells.  When the number of viruses plated out are muchfewer than the number of host cells available for infection andwhen the viruses are distributed evenly, each plaque in a layer ofbacterial or animal cells is assumed to have arisen from the reproductionof a single virus particle.  Therefore a count of theplaques produced at a particular dilution will give the number ofinfectious virions or plaque-forming units (PFU), and the concentrationof infectious units in the original sample can be easilycalculated.  Suppose that 0.10 ml of a 10–6 dilution of the viruspreparation yields 75 plaques.  The original concentration ofplaque-forming units is, PFU/ml = No. of plaques / Dilution factor x Volume = 75 / 10–6 x 0.1 = 7.5 x 108

Plaque assay

 Viruses producing different plaque morphology types on thesame plate may be counted separately.  The same approach employed in the plaque assay may beused with embryos and plants.

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 Chicken embryos can be inoculatedwith a diluted preparation or plant leaves rubbed with a mixtureof diluted virus and abrasive.  The number of pocks on embryonicmembranes or necrotic lesions on leaves is used to obtainthe concentration of infectious units.  When biological effects are not readily quantified in theseways, the amount of virus required to cause disease or death canbe determined by the endpoint method.  Organisms or cell culturesare inoculated with serial dilutions of a virus suspension.  The results are used to find the endpoint dilution at which 50% ofthe host cells or organisms are destroyed.  The lethaldose (LD50) is the dilution that contains a dose large enough todestroy 50% of the host cells or organisms.  In a similar sense, theinfectious dose (ID50) is the dose which, when given to a numberof test systems or hosts, causes an infection of 50% of the systemsor hosts under the conditions employed. 4. Focus forming assay (FFA)  The focus forming assay (FFA) is a variation of the plaque assay, but instead ofrelying on cell lysis in order to detect plaque formation, the FFA employsimmunostaining techniques using fluorescently labelledantibodies specific for a viralantigen to detect infected host cells and infectious virus particles before an actualplaque is formed.  The FFA is particularly useful for quantifying classes of virusesthat do not lyse the cell membranes, as these viruses would not be amenable to theplaque assay.  Like the plaque assay, host cell monolayers are infected with variousdilutions of the virus sample and allowed to incubate for a relatively brief incubationperiod (e.g., 24– 72 hours) under a semisolid overlay medium that restricts the spreadof infectious virus, creating localized clusters (foci) of infected cells.  Plates aresubsequently probed with fluorescently labeled against a viral antigen,and fluorescence microscopy is used to count and quantify the number of foci. TheFFA method typically yields results in less time than plaque or TCID50 assays, but itcan be more expensive in terms of required reagents and equipment. Assaycompletion time is also dependent on the size of area that the user is counting. Alarger area will require more time but can provide a more accurate representation ofthe sample. Results of the FFA are expressed as focus forming units per milliliter, orFFU/mL. 5. Endpoint Dilution assay  The maximum dilution of a virus that cannot produce an infection or disease is called end point dilution  It is used for determining virulence of a virus in animals.  Serial dilutions of virus stock are inoculated into test units.

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 Test units can be cell cultures, embryonated eggs or animals. The number of test units that have become infected is then enumerated for each virus dilution.

 50% Tissue culture Infective Dose (TCID50) is the measure of infectious virus titer.  This endpoint dilution assay quantifies the amount of virus required to kill 50% ofinfected hosts or to produce a cytopathic effect in 50% of inoculated tissue culturecells.  When used in the context of tissue culture, host cells are plated and serial dilutions of the virus areadded. After incubation, the percentage of cell death (i.e. infected cells) is manually observed and recorded for each virus dilution,and results are used

to mathematically calculate a TCID50 result.

 LD50 is the amount of virus required to kill 50% cells of the host.

