SN1 SHORT NOTE 5M ★ High-yield

Describe the methods of cultivation of viruses and the detection of viral growth.

Microbiology · 2nd Professional — Para-Clinical

Systems for Cultivating Viruses

Viruses are obligate intracellular parasites and cannot be grown on any inanimate medium. Three systems are used: cell (tissue) culture, the embryonated egg, and laboratory animals.

Type of cell culture	Nature	Uses and limitations
Primary cell culture	Taken directly from tissue; normal diploid chromosomes; survives only a few subcultures — monkey kidney, human amnion, chick embryo fibroblast	The most sensitive for isolating virus from clinical specimens, and used for vaccine production; but expensive, variable and may carry latent viruses
Diploid (semi-continuous) cell strain	Diploid, survives about 50 subcultures — human embryonic lung (WI-38, MRC-5)	Free of latent virus and safe for vaccine production
Continuous (established) cell line	Heteroploid, malignant, immortal — HeLa, HEp-2, Vero, KB, McCoy	Convenient, indefinitely propagated and easy to maintain; but their malignant nature makes them unsuitable for producing vaccines for human use

Detection of Viral Growth in Cell Culture

Cytopathic effect (CPE) — visible degeneration of the monolayer: rounding and clumping, syncytium (giant cell) formation in respiratory syncytial virus, measles and herpes; inclusion bodies; and detachment. The pattern and timing suggest the virus
Haemadsorption — infected cells expressing a haemagglutinin on their surface bind added red cells; this detects influenza and parainfluenza viruses before any cytopathic effect appears
Haemagglutination of red cells by virus in the culture fluid
Interference — a virus producing no cytopathic effect (rubella) is detected because it blocks the growth of a second challenge virus that normally does
Immunofluorescence, ELISA and PCR on the culture, which detect growth earlier and identify the virus specifically
Plaque assay — areas of dead cells under an agar overlay, allowing the virus to be quantified as plaque-forming units

CLINICAL PEARL

Continuous cell lines are convenient precisely because they are malignant, which is why they cannot be used for vaccines. HeLa and similar lines divide indefinitely because they are cancer cells, which makes them ideal for a diagnostic laboratory. But material grown in them could carry oncogenic nucleic acid into a recipient, so vaccine production uses primary or diploid cells instead — a case where the property that makes a tool useful in one setting disqualifies it in another.

Embryonated Egg and Animal Inoculation

Route in the embryonated egg	Virus grown
Chorioallantoic membrane	Poxviruses (producing visible POCKS, which were used to distinguish variola from vaccinia and herpes) and herpes simplex
Allantoic cavity	Influenza (the basis of influenza vaccine production), mumps — giving a high yield
Amniotic cavity	Primary isolation of influenza from clinical specimens
Yolk sac	Chlamydiae, rickettsiae and some viruses

Animal inoculation — the oldest method, now largely superseded but still used where no other system works: suckling mice for coxsackieviruses and arboviruses (inoculated intracerebrally); mice for rabies; the mouse footpad for Mycobacterium leprae; primates for hepatitis
Uses — primary isolation, study of pathogenesis, vaccine production and testing, and antibody production
Limitations — cost, ethical constraints, animal facilities, and the fact that many human viruses do not infect animals at all

Applied Aspects

Cell culture is no longer routine in most laboratories, having been displaced by PCR and antigen detection, which are faster, cheaper and safer; it is retained in reference laboratories for isolation, vaccine work and antiviral susceptibility testing
Egg-based influenza vaccine production explains two practical points — the several months required to produce a new vaccine each season, and the caution formerly advised in egg allergy
Vero cells are used for rabies vaccine, which replaced the older nerve-tissue vaccines that caused neuroparalytic complications; this is an important improvement in Indian practice
Culture remains the only method that proves the virus is viable, which PCR cannot do; this matters when deciding whether a person is still infectious
Handle cultures at the appropriate biosafety level, since propagating a virus greatly increases its concentration and therefore the hazard
Some important viruses have never been cultured conventionally — hepatitis B, HPV and norovirus among them — which is why their study depended on molecular methods and why vaccines against them are recombinant

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