Lecture Of Viruses PDF

What is a Virus? A virus is a small filterable and obligate intracellular parasite requiring a living host for its multiplication Reasons for studying Viruses Viruses are important agents of human, animal and plant diseases eg rice yellow mottle virus of rice , foot and mouth disease of domestic ani...

What is a Virus? A virus is a small filterable and obligate intracellular parasite requiring a living host for its multiplication Reasons for studying Viruses Viruses are important agents of human, animal and plant diseases eg rice yellow mottle virus of rice , foot and mouth disease of domestic animals and measle virus. Potential application of viruses  Phage typing of bacteria  Sources of enzymes  As pesticides  Antibacterial agents  Anticancer agents  Gene vectors for protein production  Gene vectors for treatment of genetic diseases Origin of viruses Evidence for the existence of virus was first provided in the late 19th century by Martinus Beijerinck and Dimitri Ivanovski. They made extracts from disease plants infected with tobacco mosaic virus (tmv) and passed the extracts through fine filter. The filtrates contained an agent that was able to infect new plants but no bacteria could be cultured from the filtrates. Characteristics of viruses  They are all potentially infectious  Presence of single nucleic acid  Incapability to grow  Reproduction from the genetic material only  Absence of enzymes for energy metabolism  Absence of ribosomes  Absence of information for the production of enzymes in the energy cycle  Absence of information for the synthesis of ribosomal proteins  Absence of information for the synthesis of ribosomal RNA and soluble tRNA occurrence  Viruses occur in a wide hosts  Plants--- angiosperms, gymnosperms, ferns, algae, bacteria and fungi  Animals--- protozoans, insects, fish, amphibians, birds, mammals and human Morphology Shapes– viruses have different shapes such as:  Spheroid or cubiod eg adenoviruses  Elongated eg potato viruses  Flexuous or coiled eg beet yellow virus  Bullet shaped eg rabies virus  Filamentous eg bacteriophage M13  0Pleomorphic eg alfalfa mosaic virus size Viruses have variable sizes but sizes vary from 20nm to 300nm in diameter Viral structure A complete viral structure is made up nucleic acid core surrounded by a protein coat or capsid nucleic acid * capsid = nucleocapsid nucleocapsid can be naked or enveloped Function of protein coat is for protection and attachment Function of nucleic acid is for the synthesis of viral material Classification of viruses International Committee on Taxonomy of Virus (ICTV) was formed in 1966. ICTV laid down rules for the classification and nomenclature of viruses. EVOLUTION OF VIRUSES Three hypothesis have been proposed to explain the origin of viruses. 1. Viruses are descendants of ancient precethilar urgenisins that became parasites of the first cellular organisms. As organisms and animals evolved, viruses evolved with them. Many viruses do not cause any damage but may remain alent during the life of the host.  2.Viruses have evolved from pathogenic bacteria through a retrograde evolutionary process. Although rickettsiae and chlemydiae are examples of intracellular organisms that have undergone parasitic degeneration. There is at present no evidence to support the theory that true viruses have evolved from bacteria. 3. Viruses are components of normal cells that sometimes become autonomous. Within the cells the virus might exert an antrocatalytic influence so that replicas of itself are formed from the materials within the cell. Viruses might be said to resemble genes that have escaped regulatory control and continue to multiply as long as there is building material. For example, normal-appearing, apparently healthy cell cultures derived from infants who acquired reubella infection in utero produce rubella virus that is cytopathic for other cell limes. Viruses vs Bacteria Viruses differ fundamentally from bacteria in the following characteristics: 1. Small size and filterability Viruses are measured in millimicrons (one thousandth of a micron) and (for small viruses) in Angstrom wait (Ao) (one tenth of a millioncron). Virus sial is usually determined by direct observation under the electron microscope. 