Cultivation of viruses

 
Cultivation of viruses

Introduction of virus

·         Viruses are extremely small infectious agents that invade cells of all types.
·         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.
·         Viruses have been the culprits in many human diseases, including smallpox, flu, AIDS,   and the ever-present common cold as well as in plant, bacteria and Archaea also.

Techniques in cultivating and identifying animal viruses

·         Viruses require living cells as their “medium”.
o   In vivo – laboratory-bred animals and embryonic bird tissues.
o  In vitro -  cell or tissue culture methods.

The primary purpose of virus cultivation is:-

·         To isolate and identify viruses in clinical samples.
·         To do research on viral structure, replication, genetics and effects on host cell.
·         To prepare viruses for vaccine production.

Methods of cultivation of Virus

·         Generally three methods are employed for the virus cultivation.
o   Inoculation of virus into animals
o   Inoculation of virus into embryonated eggs
o  Tissue culture

 Animal Inoculation

·         Viruses which are not cultivated in embryonated egg and tissue culture are cultivated in   laboratory animals such as mice, guinea pig, hamster and rabbits are used.
·         The selected animals should be healthy and free from any communicable diseases.
·         Mice(less than 48 hours old) are most commonly used.
·         Mice are susceptible to togavirus and coxsackie virues, which are inoculated by   intracerebral and intranasal route.
·         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.

Inoculation of Virus in Animals

·         Laboratory animals play an essential role in studies of viral pathogenesis.
·         Live animals such as monkeys, mice, rabbits, guinea pigs, ferrets are widely used for   cultivating virus.
·         Mice are the most widely employed animals in virology.

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.
·         Issues related to animal welfare systems.

 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.
·         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 , 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.
·         They are free from contaminating bacteria and many viruses.
·         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.

Tissue Culture

·         Cultivation of tissue or organ for the growth of viruses
·         Three types of tissue 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 cultureFragments of tissue can be grown as explant in plasma clot. This     method is rarely used.
·         Cell culture – it is mostly used for cultivation of viruses.
·         Itis 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
Based on the origin and the chromosome property the tissue culture are classified into 3 types:-
1.      Primary cell culture

·          These are normal cells freshly taken from animal or human body.
·          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 embryonic kidney, chick embryo       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.
·         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. Continuous cell lines

·         They are derived from cancer cells.
·         They can be serially cultured 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), BHK-21 (Baby Hamster Kidney cell line).

Advantages of cell culture


·         Relative ease, broad spectrum, cheaper and sensitivity.

Disadvantage of cell culture

·         The process requires trained technicians with experience in working on a full time basis.
·         State health laboratories and hospital laboratories do not isolate and identify viruses in   clinical work.
·         Tissue or serum for analysis is sent to central laboratories to identify virus.

Detection of virus growth in cell culture

·         Cytopathic effects – Many viruses causes morphological changes in cultured cells in which they grow. These changes can be observed microscopically and are known as cytopathic effects.
·         The  cytopathic effect are characteristic for a particular group of viruses and helps in the presumptive identification of viruses.
·         Example-entroviruses causes orientation of cells.
·        Metabollic inhibition – In normal cell cultures the medium turns acidic due to cellular metabolism. Growth of viruses inhibits metabolism and hence no acidic production. This can be made out by the colour of the indicator (phenol red).
·        Haemadsorption  - When Haemogglutinating viruses (influenza and Para influenza) grow in cell culture. They adsorb Guinea pig RBCs onto the surface of cell cultures.

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