Introduction:
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism, having the capacity to infect all types of life forms, from animals and plants to microorganisms.
It has the smallest size in the microorganisms 20-300nm.
They lack essential metabolic machinery to synthesize their own essential proteins hence has to depend on the hosts.
Viruses hence are called obligate intracellular parasites.
Cultivation of Viruses:
Viruses being obligate intracellular parasites can not be grown on inanimate culture media, they strictly require living cells for their cultivation.
Following are the methods used commonly for cultivation of viruses,
Laboratory Animals.
Embryonated eggs.
Tissue Culture.
Laboratory Animals:
One of the oldest methods used for cultivation of the viruses in the laboratories.
Reed and his colleagues (1900) used human volunteers for their work on Yellow Fever.
Due to serious risks involved nowadays humans are not used and are only used when there is no alternative present.
Landsteiner and Popper (1909) used monkeys for isolation of Poliovirus.
The use of monkeys as a test animal in virology is now limited due to,
Their Cost.
Handling Problems.
Theiler in 1903 initiated use of white mice, the animal is highly susceptible against certain viruses.
Their cost, easy handling made them a popular laboratory test animal.
Mice is the most widely used experimental animal in virology.
Other animals which are used as experimental animals in virology are, Rabbits, Guinea pigs, Ferrets etc.
Animal inoculation is used for study of pathogenesis, immune response of the host and epidemiology.
Embryonated Eggs:
Goodpasture in 1931 first used “Embryonated hens Eggs” in cultivation of viruses.
Burnet is credited for developing the process.
The embryonated hen eggs has many sites for inoculation of the viruses.
Fertile eggs are incubated for 5 to 12 days and can be inoculated through the shell aseptically.
The opening made for inoculation can be sealed with paraffin wax and eggs are again incubated for desired virus growth period @ 36°C.
The inoculation of viruses in the eggs may cause death of the embryo or produce some symptoms like scars on chorioallantoic membrane or haemagglutinating action in allantoic or amniotic fluid.
Common Viruses and their inoculation route:
Advantages of Embryonated Eggs:
Simple handling.
Economical.
Easily available.
Clean, bacteriologically sterile.
Don't need food and cages to store.
Lacks immune response.
Offers several inoculation sites.
Disadvantages of Embryonated Eggs:
Some viruses don't show growth on primary inoculation.
Slight bacterial contamination results in death of the embryo.
Some eggs may be contaminated with “Mycoplasma” and some latent viruses like “Fowl Viruses”, which may interfere with the growth of the virus under study.
3) Tissue Culture:
It is a most important method for cultivation of viruses.
Following are the main three types of the tissue culture.
Organ Culture: For organ specific viruses like coronavirus.
Explant Culture: Not useful in virology.
Cell Culture: Very popular and routinely used technique.
Cell Culture:
Component cells are separated from tissue with help of proteolytic enzymes like trypsin and mechanical shaking.
Washed, counted and then suspended in a growth medium and then transferred to a suitable glass container.
In glass containers they stick to the glass walls amd grow out to form a monolayer sheet.
On basis of their characteristics they are divided into following types,
Primary Cell Cultures.
Diploid Cell Strains.
Continuous Cell Line.
Primary Cell Culture:
These are normal cells taken fresh from the body and cultured.
Capable of only limited growth in a culture and can not be maintained in a serial culture.
Useful for isolation of viruses and for vaccine production.
E.g. Rhesus monkey kidney cell culture.
Human embryonic kidney cell culture.
Diploid Cell Strains:
Derived from Primary cell cultures developed from a particular tissue.
Cells are from embryonic origin.
They are of single type and undergo 50 to 100 divisions before death.
They possess a normal diploid set of chromosomes.
Useful for isolation of fastidious pathogens and for production of vaccines.
E.g. Human embryonic lung cell stain (WI-38)
Rhesus embryo cell strain (HL-8)
Continuous Cell Line:
Single Type of cells derived from cancer cells.
Can be grown in successive generations by transferring from one test tube to another and hence called “Continuous Cell line”.
Maintained by serial subcultivation or storing at -70°C.
E.g. Human carcinoma of nasopharynx line (KB)
Human epithelioma of larynx cell line (Hep-2)
Virus growth in the cell cultures can be detected by “Cytopathic Effect”, metabolic inhibition, transformation, hemadsorption, immuno-fluorescence etc.