 ID50 is the amount of virus required to cause infection in 50% of the host.  The dilution at which no infection was demonstrated is known as Dilution End point (DEP) 6. Single assay  Single radial immunodiffusion assay (SRID), also known as the Mancini method, is a protein assay that detects the amount ofspecific viral antigen by immunodiffusion in a semi-solid medium (e.g. agar).  The medium contains antiserum specific to the antigenof interest and the antigen is placed in the center of the disc. As the antigen diffuses into the medium it creates a precipitate ring thatgrows until equilibrium is reached.  Assay time can range from 10 hours to days depending on equilibration time of the antigen andantibody. The zone diameter from the ring is linearly related to the log of protein concentration and is compared to zone diameters forknown protein standards for quantification. 7. Flow Cytometry  While most flow cytometers do not have sufficient sensitivity, there are a few commercially available flow cytometers that can beused for virus quantification.  A virus counter quantifies the number of intact virus particles in a sample using fluorescence to detectcolocalized proteins and nucleic acids. Samples are stained with two dyes, one specific for proteins and one specific for nucleic acids,and analyzed as they flow through a laser beam.  The quantity of particles producing simultaneous events on each of the two distinctfluorescence channels is determined, along with the measured sample flow rate, to calculate a concentration of virus particles(vp/mL).  The results are generally similar in absolute quantity to a TEM result. The assay has a linear working range of 105–109vp/mL and an analysis time of ~10 min with a short sample preparation time.

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1.4 CLASSIFICATION OF VIRUSES: PCNV, ICNV CRYPTOGRAM SYSTEM OF VIRAL CLASSIFICATION  According to the nature of the host, viruses are subdivided into plant viruses, animal viruses, and bacterial viruses or bacteriophages.  Certain plant viruses can multiply even in their insect vectors.  Each virus has a range of related organisms as host into which it can reproduce.  Viruses are generally grouped on the basis of their major hosts. 1. Bacterial viruses  There are viruses or bacteriophage for almost all the groups of bacteria.  The host range of phages are well within the taxonomic boundaries of bacterial groups. Eg. Phage active on Micrococci will not multiply in Streptococci Phage of enteric bacteria do not usually multiply in Streptococci  Phage specificity for host may be broader or narrower than the classification boundaries that separate bacterial genera and species. Eg. A phage may multiply only on a certain strain of E. coli, Whereas another phage reproduce in many strains of E. coli and closely related genus Shigella.  Bacteria can acquire phage resistance by mutation and hence can produce stable mutant resistant to one or more phages.  Phages that attack blue green algae have been discovered suggesting biochemical and taxonomic relationship between the Cyanophyceae and the bacteria. 2. Animal viruses  Virus diseases are known in a variety of vertebrates,  Eg. Fish (carp pox, infectious tumors) o Amphibia (kidney tumor of the leopard frog) o Birds (Newcastle diseases and laryngotracheitis both economically very important) o Fowl (Neoplastic like sarcoma and leukose) o In many domestic animals as well as many wild ones o Humans (Epidemiological problems as small pox, yellow fever, poliomyelitis, measles, mumps, rabies and various types of encephalitis 3. Plant viruses  There are relatively few known viruses in gymnosperms (non flowering plants), fungi or algae  The angiosperm (flowering plants) are the host to many types of viruses.  In fact, viruses rank next to fungi in causing plant diseases of economic importance including diseases of potatoes, beans, tobacco, sugarcane, cocoa and fruit crops. Viruses in Eukaryotic microorganisms  There is also evidence that cells of eukaryotic microorganism may contain viruses.

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 Some of these viruses may be associated with prokaryotic endosymbionts living in eukaryotic cells.  Virus like particles have been observed in species of  Protozoa: Leishmania, Entamoeba histolytica, Plasmodium vivax, P. bergha, Paramecium aurelia etc.  Algae:Aulacomonassubmarina, Characorralina, Oedogoniumspp etc.  Fungi:Saccharomyces cerevisiae, Ustilagomaydis, Penicilliumspp etc.

