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UNIT-1 GENERAL PROPERTIES AND CULTIVATION OF VIRUSES.

Early development of virology

Discovery of viruses

1892 – Dimitrii Ivanovsky observed that agent of tobacco mosaic disease passes through porcelain filters that retain bacteria

1898 – Marcus Beijerinck makes the same observation; concludes that the pathogen must be a distinctive agent

1898 – Friedrich Loeffler and Paul Frosch (former students of Koch), find that causative agent of foot-and-mouth disease is filterable (the first animal virus)

1901 – Yellow fever virus – Walter Reed (the first human virus)

1903 – Rabies virus (Remlinger, Riffat-Bay)

1906 – Variola virus (Negri)

1908 – Poliovirus (Karl Landsteiner and E. Popper); chicken leukemia virus (Ellerman, Bang)

1911 – Rous sarcoma virus (Peyton Rous)

1915 – Bacteriophages -Frederik Twort, Felix D’Herelle

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1931 – Swine influenza virus (Shope)

1933 – Human influenza virus (Smith)

The name virus was coined from the Latin word meaning slimy liquid or poison. It was originally used to described any infectious agent, including the agent of tobacco mosaic disease, tobacco mosaic virus. In the early years of discovery, viruses were referred to as filterable agents. Only later was the term virus restricted to filterable agents that require a living host for propagation.

Although the ancients did not understand the nature of their illnesses, they were acquainted with diseases, such as rabies, that are now known to be viral in origin. In fact, there is some evidence that the great epidemics of A.D. 165 to 180 and A.D. 251 to 266, which severely weakened the Roman Empire and aided its decline, may have been caused by measles and smallpox viruses. Smallpox had an equally profound impact on the New World. Hernán Cortés’s conquest of the Aztec Empire in Mexico was made possible by an epidemic that ravaged Mexico City. The virus was probably brought to Mexico in 1520 by the relief expedition sent to join Cortés. Before the smallpox epidemic subsided, it had killed the Aztec King Cuitlahuac (the nephew and son-in-law of the slain emperor, Montezuma II) and possibly 1/3 of the population. Since the Spaniards were not similarly afflicted, it appeared that God’s wrath was reserved for the Native Americans, and this disaster was viewed as divine support for the Spanish conquest The first progress in preventing viral diseases came years before the discovery of viruses. Early in the eighteenth century, Lady Wortley Montagu, wife of the English ambassador to Turkey, observed that Turkish women

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inoculated their children against smallpox. The children came down with a mild case and subsequently were immune. Lady Montagu tried to educate the English public about the procedure but without great success. Later in the century an English country doctor, Edward Jenner, stimulated by a girl’s claim that she could not catch smallpox because she had had cowpox, began inoculating humans with material from cowpox lesions. He published the results of 23 successful vaccinations in 1798. Although Jenner did not understand the nature of smallpox, he did manage to successfully protect his patients from the dread disease through exposure to the cowpox virus. Until well into the nineteenth century, harmful agents were often grouped together and sometimes called viruses [Latin virus, poison or venom]. Even Louis Pasteur used the term virus for any living infectious disease agent. The development in 1884 of the porcelain bacterial filter by Charles Chamberland, one of Pasteur’s collaborators and inventor of the autoclave, made possible the discovery of what are now called viruses. Tobacco mosaic disease was the first to be studied with Chamberland’s filter. In 1892 Dimitri Ivanowski published studies showing that leaf extracts from infected plants would induce tobacco mosaic disease even after filtration to remove bacteria. He attributed this to the presence of a toxin. Martinus W. Beijerinck, working independently of Ivanowski, published the results of extensive studies on tobacco mosaic disease in 1898 and 1900. Because the filtered sap of diseased plants was still infectious, he proposed that the disease was caused by an entity different from bacteria, a filterable virus. He observed that the virus would multiply only in living plant cells, but could survive for long periods in a dried state. At the same time Friedrich Loeffler and Paul Frosch in Germany

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found that the hoof-and-mouth disease of cattle was also caused by a filterable virus rather than by a toxin.

General properties of virus

1. Size:

The size of virus ranges from (20-300) nm in diameter. Parvovirus is the smallest virus with size 20nm whereas Poxvirus is

largest being 400nm.

2. Shape:

The overall shape of virus varies in different groups of virus. Most of animal viruses are spherical shape, Pox virus is

rectangular shape, TMV is rod shape, Poliovirus is bullet shape etc Some virus are irregular and pleomorphic in shape.