2. Growth only in living cells Host cells are usually provided in one of three forms: a) The experimental animal b) Chick embryos c) Tissue cultures 3. Resistant to the action of antibiotics and other agents that destroy bacteria. a) Heat – generally inactivated by temperature of 56-60oC for 30 minutes b) pH – are usually destroyed at and pH below 5 and alkaline pH above 9 c) Glycerol – most viruses survive 50% glycerol whereas bacteria are destroyed d) Bactericidal Agents – e.g. Phenol is not efficient as a viral disinfectant oxidizing agents are most effective with viruses  e) Antibiotics and Chemotherapeutic Agents – Sulfonamides, penicillin, streptomycin and the tetracyclines have little effect on the viruses 4. Viral reproductive processes differ from the simple binary fission of bacteria. 5.Viruses contain only one kind of nucleic acid and it is covered by a protein coat. What is Virology? This is the branch of microbiology which is concerned with viruses and viral diseases. NATURE OF VIRUSES Virus is one of a group of minutes infections agents with certain exceptions (e.g. Pox viruses) not resolved in the light microscope, and characterized by a lack of independent metabolism and by the ability to replicate only within living host cells. Like living organisms, they are able to reproduce with genetic continuity and the possibility of mutation. They are morphologically heterogeneous, occurring as rod shaped, spherical or polyhedral and tadpole-shaped form. The individual particle or virion, consists nucleic acid (the nucleoid), DNA or RNA (but not both) and a protein shell or Capsid, which contains and protects the nucleic acid. Viruses are customarily separated into three subgroups on the basis of host specificity namely – bacteria viruses, animal viruses and plant viruses. They are classified as to their origin (e.g. reoviruses), mode of transmission (arborviruses, tick born viruses) or the manifestation they produce (polioviruses, polymaviruses, poxviruses). They are sometimes warmed for the geographical location in which they were first isolated (e.g. coxsackevirus). Viruses are the smallest infectious agents containing a molecule of nucleic acid (RNA or DNA) as their genome. The nucleic acid is encased in a protein shell and the entire infectious unit is termed a virion. Viruses infectious unit is termed a virion. Viruses replicate only in living cells. The viral nucleic acid contains information necessary for programming the infected host cell to synthesize a number of virus specific Macoomolecules required for the production of virus progeny. During the replicative cycle, numerous copies of viral nucleic acid and coat proteins are produced. The coat protein assemble together to form the capsid, which encases and stabilized the viral nucleic acid against the extra cellular environment and facilitates the attachment and penetration of virions upon contact with new susceptible cells. The nucleic acid, once isolated from the virion, can be hydrolyzed by either ribo- or deoxyribonuclease whereas the nucleic acid within the intact virus is not affected by such treatment. In contrast, viral antiserum will neutralize the virion because it reacts with the antigens of the protein coat. However, the same antiserum has no effect on the free infectious nucleic acid isolated from the virion. The host range for a given virus may be extremely limited, but viruses are known to infect unicellular organisms such as mycoplasma, bacteria and algae and all higher plants and animals. Capsid: The symmetric protein shell which encloses the nucleic acid genome. Often, empty Capsids are by-products of the viral replicative cycle. Nucleocapsid is the capsid together with enclosed nucleic acid. A virion (virus particle) lacks certain components absolutely essential for its own replication and must depend on the host cell in which it is replicating to provide these missing factors. One component missing from all viruses is ATP – generating system. For independent life, a cell must carry out oxidations to provide energy to regenerate those high energy phosphate bonds used for biosynthetic reactions. No virion possess this regeneration system and hence, it must rely on the ATP-generating system present in the infected host cell. A second component that viruses lack and that the host cell must provide is the structural component for protein synthesis, that is ribosomes. The synthesis of any protein require that a ribonucleic acid (message RNA) be attached to a ribosome so that the individual amino acid can be