NOMENCLATURE AND CLASSIFICATION OF VIRUSES  Virus nomenclature and classification are a troublesome area of virology.  At present there is no reason to believe that viruses form a single group of organisms having a common ancestry and a common evolutionary history.  Any classification of viruses is bound to be mainly a “determinative key” of practical value for use by workers in the field.  Viruses have been traditionally named by adding the word “virus” after the disease caused in the major host, eg. Polio virus the causative agent of poliomyelitis.  Bacteriphages were named after laboratory code system eg. ɸX174, P22, T7 etc.  This method of nomenclature was good for day to day needs of researchers.  But as more information about viruses became available the need for a more systematic nomenclature was felt.  As soon as one attempts to classify viruses, however, one faces the problem of the choice of criteria, whether to choose the nature of the major host, or the type of disease produced or the properties of the virions or the features of the reproductive cycle.  A good system should employ several or all of these criteria, with either equal or hierarchical prominence.  In 1948, Holmes proposed a latin binomial system analogous to plant nomenclature in which the viruses formed the order – virales, with three suborders Suborder: Phagineae – Bacteriophage Phytophagineae – Plant viruses Zoophagineae - Animal viruses  These suborders further divided into families, genera and species.  This system is not very popular and is even not used in plant .  In last 50 years progress in Electron microscopy has given sufficient data about the size and morphology of most viral particles. Purification techniques have made possible the identification of nucleic acid type they contain and to compare them while serological tests have characterized specific virion proteins whose relations indicate chemical and genetic relatedness.

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 Hence it became clear that morphological and serological properties of virion and the properties of their nucleic acid are convenient and meaningful criteria of classification.  Such a system was proposed by a group of virologists Andre Lwoff, Robert Horne and Paul Tournier (1962), which was adapted by the Provisional Committee on the Nomenclature of Viruses (PCNV)of the International Association of Microbiological Societies.  This system is known as the LHT system and is based on the following characteristics: 1. Nucleic acid: DNA or RNA 2. Symmetry: Helical, cubic, cubic-tailed 3. Prsence or absence of envelops 4. Diameter of helical capsid 5. Number of morphological units (capsomers) in cubic types  In recent years, several system of classification have been proposed (Lwoff et al., 1962, Bellet 1967, Gibbs 1969)  All of them have proved to be controversial and have failed to find general acceptance.  To resolve the problem, a separate agency the International Committee on Nomenclature of Viruses (ICNV) was set up at the International Congress for Microbiology held in Moscow in 1966.  Its job was to look into the various aspects of classification and nomenclature of viruses and to devise universally acceptable norms for both.  The ICNV specifically laid down that: 1. Groups (or genera) of viruses must be defined and listed. 2. Species belonging to these genera be listed 3. Names for the groups be provided. 4. Taxonomic development in various broad branches of virology be summarized and be based on uniform set of principles. 5. Norms for description and identification of viruses be set. LHT system  The above mentioned five “Essential Integrates” or characteristics were utilized singly or in correlated combination to classify viruses into groups, sub-group and intra sub-groups.  LHT system has been accepted because 1. It is the first attempt to classify viruses as a whole 2. It is based on the structure and composition of viruses 3. It attempts to classify on the basis of correlation amongst characters.

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 LHT system has been widely criticized also but still it is getting more and more attention in recent years.  Bellet System classifies viruses on the basis of molecular weight and % G+C ration along with serological reaction and phenotypic properties.  Gibbs system for the classification of plant viruses on the basis of (1) shape of the capsid, (2) the mode of transmission, (3) the type of vector, (4) the symptoms of infection, and (5) the nature of the accessory particles DESCRIPTION AND IDENTIFICATION OF VIRUS  Gibbs and associates proposed in 1966 that all viruses be technically identified on the basis of certain approved parameter.  They suggested following eight characters: 1. The nucleic acid type 2. The number of strand in a nucleic acid 3. The molecular weight 4. % of nucleic acid in a virion 5. The form of the particle 6. The form of the nucleocapsid 7. The host 8. The vector  They further suggested that these parameters be defined by abbreviations and that the abbreviation presented in the form of a formula describing a virus.  They renamed such formula as Cryptogram  This proposal was accepted with certain modification by the ICNV.