3. Symmetry:

Morphological protein subunits of capsid are arranged together to from a symmetrical structure of the virus.

Two basic symmetry are recognized in virus, they are helical symmetry and icosahedral symmetry.

In some virus, symmetry is more complex, which is other than helical or icosahedral.

4. Structure and Chemical composition:

i. Genome:

Viral genome or nucleic acid contains either DNA or RNA but not both.

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The genome can be either ds DNA or ss DNA or ds RNA or ss RNA The genome can exist as single piece or segmented. Eg, Influenza

virus contains 8 segments of ss RNA genome. The genome may be linear or circular. Most virus possess linear

genome except Papova virus which contains circular ss DNA. Genome helps replication of virus in host cell.

ii. Capsid:

Capsid is the outer shell of a virus. It is chemically a viral protein. Capsid is composed of capsomere. Structure of capsid gives the symmetry of virus. Capsid protects the nuceic acid and also helps in attachments on

host cell surface during infection.

iii. Envelope:

Some virus contains phospholipid bilayer known as envelope. Virus lacking envelope is called naked virus. Envelope is a lipid bilayer which is acquired from host cell

membrane

iv. Glycoprotein spike:

Envelope of some virus contains viral coded spike projected outside the envelope called glycoprotein spike or peplomers.

Glycoprotein spike are viral coded protein with carbohydrate head.

Glycoprotein spikes is an important antigenic structure.

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Neuraminidase and Haemagglutinin are glycoprotein spikes which helps in virus attachment to cellular receptor on host cell to establish infection.

v. Enzymes:

Some virus possess their own enzymes. Retrovirus possess reverse transcriptase

5. Viral replication:

Virus only replicates inside host cell

6. Metabolism:

Viruses are metabolically inert outside host cell. They are also called as obligate intracellular parasite

7. Resistance:

i. Temperature:

Most viruses are heat labile. Viruses are inactivated by heating at 60°C for 30 minutes or 100°C

for few seconds.

ii. Cold:

Viruses are stable and resistant to cooling. Virus can be stored for long duration at -40°C to -70°C by

lyophilization or freeze drying.

iii. Radiation:

Both non-ionizing and ionizing radiation can kill virus.

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UV rays causes pyrimidine dimer formation while ionizing radiation eg, X-rays causes lethal break of viral genome.

iv. Organic solvent: Chloroform, ether and bile salt can destroy all viruses by lipid

solubiliation.

v. Disinfectant:

Most viruses are destroyed by oxidizing agents such as chlorine, H2O2, iodine etc.

Many viruses are resistant to phenol and chlorination. The phenol and chlorine do not always inactivates enterovirus, particularly if they are present in faecal materials.

vi. Antibiotics:

Viruses are resistant to antibiotics.

Viruses are the smallest infectious agents (ranging from about 20 nm to 300 nm in diameter) and contain only one kind of nucleic acid (RNA or DNA) as their genome. The nucleic acid is encased in a protein shell, which may be surrounded by a lipid-containing membrane. The entire infectious unit is termed a virion. Viruses are inert in the extracellular environment; they replicate only in living cells, being parasites at the genetic level. The viral nucleic acid contains information necessary for programming the infected host cell to synthesize virus-specific macromolecules required for the production of viral progeny. During the replicative cycle, numerous copies of viral nucleic acid and coat proteins are produced. The coat proteins assemble together to form the capsid, which encases and stabilizes the viral nucleic acid against the extracellular environment and facilitates the attachment and penetration by the virus upon contact with new susceptible cells. The virus infection may have little or no effect on the host cell or may result in cell damage or death.

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The universe of viruses is rich in diversity. Viruses vary greatly in structure, genome organization and expression, and strategies of replication and transmission. The host range for a given virus may be broad or extremely limited. Viruses are known to infect unicellular organisms such as mycoplasmas, bacteria, and algae and all higher plants and animals.

Structural properties of virus

Capsid: The protein shell, or coat, that encloses the nucleic acid genome.

Capsomeres: Morphologic units seen in the electron microscope on the surface of icosahedral virus particles. Capsomeres represent clusters of polypeptides, but the morphologic units do not necessarily correspond to the chemically defined structural units.

Defective virus: A virus particle that is functionally deficient in some aspect of replication.

Envelope: A lipid-containing membrane that surrounds some virus particles. It is acquired during viral maturation by a budding process through a cellular membrane. Virus-encoded glycoproteins are exposed on the surface of the envelope. These projections are called peplomers.

