COMMON CHARACTERISTICS OF VIRUSES
1. Viruses are the smallest known infective agents, ultramicroscopic, and can only be viewed with an electron microscope (the smallest known virus is merely 0.002 um in diameter, while the largest viruses are typically about 0.8 µm in diameter).
2. They are obligate parasites.
3. They multiply only within their living host’s cells and remain metabolically inert outside the host cell.
4. Viruses are actually, nucleoproteins. The proteinaceous coat (capsid) surrounds the nucleic acid, which forms the central core of the virus particles.
5. The viral genetic material or the nucleic acid may be either DNA or RNA. The two nucleic acids cannot be present in a given virus.
6. Viruses are smaller than other organisms. They vary in size from 10-300 nm. They are so minute that they can easily pass through bacterial filters.
7. Viruses communicate easily from an infected host to healthy ones through various agencies.
8. Viruses are so effective that even their smallest amount can cause infection on the host successfully.
9. Viruses can turn into crystal form easily.
10. Viruses have no metabolic activities of their own and utilize the metabolism of host cells hence antibiotics do not affect them.
STRUCTURE OF VIRUSES
A fully developed infectious virus is called a virion. The simplest virions contain two basic components: nucleic acid (single- or double-stranded RNA or DNA) and a protein coat, the capsid, which functions as a shell to protect the viral genome from nucleases and during infection attaches the virion to specific receptors exposed on the host cell. The capsid is made up of a large number of protein subunits known as capsomeres. The capsid together with NA is known as the nucleocapsid. Some virus families have an additional covering, called the envelope, which is usually acquired during the release of progeny virus by the budding process through the host cell membrane.
Viruses containing envelopes are called enveloped viruses and those lacking envelopes are generally called naked viruses. On the outer surface of the envelope glycoprotein subunits are exposed in the form of projecting spikes known as peplomers (peplos meaning envelope).
SYMMETRY IN VIRUSES
The capsid of the virus particles shows three types of symmetry:
- Helical symmetry
- Icosahedral (cubical) symmetry
- Complex symmetry
1. Helical symmetry
These viruses are made up of a single type of capsomere. These capsomers and NA are wound together around a central axis to form a helical or spiral tube. This structure results in rod-shaped or filamentous virions: these can be short and highly rigid, or long and very flexible. Tobacco mosaic virus (TMV) is a helical virus.
2. Icosahedral (cubical) symmetry
Most animal viruses are icosahedral. An icosahedron! is a polygon with 12 vertices or corners and 20 facets or sides (each facet is an equilateral triangle) with 30 edges. The icosahedral capsids are of two types, pentagonal capsomeres at the vertices called pentons and hexagonal capsomeres called hexons. Minimum twelve identical capsomeres are required, each composed of five identical sub-units. Many viruses, such as rotavirus, have more than twelve capsomeres and I appear spherical but they retain this symmetry.
3. Complex symmetry
These viruses possess a capsid that is neither purely helical nor purely icosahedral due to the complexity of their structure. The Poxviruses and some bacteriophages are large, complex viruses that have an unusual morphology and are assigned to have complex symmetry.
REPLICATION IN VIRUSES
Due to the lack of biosynthetic enzymes, viruses replicate by utilizing the biochemical machinery of host cells to synthesize the various macromolecules required for the production of a new virion/ virus particle.
The replication is divided into six sequential stages
- 1. Adsorption
- 2. Penetration
- 3. Uncoating
- 4. Biosynthesis
- 5. Virion assembly
- 6. Release
1. Attachment (adsorption)
It is the first event in any viral infection in which the virus comes in contact with cells by random collision and gets attached to the cells. This attachment or adsorption is specific and depends on the presence of specific receptors on the host cell surface (called the host cell receptors). Specialized attachment sites are found distributed over the surface of the virion in animal viruses. e.g.Orthomyxo viruses and Paramyxoviruses attach through glycoprotein spikes, and adenoviruses attach through the penton fibers.
2. Penetration
Penetration of the virus occurs either by engulfment of the whole virus or by fusion of the viral envelope with the cell membrane. It allows only the nucleocapsid of the virus to enter the cell. Thus, the virus particle is taken inside the cell by one of the following mechanisms;
a.non-enveloped viruses enter the cell by engulfment of virion by invagination of the plasma membrane with an accumulation of virus particles in cytoplasmic vesicles called phagosomes. This is known as viropexis, a mechanism resembling phagocytosis.
b.In enveloped viruses, the envelope may fuse with the host cell’s plasma membrane, re the nucleocapsid into the cytoplasm.
3. Uncoating
The process involves the physical separation of nucleic acid from its outer structural components. It is assumed that host components and proteolytic enzymes within the lysosomes cause this uncoating process. The enveloped viruses get uncoated by the action of lysosomal enzymes. In Adenoviruses and Parvoviruses, uncoating is accomplished by cellular proteases present in the cytosol.