Cryptogram Virus 1st pair 2nd pair 3rd pair 4th pair Pox virus (Vaccinia) D/2 160/5-7.5 X/* V/O Coliphage D/2 130/40 X/X B/O Herpes Simplx D/2 67/7 S/S V/O ColiphageɸX174 D/1 1.7/25 S/S B/O Tobacco Mosaic R/1 2/5 E/E S/O Turnip Yellow Mosaic R/1 1.9/37 S/S S/Cl Tobacco Necrosis R/1 1.5/19 S/S S/fu Cauliflower Mosaic D/2 5/15 S/S S/AP Yellow Fever Virus R/* */* S/* V,I/Di Poliovirus R/1 2.5/30 S/S V/O

Abbreviations 1st pair: Nucleic acid type/strand (D/R; 2/1)

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2ndpair: Mol. Wt (106) of NA/percentage 3rdpair: Outline of particle/nucleocapsid (S: Spherical, E: Elongated, X: Complex) 4th pair: Host/Vector (B: Bacterium, S: Seed plant, I: Invertebrate, V: Vertebrate, Di: Diptera Cl: Coleoptera, Ap: Aphid, Fu: Fungi, *: Unknown, O: Spread without vector)  Lwoff &Tournier (1969) proposed a system for identification of viruses on the basis of symbolic description called Phanerogram.  It uses four parameters namely nucleic acid type, symmetry of capsid, naked or enveloped nature of the nucleocapsid and number of capsomers/diameter of nucleocapsid.  The approach was similar to that for cryptogram formulated.

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NOMENCLATURE OF VIRUSES  Nomenclature is the process of giving Names to the living systems or any object.  No system of classification can be full proof without assigning definite nomenclature to the various categories.  In recent years, virologist have agreed to use and accept the various categories used in biological nomenclature of organisms.  But some virologist still prefer to use the term “group”  The International Committee for Nomenclature of Viruses established rules guiding the nomenclature of viruses. Some of these are: 1. The species includes identical viruses. 2. The genus is a group of species having common characteristics 3. The name of the genus must terminate in the suffix “virus” and an effort should be made towards binomial nomenclature. 4. Each genus must have a type species. 5. A group of genera are to be referred to as family which shall have a name terminating in “idae”  These groups have been used and nomenclature of virus groups suggested.  But many virologist are still not convinced about using binomial nomenclature.  Binomials for some important viruses are as follows: Virus Binomial Pox (Variola) Poxvirus variolae Polyoma Polyomavirus neoformans Herpes Herpesvirus hominis Phage T2 Phagovirus (coli) tsecundus Tobacco Mosaic Protovirus tobacco Influenza Myxovirus influenza Rabies Rabiesviruscanis Polio Poliovirus primus Arbo occidentalis

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Phylum - Vira

Subphylum Subphylum Deoxyvira Ribovira Class

Deoxyhelica Deoxycubica Deoxybinales Ribohelica Ribocubica Order

Chaetovirales Enidovirales Urovirales Without envelope Rhabdovirales Sagovirales Gymnovirales Togovirales with envelope withoutenvelop With envelope Without envoleope

Phagoviridae Napoviridae Arbidoviridae Pox viridae Enidoviridae Dolichoviridae Myxoviridae Encephaloviridae Protoviridae Paramyxoviridae

Mesoviridae Stomatoviridae Leptoviridae Thylaxoviridae Adroviridae Peplovirales Haplovirales with envelope with envelope

Herpesviridae Microviridae Parvoviridae Densoviridae Papilloviridae Adenoviridae Iridoviridae

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