CULTIVATION OF VIRUSES
⇒ Viruses are the obligate intracellular parasites i.e. they strictly require a living host or living cells for the growth and multiplication so they cannot be grown on an inanimate culture medium.
⇒ Three methods are employed for the cultivation of viruses:-
- Animal inoculation
- Embryonated egg inoculation
- Tissue culture
- This is the earliest method for the cultivation of viruses & mice are the most widely used animals in virology.
- For e.g. – Infant (suckling) mice are used in the isolation of arbovirus and coxsackieviruses, which don’t grow in any other system.
- Mice may be inoculated by several routes: intracerebral, subcutaneous, intraperitoneal or intranasal.
- Other animals such as Guinea pig, Rabbit & ferrets are used in some situations.
- The growth of virus in inoculated animals may be indicated by Death, Disease or visible Lesions.
- Later on, the test organism is sacrificed and tissues are tested for the presence of the virus.
EMBRYONATED EGG INOCULATION
⇒ Embryonated hen’s eggs (7-12 days old) are used for the cultivation of viruses in the laboratory.
⇒ It offers several sites for the cultivation of viruses as – Chorioallantoic membrane (CAM), Allantoic cavity, Amniotic Sac & Yolk Sac.
- Chorioallantoic membrane (CAM) is inoculated for growing poxviruses. It produces visible lesions called as pocks. Pocks produced by different viruses have different morphology.
- Allantoic cavity inoculation is employed for growing influenza virus for vaccine production.
- Amniotic sac inoculation is mainly used for the primary isolation of influenza virus.
- Yolk sac inoculation is used for the cultivation of some viruses and some bacteria (Chlamydia & rickettsiae).
⇒ After inoculations, eggs are incubated for 2-9 days.
⇒ Tissue culture is the most common method employed for the propagation of viruses in the laboratory.
⇒ Tissue culture & cell culture are the terms used interchangeably for this method.
⇒ Three types of tissue cultures are available:-
- Organ culture
- Explants culture
- Cell culture
A.) ORGAN CULTURE
- In this, small bits of organs are maintained in tissue culture growth medium in vitro (in labs) for days & weeks.
- Organ cultures are useful for the isolation of some viruses which appear to be highly specialized parasites of certain organs.
- For e.g. – trachea ring culture for the isolation of coronavirus.
B.) EXPLANT CULTURE
- In this, fragments of minced tissue can be grown as explants. It was originally known as tissue culture.
- For e.g. – Adenoid tissue explants cultures were used for the isolation of adenoviruses.
- This method is rarely employed nowadays.
C.) CELL CULTURE
- This is the type of culture technique that is routinely used for the cultivation of viruses in the laboratory.
- In this, the tissues are dissociated into component cells by the action of proteolytic enzymes e.g. trypsin or collagenase.
- Based on their origin, chromosomal characters and no. of cell divisions, they are classified into three types as:-
- Primary cell culture
- Diploid cell strains
- Continuous cell lines
1.) Primary cell culture
- In this, normal cells are freshly taken from the body & cultured.
- They are capable of very limited growth in culture (5-10 divisions at most)
- Primary cell cultures are useful for the isolation of viruses and their cultivation for vaccine production.
- For e.g. – monkey kidney, the human kidney, human amnion, chick embryo.
2.) Diploid cell strain
- These are the cells of the single type that retains the original diploid chromosome number during serial subcultivation for a limited number of times.
- After about 50 serial subcultures they undergo ‘senescence’ and the cell strain is lost.
- For e.g. – WI-38 human embryonic lung cell strain
3.) Continuous cell line
- These are the cells of the single type, usually derived from cancer cells that are capable of indefinite growth in-vitro.
- Some cell lines are also being used for vaccine manufacture e.g. Vero cell for rabies vaccine.