joined to form the protein. The virion does not carry its own ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), but all viruses must use host cell ribosomes for protein synthesis. Another characteristic that is peculiar only to viruses is that, whereas all other forms of life contain both RNA and DNA. Viruses contain only one type of nucleic acid (RNA or DNA) but not both. Many viruses are capable of producing cancer (tumor) in certain animals. There is a strong evidence that a human maliquancy called BURKITT’S LYMPHOMA is the result of a virus infection. DNA viruses accomplish this by incorporating their DNA into the host cell chromosomes. RNA viruses must first transcribe their RNA into DNA in order to render a cell malignant. Transcription:- The mechanism by which specific information encoded in a nucleic acid chain is transferred to messenger RNA Translation:- The mechanism by which a particular base sequence in messenger RNA results in production of a specific amino acid sequence in protein. MICROSCOPY Although the majority of viruses cannot be seen by ordinary microscopic methods, the light microscope nevertheless has some important applications in virology The large viruses of the pox group are just above the limit of resolution with ordinary light and can be demonstrated by the use of staining procedures which, as a result of deposition of stain on the surface of the particle, increase apparent size The fluorescent antibody technique has been of considerable value for the demonstration of viral antigens in tissues. Antigenic components of the smallest viruses can be demonstrated by this technique. For the recognition of virus induced degenerative changes (cytopathogenic effect, CPE) in infected tissue cultures. For the recognition of characteristic histological and cytological changes in infected animals or man e.g. Poliomyelitis glandular fever and the characteristic myositis produced in body mice by Coxsackie viruses. The greatest advances of recent years in our knowledge of the structure of virus particles have been achieved through the use of the electron microscope. In this instrument a beam of electrons is used instead of light rays and because of the very short wave length of high velocity electrons, the limit of resolution is enormously increased. The electron microscope is capable of resolving particles with diameters of less than ι nm and its use has consequently brought even the smallest viruses into visible range. Electron microscopy has also been used for counting the number of virus particles in a suspension. For this purpose the suspension mixed with a suspension of polystyrere latex particle of a known concentration. From the relative numbers of latex particles and of virus particle seen in droplets of the mixture the concentration of virus in the original suspension can be estimated. CHEMICAL COMPOSITION OF VIRUSES Viral Protein: The structured proteins of viruses have several important functions. They serve to protect the viral genome against inactivation by nucleases, participate in the attachment of the virus particle a susceptible cell and are responsible for the structural symmetry of the virus particle. Also, proteins determine the antigenic characteristics of the virus. The structural proteins of many viruses could be studied by dissociating the proteins of the virus particle with a detergent (sodium dodecyl sulfate) and then separating them by electrophoresis through polyacrylamide gel matrix. Virus structural proteins may be very specialized molecules designed to perform a specific task (a) vaccinia virus carries many enzymes within its particle to perform certain functions early in the infection cycle; (b) some viruses have specific proteins for attachment to cells e.g. influenza virus hemagglutinin and (c) RNA tumor virus contain the enzyme, reverse transcriptase that makes a DNA copy of virus RNA, which is an important step in the transformation of these viruses. Viral Nucleic Acid: Viruses contain a single kind of nucleic acid, either DNA or RNA, that encodes the genetic information necessary for the replication of the virus. The RNA or DNA genome may be single-stranded or double-stranded. Most major families of RNAcontaining animal viruses have single- stranded RNA genomes except reoviruses, which have double-stranded RNA. NOTE Major families of DNA-containing animal viruses have double- stranded DNA genomes