4. Biosynthesis (tiral synthesis)
After uncoating, the viral genome directs the biosynthetic machinery of the host cell to shut down the normal cellular metabolism and to produce its own viral components
In general, the NA genome of most of the DNA viruses is synthesized in the host cell nucleus except Poxvirus, which synthesizes all its components in the cytoplasm. The viral leasingDNA is released into the host cell’s nucleus where it is transcribed into early mRNA for transport into the cytoplasm. And there it is translated into early viral proteins.
The early viral proteins are concerned with the replication of the viral DNA, so they are transported back into the nucleus, where they are involved in the synthesis of multiple copies of viral DNA. These copies of the viral genome act as templates for transcription into late mRNAs, and they are also transported back into the cytoplasm for translation into late viral proteins. The late proteins are structural proteins (e.g. coat, envelope proteins) or core proteins (certain enzymes) which are transported back into the nucleus for the next stage of the replication cycle.
In the case of some RNA uses (e.g. Picoma viruses), the viral genome (RNA) stays in the cytoplasm where it mediates its own replication and translation into viral proteins. In other cases (e.g. Orthomyxo, Paramyxo, and Retroviruses), the infectious viral RNA enters into the nucleus where it is replicated and then they are transported back to the cytoplasm for translation into viral proteins.
5. Assembly (virion assembly)
Assembly of various viral components into a complete virus particle occurs shortly after replication of viral NA and may take place in either nucleus (Adeno and Herpes viruses) or cytoplasm (Pox and Picorna viruses). The viral nucleocapsid is formed by enclosing the nucleic acid by capsomere proteins enclose. The process is called encapsidation. In the case of enveloped viruses, the envelope is derived from the nucleus (Herpes virus) and from the plasma membrane if they assemble in the cytoplasm of the host cell (Paramyxo and Orthomyxo viruses).
6. Release
It is the final event in viral replication, and it results in the release of the mature virions from their host cell. Virus maturation and release occur over a considerable period. Non-enveloped (naked) viruses may be released by the lysis of the host cell or released by a process which may be called reverse phagocytosis without affecting the host cell, except poliovirus which damages the host cell. In enveloped viruses, the nucleocapsid acquires its final envelope from the nuclear or cell membrane by the budding-off process (envelopment) before being released out of the host cell.
CULTIVATION OR ISOLATION OF VIRUSES IN LABORATORY
Viruses are obligate intracellular parasites. Viruses totally depend on their host cells for their existence. They can replicate (multiply) only in living cells & cannot be grown on any of the inanimate (non-living) culture media. There are three methods employed for virus cultivation:
A. ANIMAL INOCULATION
Earlier viruses causing human diseases were inoculated into human volunteers. However, due to the serious risk involved, human volunteers are involved only when no other method is available & the virus inoculation is relatively harmless. Due to much simpler methods of isolation such as cell culture, animal inoculation is less commonly preferred by most diagnostic laboratories. However, white mice, particularly suckling ones (<48 hours old) are still used in the isolation of Arboviruses &Coxsackie viruses. They are inoculated by intracerebral, intranasal, intraperitoneal, or subcutaneous routes.
Other animals like hamsters, rabbits, guinea pigs & monkeys are sometimes used in certain situations. After inoculation, these animals are observed for signs of illness or death. The viruses are identified by microscopy (for inclusion bodies) & by neutralization (Nt) & haemagglutination inhibition (HI) tests.
B.EMBRYONATED EGGS (CHICK EMBRYO)
The embryonated hen’s egg was first used for the cultivation of virus by Woodruff, Good Pasteur & Burnett (1931). The egg used for inoculation must be sterile & the shell should be intact & healthy. Eggs are kept in an incubator & embryos of 7-12 days old are used. After inoculation, the eggs are incubated for 2-9 days & examined daily for virus growth.
The routes of inoculation include
- 1. Chorioallantoic membrane (CAM):
- 2. Allantoic cavity
- 3. Amniotic cavity
1. Chorioallantoic membrane (CAM)
CAM is inoculated mainly for growing Pox viruses& sometimes for Herpes simplex virus. The replication of the virus produces visible lesions (pocks), a grey-white area in transparent CAM. Each pock is derived from a single virion. Therefore, the number of pocks formed indicates the number of viruses present in inoculums.
2. Allantoic cavity
The allantoic cavity is mainly inoculated for a rich yield of Influenza & some Paramyxoviruses. The inoculation is done mainly for growing influenza virus for vaccine production. Other allantoic vaccines include yellow fever (17 D strain) & rabies vaccine. Duck eggs are bigger & have a longer incubation period than hen’s eggs. Therefore, they provide a better yield of rabies virus & thus, they were used for the preparation of inactivated non-neural rabies vaccines (duck egg vaccine).
