MI 308 Virology & Mycology · 2020. 4. 16. · General Characteristics of Viruses Viruses are a...

Sheetal Pithva, Dept. of Microbiology, Government Science College, Gandhinagar

MI – 308 Virology & Mycology

Unit I Viruses: General

1.1 General characteristics and structural organization of virus

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 bacteriophage

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 nucleic acid, 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 smallpox, 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 bacteria proof filter.


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.

Frederick Twort (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 Norman Pirie 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 host metabolism to synthesize virion components; eventually complete virus

particles or virions are released.

In summary, viruses differ from living cells in at least threeways:


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 nature 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

vaccinia, 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 capsid, which protects viral genetic material and aidsin its

transfer between host cells.

There are four general morphological types of capsids 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 bacteriophages)or have

complex, multilayered walls surrounding the nucleicacid (e.g., poxviruses such as

vaccinia).


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.

Influenza virus RNAs 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.


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.


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.


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,mumps, 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

single chain breaks

T5 coliphage

Double strand with cross-

linked ends

Vaccinia, smallpox

Closed circular double

strand

Polyomaviruses (SV-40), papillomaviruses, PM2

phage,cauliflower mosaic

RNA

Single-Stranded Linear, single stranded,

positive strand

Picornaviruses (polio, rhinoviruses), togaviruses,

RNA bacteriophages, TMV, and most plant

viruses

Linear, single stranded, Rhabdoviruses (rabies), paramyxoviruses


negative strand (mumps, measles)

Linear, single stranded,

segmented, positive strand

Brome mosaic virus (individual segments

inseparate virions)


segmented, diploid(two

identical single strands),

positive strand

Retroviruses (Rous sarcoma virus,

humanimmunodeficiency virus)


segmented, negative strand

Paramyxoviruses, orthomyxoviruses (influenza)

Double-Stranded Linear, double stranded,

segmented

Reoviruses, wound-tumor virus of plants,

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.

Animal virus 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 hemagglutinin proteins, so named because they can bind thevirions

to red blood cell membranes and cause hemagglutination.

Hemagglutinins 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.


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.


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


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 growth medium 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 vaccine virus.

Vaccines 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:


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 antiserum and viewed under UV microscope. Viral

antigen present on the cell surface binds with the antiserum. Fluorescence 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.


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 petri dish 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.)


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 ASSAY 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.


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.


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 antibodies 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.


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 radial immunodiffusion 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.


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 viral disease 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.


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 pathology.

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.


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.


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.

Virus Cryptogram

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)


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.


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 Arbovirus occidentalis


Phylum - Vira

Subphylum

Deoxyvira Subphylum

Ribovira

Deoxyhelica Deoxycubica Deoxybinales

Chaetovirales

with envelope Enidovirales

withoutenvelop

Class

Order

Pox viridae Enidoviridae

Peplovirales

with envelope

Haplovirales

with envelope

Herpesviridae Microviridae

Parvoviridae

Densoviridae

Papilloviridae

Adenoviridae

Iridoviridae

Urovirales Without envelope

Phagoviridae

Ribohelica

Ribocubica

Rhabdovirales With envelope

Sagovirales Without envoleope

Dolichoviridae

Protoviridae

Mesoviridae

Leptoviridae

Adroviridae

Myxoviridae

Paramyxoviridae

Stomatoviridae

Thylaxoviridae

Gymnovirales

Togovirales

Napoviridae

Reoviridae

Arbidoviridae

Encephaloviridae

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