Nucleocapsid: The protein–nucleic acid complex representing the packaged form of the viral genome. The term is commonly used in cases in which the nucleocapsid is a substructure of a more.

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Morphology (Symmetry)

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

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

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

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Baltimore system of classifiaction

Viral genomes can be broken down into seven fundamentally different groups, which obviously require different basic strategies for their replication depending on the nature of their genome. When originally conceived, the Baltimore scheme encompassed six classes of viral genome. Subsequently the gapped DNA genome of hepadnaviruses (e.g. hepatitis B virus) was discovered and incorporated in a new scheme.

Viruses can be classified into seven (arbitrary) groups:

Baltimore System of Classification of Virus.

I. Double-stranded DNA viruses

Some replicate in the nucleus e.g adenoviruses using cellular proteins. Poxviruses replicate in the cytoplasm and make their own enzymes for nucleic acid replication. e.g. Adenoviruses; Herpesviruses; Poxviruses, etc

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II. Single-stranded (+) sense DNA viruses

Replication occurs in the nucleus, involving the formation of a (-) sense strand, which serves as a template for (+)strand RNA and DNA synthesis. e.g., Parvoviruses

III. Double-stranded RNA viruses

These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mRNAs. e.g., Reoviruses

IV. Single-stranded (+)sense RNA viruses (Picornaviruses; Togaviruses, etc)

Polycistronic mRNA: Genome RNA = mRNA. Since the RNA is the same sense as mRNA, the RNA alone is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins. e.g. Picornaviruses (poliovirus, rhinovirus); Hepatitis A virus

Complex Transcription: Two or more rounds of translation are necessary to produce the genomic RNA. e.g. Picornaviruses; Hepatitis A.

V. Single-stranded (-) sense RNA viruses

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The virion RNA is negative sense (complementary to mRNA) and must therefore be copied into the complementary plus-sense mRNA to make proteins. This group of viruses must code for RNA-dependent RNA-polymerase and also carry it in the virion so that they can make mRNAs upon infecting the cell. e.g. Orthomyxoviruses, Rhabdoviruses, etc

Segmented e.g. Orthomyxoviruses. First step in replication is transcription of the (-) sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for genome replication.

Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mRNAs are produced.

VI. Single-stranded (+) sense RNA viruses with DNA intermediate in life-cycle

RNA genome is (+) sense but unique among viruses in that it is DIPLOID, and does not serve as mRNA, but as a template for reverse transcription. e.g. Retroviruses.

Retroviruses therefore encodes an RNA-dependent DNA polymerase (reverse transcriptase) to make the DNA provirus which then is transcribed to genomic RNA by a host enzyme, RNA polymerase II.

VII. Double-stranded DNA viruses with RNA intermediate

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This group of viruses also relies on reverse transcription, but unlike the Retroviruses, this occurs inside the virus particle on maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription. e.g., Hepadnaviruses.

Cultivation of virus Viruses are obligate intracellular parasites so they depend on host for their survival.

They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication.

The primary purpose of virus cultivation is:

1.To isolate and identify viruses in clinical samples.

2.To do research on viral structure, replication, genetics and effects on host cell.

3.To prepare viruses for vaccine production.

Cultivation of viruses can be discussed under following headings: Animal Inoculation Inoculation into embryonated egg Cell Culture

1. Animal Inoculation

Viruses which are not cultivated in embryonated egg and tissue culture are cultivated in laboratory animals such as mice, guinea pig, hamster, rabbits and primates are used.

The selected animals should be healthy and free from any communicable diseases. Suckling mice are susceptible to togavirus and coxsackie virues, which are inoculated

by intracerebral and intranasal route. Viruses can also be inoculated by intraperitoneal and subcutaneous route.

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After inoculation, virus multiply in host and develops disease. The animals are observed for symptoms of disease and death.

Then the virus is isolated and purified from the tissue of these animals.

Live inoculation was first used on human volunteers for the study of yellow fever virus

Advantages of Animal Inoculation

Diagnosis, Pathogenesis and clinical symptoms are determined.

Production of antibodies can be identified.

Primary isolation of certain viruses. Mice provide a reliable model for studying viral replication. Used for the study of immune responses, epidemiology and oncogenesis. Disadvantages of Animal Inoculation Expensive and difficulties in maintenance of animals. Difficulty in choosing of animals for particular virus Some human viruses cannot be grown in animals,or can be grown but do not cause

disease. Mice do not provide models for vaccine development. It will lead to generation of escape mutants Issues related to animal welfare systems.