with exception of single-stranded DNA- containing parvovirus. The nucleic acid can be determined using either the intact virus particle or the free nucleic acid. Both the type of nucleic acid and the strandedness can be determined in the fluorescence microscope by staining with acridine orange and the identification of the nucleic acid by color reaction and enzyme digestion tests. Viral Lipids: A number of different viruses com lipids as part of their structure. Lipid-contain Nucleic Acviruses are sensitive to treatment with ether and other organic solvents, indicating that disruption or loss of lipid results in loss of infectivity. Non lipid-containing viruses are generally resistant to the action of ether. Viral Carbohydrates: The viral envelope also has a significant amount of carbohydrates mainly in glycoproteins. These glycoproteins may contain glucosamine, fructose, galactose, and mannose. The glycoproteins are important components of viral antigenic determinants. Glycoprotein synthesis may be partially controlled by the virus but is determined also by the host cell genome. CLASSIFICATION OF VIRUSES Basis of Classification The following properties, listed in the order of importance, have been used as a basis for the classification of viruses. The amount of information available in each category is not uniform for all viruses: 1. Nucleic acid type: RNA or DNA, single-stranded or double stranded i.e. strategy of replication. 2. Size and morphology including type of symmetry, number of capsomeres and presence of membranes. 3. Susceptibility to physical and chemical agents. 4. Immunologic properties. 5. Natural method of transmission 6. Host, tissue and cell tropism. 7. Pathology, including inclusion body formation. 8. Symptomatology. Classification by Symptomatology The oldest classification of viruses is based on the diseases they produce, and this system offers certain conveniences for the clinician. However, it is not satisfactory for the biologist because the same virus may appear in several groups, since it causes more than one disease depending upon the organ attacked. Generalized Diseases: Disease in which virus is spread throughout the body via the bloodstream and in which multiple organs are affected. These include smallpox, vaccinia measles, rubella, chickenpox, yellow fever, dengue, enteroviruses. II. Diseases Primarily Affecting Specific Organs: The virus may spread to the organs through the bloodstream, along the peripheral nerves or by other routes. 1-Diseases of the nervous system – 1) poliomyelitis, 2) aseptic meningitis (polio – coxsacke – and echoviruses), 3) rabies, 4) arthropod-borne encephalitis, 5) lymphocytic choriomeningitis, 6) herpes simplex meningoencephalitis of mumps, 7) measles, 8) vaccinia and “slow” virus infections. 2) Diseases of the respiratory tract – 1) Influenza, parainfluenza, 2) RJ pneumonia and bronchiolitis, 3) adenovirus pharyngitis, 4) common cold (caused by many viruses). 3) Localized diseases of the skin or mucous membranes – 1) Herpes simplex, 2) molluscum contagiosum, 3) warts, 4) herpangina, 5) herpes zoster. 4) Diseases of the eye:- 1) Adenovirus conjunctivitis, 2) New-castle virus conjunctivitis, 3) herpes keratoconjunctivitis 4) epidemic hemorrhagic conjunctivitis (enterovirus – 70). 5) Diseases of the Liver – 1) Hepatitis type A (infectious hepatitis) and 2) type B (serum hepatitis), 3) yellow fever. In neonate, enteroviruses, herpes v is used and rubella virus. 6) Diseases of the salivary of glands – mumps and cytomegalovirus. 7) Diseases of gastrointestinal tract – Gastroenteritis A virus and gastroenteritis B virus (rotavirus), poliovirus, Hepatitis A, Hepatitis B. Classification by Biologic, Chemical and Physical Properties Viruses can be clearly separated into families on the basis of the nucleic acid genome and the dice shape, substructure and mode of replication of the virus particle within each family, general are usually based on antigenicity. DNA-containing Viruses 1-PARVOVIRIDAE FAMILY – 1) The members of this family are very small viruses. 2) They contain single-stranded DNA. 3) They have no envelope and are either-resistant. 4) Replication take place in the nucleus of the infected cell. 5) Genus PARVOVIRUS includes the autonomously replicating parvoviruses of harmster, rats, mice and swine. 