3. Amniotic cavity
It is mainly inoculated for primary isolation of the influenza virus. 4. Yolk sac: It is inoculated for the cultivation of some viruses as well as for some bacteria like Chlamydiae &Rickettsiae.
C. TISSUE CULTURE
Steinhardt et al (1913), maintained the vaccine virus in fragments in rabbit cornea and demonstrated the application of tissue culture in virology for the first time.
There are three types of tissue cultures
- Organ culture
- Explant culture
- Cell culture
1. Organ culture
Small bits of organs can be maintained in vitro for days to weeks, preserving their original morphology & physiology. Formalin is used for preservation. Such cultures are useful for the isolation of some viruses which are highly specialized parasites of certain organs e.g. tracheal ring organ culture used for isolation of Corno virus which is a respiratory pathogen.
2. Explant culture
Fragments of minced tissue can be grown as explants embedded in the plasma clots. They may also be cultivated in suspension e.g., Adenoid tissue explant culture used for the isolation of Adenoviruses.
3. Cell culture
The cell culture is the method routinely employed nowadays for the identification and cultivation of viruses. Cells of various tissues of animals can be cultivated. But more commonly, fibroblast and muscle epithelial cells are used for the propagation of the virus.
The tissue is first removed from the organism concerned, broken down into its constituent cells by utilizing suitable physical means like homogenization. The complete tissue is then converted into many small pieces. The tissue fragments are washed with a salt solution like sterile physiological saline or other types of solution like Hank’s solution or Eagle’s solution. Using the process dispersion the pieces are converted into their constituent cells from tissue. The proteinaceous cementing material (i.e. hyaluronic acid) is broken down by joining the cells with the help of proteolytic enzymes like trypsin, and mechanical shaking, a process called trypsinization.
The washed tissue fragments are then placed in a flask with sterile trypsin solution at 4°C for about 18 hours. During this period, the tissue fragments are gradually dispersed into their cellular components in presence of chemicals like EDTA which helps in the dispersion of cells. The cells are then centrifuged and re-suspended in a washing medium. It is done repeatedly. The washed suspended cells are then cultivated in a suitable growth medium containing essential amino acids, vitamins, fatty acids, carbohydrates, salts, and glucose. 5 10% calf or fetal calf serum is added to provide several growth factors. Antibiotics are added to prevent bacterial contaminants and phenol red as an indicator.
Most cell types multiply with a division time of 24-48 hrs in such media. The cell suspension is dispensed in glass or plastic bottles, tubes, or Petri dishes. The cells of epithelial or fibroblastic nature adhere to glass or plastic surfaces. On incubation at 36 °C for 72 hours these cells divide and spread out on the glass/plastic surface to form a thin but continuous layer, which is often one cell thick. Such cell layers are called a monolayer.
Types of Cell Cultures
Based on origin, chromosomal characters, and the number of generations through which they can be maintained.
Cell cultures are classified into three types.
- Primary cell culture
- Semi-continuous cell culture or Diploid cell culture
- Continuous cell culture
i. Primary cell culture
These are normal cells obtained from fresh organs of animals and cultured. Once the cells are attached to the vessel surface, they undergo mitosis until a confluent monolayer of cells covers the surface. These layers are capable of limited growth in culture and cannot be maintained in serial culture. They are commonly employed for the primary isolation of viruses and in the preparation of the vaccine. e.g, Rhesus monkey kidney cell culture and human amnion cell culture.
ii. Semi-continuous cell culture or Diploid cell culture
These are subsequent cultures derived from primary cell cultures. These are cells of a single type, usually fibroblasts, containing the same number of chromosomes as the parent cells and are diploid. There is a rapid growth rate and after 50 serial subcultures, they undergo senescence and the cell strain is lost. The diploid cell strains are susceptible to a wide range of human viruses. They are also used for the isolation of some fastidious viruses and the production of virus vaccines. e.g, Human embryonic lung strain (W1-38) and Rhesus embryo cell strain (HL-8).
iii. Continuous cell culture
These are cells of a single type, usually derived from the cancer cells that are capable of indefinite growth in vitro; therefore, they are known as continuous cell lines. In these cells chromosomes are haploid and they grow faster. They are also called permanent cell lines and the permanent cell lines derived from a single separated cell are called clones. One common example of such a clone is the Hela strain derived from the cervical cancer of lady Hela, by name. Continuous cell lines are maintained either by the serial subculture or by storing in deep freeze at -70°C so that these can be used when necessary. Some cell lines are now permitted to be used for vaccine manufacture. eg, Vero i.e. Vervet monkey kidney cell line, BHK, i.e. Baby Hamster kidney cell line.