2. Inoculation into embryonated egg

Good pasture in 1931 first used the embryonated hen’s egg for the cultivation of virus.

The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used.

Viruses are inoculated into chick embryo of 7-12 days old.

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For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill.

After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days.

After incubation, the egg is broken and virus is isolated from tissue of egg. Viral growth and multiplication in the egg embryo is indicated by the death of the

embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes

Viruses can be cultivated in various parts of egg like chorioallantoic membrane, allantoic cavity, amniotic sac and yolk sac.

Chorioallantoic Membrane (CAM): Inoculation is mainly for growing poxvirus. After incubation and incubation, visible lesions called pocks are observed, which is grey

white area in transparent CAM. Herpes simplex virus is also grown. Single virus gives single pocks This method is suitable for plaque studies.

Allantoic cavity: Inoculation is mainly done for production of vaccine of influenza virus, yellow fever,

rabies. Most of avian viruses can be isolated using this method.

Amniotic sac: Inoculation is mainly done for primary isolation of influenza virus and the mumps virus. Growth and replication of virus in egg embryo can be detected by haemagglutination

assay. Yolk sac inoculation:

It is also a simplest method for growth and multiplication of virus. It is inoculated for cultivation of some viruses and some bacteria (Chlamydia,

Rickettsiae) Immune interference mechanism can be detected in most of avian viruses. Advantages of Inoculation into embryonated egg Widely used method for the isolation of virus and growth. Ideal substrate for the viral growth and replication. Isolation and cultivation of many avian and few mammalian viruses. Cost effective and maintenance is much easier. Less labor is needed. The embryonated eggs are readily available.

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Sterile and wide range of tissues and fluids They are free from contaminating bacteria and many latent viruses. Specific and non specific factors of defense are not involved in embryonated eggs. Widely used method to grow virus for some vaccine production. Disadvantages of Inoculation into embryonated egg The site of inoculation for varies with different virus. That is, each virus have different

sites for their growth and replication.

3. Cell Culture (Tissue Culture)

There are three types of tissue culture; organ culture, explant culture and cell culture. Organ cultures are mainly done for highly specialized parasites of certain organs e.g.

tracheal ring culture is done for isolation of coronavirus. Explant culture is rarely done. Cell culture is mostly used for identification and cultivation of viruses. Cell culture is the process by which cells are grown under controlled conditions. Cells are grown in vitro on glass or a treated plastic surface in a suitable growth

medium. At first growth medium, usually balanced salt solution containing 13 amino acids, sugar,

proteins, salts, calf serum, buffer, antibiotics and phenol red are taken and the host tissue or cell is inoculated.

On incubation the cell divide and spread out on the glass surface to form a confluent monolayer.

Types of cell culture

1. Primary cell culture:

These are normal cells derived from animal or human cells. They are able to grow only for limited time and cannot be maintained in serial culture. They are used for the primary isolation of viruses and production of vaccine. Examples: Monkey kidney cell culture, Human amnion cell culture

2. Diploid cell culture (Semi-continuous cell lines):

They are diploid and contain the same number of chromosomes as the parent cells. They can be sub-cultured up to 50 times by serial transfer following senescence and the

cell strain is lost.

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They are used for the isolation of some fastidious viruses and production of viral vaccines.

Examples: Human embryonic lung strain, Rhesus embryo cell strain

3. Heteroploid cultures (Continuous cell lines):

They are derived from cancer cells. They can be serially cultured indefinitely so named as continuous cell lines They can be maintained either by serial subculture or by storing in deep freeze at -70°c. Due to derivation from cancer cells they are not useful for vaccine production. Examples: HeLa (Human Carcinoma of cervix cell line), HEP-2 (Humman Epithelioma of

larynx cell line), Vero (Vervet monkey) kidney cell lines, BHK-21 (Baby Hamster Kidney cell line).

Assay of virus

Virus quantification

Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications.

One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay. This technique was first developed to calculate the titers of bacteriophage stocks. Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has since been used for reliable determination of the titers of many different viruses.

To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. When the plates are incubated, the original infected cells release viral progeny. The spread of the new viruses is restricted to neighboring cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes large

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enough to be visible to the naked eye. Dyes that stain living cells are often used to enhance the contrast between the living cells and the plaques. Only viruses that cause visible damage of cells can be assayed in this way. An example of plaques formed by poliovirus on a monolayer of HeLa cells is shown at left. In this image, the cells have been stained with crystal violet, and the plaques are readily visible where the cells have been destroyed by viral infection.

The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. To determine the virus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used.