6) Genus ADENOSATELLOVIRUSES consist of the adeno- associated satellite viruses which are defective and cannot multiply in the absence of a replicating adnovirus which serves as a “helper virus”. (Herpesvirus can act as a partial helper). 7) Genus DENSOVIRUS include the arthropod parvoviruses. 2-PAPOVAVIRIDAE FAMILY: 1. these are small, either-resistant viruses 2. contain double-stranded circular DNA. 3. The human representative are the pilloma or wart, virus and SV40 like viruses. 4. Isolated from the brain tissue of patients with progressive multifocal leukoencephalopathy (PML) (JC virus) or from the urine of immunosuppressed renal transplant recipients (BK virus). 5. Ethers are papilloma viruses of rabbits and cattle, polyoma virus of mice and vacuolating viruses of monkeys (SV40) and of rabbits. 6. These viruses have relatively slow growth cycles characterized by replication within the nucleus. 7. Papovaviruses produce latent and chronic infections in their natural hosts. 3-ADENOVIRIDAE FAMILY: 1. These are medium-sized viruses 2. containing double-stranded DNA. 3. They are not enveloped and are ether-resistant. 4. Thirty-one types are known to infect humans. 5. They have predilection for mucous membranes and may persist for years in lymphoid tissue.  6. Some of these agent cause acute respiratory diseases, febrile catarrhs, pharyngitis and conjunctivity. 7. Human adenoviruses rarely cause disease in laboratory animals. 4-HERPETOVIRIDAE FAMILY: 1. These are medium sized viruses 2. containing double-stranded DNA. 3. Latent infections may occur and last for the life span of the host even in the presence of ciruculating antibodies. 4. In human, herpes simplex virus types 1 and 2 cause oral and genital lesions (cold sores). 5. Varicella – zoster viruses causes zoster and chickenpox. 6. Cytomegalovirus causes cytomegalic inclusion disease. 7. EB virus causes infections mononucleosis. 8. EB virus has also been associated with several human malignanties. 9. Other members occur in monkey, rabbits, cattle, horses, pigs, dogs, frogs, shrews, fowl and snakes. 5-POXYVIRIDAE FAMILY: 1. These are relatively large, brick-shaped or ovoid viruses 2. containing a double-stranded DNA genome and protein, 3. enveloped by double membranes. 4. This is the major DNA-containing virus family that replicates solely within the cytoplasm. This family includes members that are chiefly pathogenic for the skin in human (small pox, vaccine, milluscum contagiosum) and in animals (e.g. cowpox, monkeypox and myxoma of rabbits). Some of the animal poxvirus (e.g. cowpox and monkeypox) can infect humans.  RNA-containing Viruses 1-Picornaviridae Family: 1. The genera whose members commonly infect humans are ENTEROVIRUS and PHINOVIRUS. 2. At least to human enteroviruses are known – these include polio, coxsackie and echo-viruses. 3. Other types exist throughout the animal kingdom. More than 100 rhinoviruses exist and are the most common cause of colds in humans. 4. Two other genera are APHTHOVIRUS (foot- and mouth-virus of cattle) and CARDIOVIRUS (encephalomyocarditis virus, which rarely infects humans). 5. Picornaviruses are small, ether resistant viruses that contain single- stranded. 2-Reoviridae Family: 1. Reoviruses were the first organisms shown to have double-stranded RNA. They are ether-resistant viruses. 2. Reovirus strains from animals are similar to those of humans. 3. Diseases associated with them not really clear. 4. The family Reoviridae contains two genera. (REOVIRUS and ORBIVIRUS). 5. A probable new genus is ROTAVIRUS, the agent of infantile gastroenteritis. 6. Rotaviruses are major pathogens of humans, causing infantile diarrhea in all parts of the world. 7. This is one of the most common childhood illness and in developing countries is a leading cause of infant death. 3-Togaviridae Family: 1. This family includes most arbiviruses of antigenic groups A and B, rubella virus, and LDH (Lactic dehydrogenase) virus of mice. 2. These viruses possess a lipid containing, 3. ether-sensitive envelope and have a genome of single-S RNA. 4. Togaviruses include 4 genera: a) Alphavirus (group A arboviruses), with sindbis virus as the type species. b) Flavivirus (group B arboviruses), with yellow fever virus as the type species. c) Rubivirus, with rubella virus as the type species and d) Pestivitus, with mucosal diarrhea virus as the type species. 4-Coronaviridae Family: 1. This members are enveloped and contains a genome of single- stranded RNA. 2. The human coronaviruses have been isolated from patients with acute upper respiratory tract illness. 5-Bunyaviridae Family: 1. They are ophevila (spherical) and enveloped. Single stranded 2. These viruses replicate in the cytoplasm and acquire an envelope by budding through the cytoplasmic membrane. 3. They are sensitive to ether, acid and heat. 6-Orthomyxoviridae Family: 1. These are medium-sized enveloped viruses containing single stranded RNA. 2. The orthomyxoviruses are sensitive to diactinomycin. 3. A;; orthomyxoviruses are influenza viruses. 4. They are classed as types A, B or C on the basis of these RNA antigen, which does not cross-react between types. 7-Rhabdoviridae Family: 1. Members of this family have enveloped virion that are rod-shaped, resembling a bullet. 2. The genome is single-stranded RNA. 3. Virus particles are formed by budding from the cell surface membrane. 4. include Rabies virus and other viruses of cattle, fish and plants. Other Viruses: For some viruses there are insufficient data to permit their classification. These include viruses that cause hepatitis (hepatitis A, and hepatitis B) and viruses responsible for certain immune complex disease and for neurologic disorders with a long laent period (“slow” virus diseases). Viroids: A class of infectious agents smaller than viruses, termed viroids. Viroids exhibit the characteristics of nucleic acids in crude extracts i.e. they are insensitive to heat and organic solvents but sensitive to nuclease, and they do not appear to possess a protein coat. Presently known viroids consist solely of a short strand of RNA. Cultivation; Quantification, Inclusion Bodies; Chromosome Damage Cultivation Of Viruses In the early years of virus research, the use of animals was mandatory for the recognition of viruses, and rapid, quantitative results were often difficult. For example, poliomyphitis research was limited as long as the presence of the virus could be detected only by monkey inoculation. At present, many viruses can be grown in cell cultures or infertile eggs under strictly controlled conditions. Growth of virus in animals is still used for the primary isolation of certain viruses and for the study of pathogenesis of viruses. Chick embryo: Virus growth in an embryonated egg may result in the death of the embryo (e.g. encephalitis virus), the production of plaques on the chorioallantoic membrane (e.g. herpes, smallpox, vaccinia), the development of hemagglutinins in embryonic fluids or tissues (e.g. influenza) or the development of infective virus (e.g. poliovirus type 2). Inoculation may be into the allantoic or amniotic cavities into the yolk sac or on to the chorioallantoic membrane, the precise route used depending on the particular virus being cultivated. The amniotic route is the method of choice for the isolation of the influenza and mumps viruses. The alantoic route, which is technically the simplest, is used mainly for the passage of influenza viruses that have already been established in embryo. Yolk sac inoculation is of particular value for the propagation of rickettsince. Inoculation on to the chorioallantoic and characterization of the poxviruses and herpes simplex. Tissue Cultures: Most of the humans pathogenic viruses may be propagated in tissue cultures derived from a variety of animal species. Since, however, a number of common viruses e.g. the enteroviruses and the adenoviruses will grow only in the cells of primate tissues, the tissue cultures used in diagnostic virology are generally derived from monkey or human sources. Tissue culture techniques may be divided broadly into three groups: (1) Fragment cultures, (2) cell cultures and (3) organ cultures. 1. Fragment Cultures: The simplest form of fragment culture is Maitland type of culture which consists of fragments of tissue suspended in a fluid medium. The cell remain viable for several days-sufficiently long to permit virus growth but do not multiply. In plasma clot cultures, the tissue fragments are fixed by a plasma clot to the sides of tubes on rattler; new cells, mostly fibroblast, then grow out from the tissue fragments. Fragment cultures have no application in routine diagnostic virology. 2.Cell Cultures: These are prepared from cell suspensions obtained from intact tissue or from a prior tissue culture. Dispersal of cells is usually achieved by treatment of the tissue or tissue culture with a proteolytic enzyme usually trypsin or with the chelating agent versene (EDTA, ethylenediamine tetra-acetic acid, sequenstrene). This results in the release of single cells and small aggregates of cells capable of initiating growth. Cell culture may be prepared as suspended cultures or as monolayer cultures. (a)-Suspended cell cultures resemble Maitland type culture in that the cells are simply suspended in nutrient medium. They are used for metabolic inhibition tests which may, in diagnostic work, be applied to the estimation of antiviral antibody. Uninfected cells metabolize and actively produce acid. This causes a colour change in an appropriate pH indicator incorporated in the nutrient medium. If the cells are infected by virus, their metabolism is interfered with and the indicator change does not occur. If however the virus is neutralized by antibody the indicator change occurs as in a normal uninfected culture. (b)-In monolayer cell cultures, the cells are allowed to settle on the sides of a tube or flat bottle and are covered with nutrient medium. Antibiotics are incorporated in order to control, bacterial and fungal contamination. After two to seven days incubation at 37oC the growing cells will have formed into a continuous sheet or monolayer, no called because it is one cell thick, adherent to the glass of the tube or bottle. The cell growth medium is then removed and replaced with a maintenance medians which is nutritionally less rich than the initial growth medium but adequate to maintain the viability of the tissue cells. Then the virus inoculation is introduced and culture again incubated. During incubation tubes are maintained in a slightly sloped and bottles in a horizontal position. Many viruses when propagated in a monolayer cultures produce degenerative changes in the tissue cells readily visible under the lower power objective or if the area involved is sufficiently large, to the naked eye. This is known as a cytopathogenic effect (cytopathic effect, CPE). The type of cellular change produced differs with different viruses. Cell cultures may be divided into three types according to the damage history of cells used for their preparation: i) Primary and secondary cell cultures – primary cell cultures are prepared from cells obtained directly from the tissues. A secondary cell culture is the first subculture of a primary cell culture, the cells of which are dispersed by treatment with typsin or versene for the preparation of the secondary culture. This procedure is particularly convenient for the preparation of monkey Kichey cell cultures. ii) Continuous cell lines – As a rule when tissue cultures are prepared directly from an animal tissue, the cultures cannot be serially propagated, the cells dying out after a few subcultures. A number of lines of mammalian cells are, however, available (mainly derived from fetal and malignant tissues) which can serially be propagated more or less indefinitely. These established cells lines have the advantage of the reccorsity for procuring fresh animal tissue for each set of cultures. They have the disadvantage, on the other hand, that on prolonged subculture the tissue is liable to undergo spontaneous changes in its susceptibility to infection. The established cell lines most frequently employed are Hela cell culture which was derived originally from a human cervical caocinoma, and the HEp-2 cell derived from a carcinoma of the larynx. iii) Diphoid cell cultures – The cells of certain tissues, notably human embryo cells can be serially propagated for about 50 subcultures without transformation. Genetically, they differ from continuous cell lines and resemble normal cells. Diphoid cell cultures are highly susceptible to infection by viruses such as the H rhinoviruses and the cytomegalovirus which may be difficult to propagate in other systems. 3. Organ Culture: Organ culture has been recommended for the isolation of cold viruses which cannot be propagated by other procedures. This is done by use of tissue from appropriate organ for the isolation of viruses. III. Animal Inoculation: Of the ordinary laboratory animals the mouse is the most generally useful and depending on the virus, mouse can be infected by nasal instillation or by intraperitoneal (stomach) or intracerebral inoculation. The presence of virus in the inoculated material is shown by the development of appropriate symptoms, diagnostic pathological changes or specific antibody.