Virus Cultivation Methods
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Direct answer
Virus cultivation methods are the laboratory systems used to grow viruses, namely cell cultures, embryonated hen's eggs and experimental animals. Cell culture is the commonest route, and viral growth is recognised by cytopathic effect, haemadsorption or plaque formation. Viruses that resist cell lines may still need eggs or suckling mice, and cytopathic effect alone never identifies the virus.
What you must remember
- Primary cell culture: Cells taken straight from tissue, such as monkey kidney cells, have a finite lifespan and must be prepared afresh for each use.
- Continuous cell line: Immortal lines such as HeLa and Vero cells are easier to maintain and are standard in diagnostic and research laboratories.
- Cytopathic effect: Cell rounding, syncytium formation, inclusion bodies and lysis indicate that a virus has replicated, but many viruses produce similar changes.
- Haemadsorption: Red cells adsorb to infected cells expressing viral haemagglutinin, a test used for influenza and parainfluenza viruses.
- Embryonated egg routes: Influenza is isolated in the amniotic cavity and then passaged in the allantoic cavity, pox viruses form pock lesions on the chorioallantoic membrane, and the yolk sac suits some other viruses.
- Suckling mice: These animals are highly susceptible to coxsackieviruses, so inoculating clinical material allowed isolation before cell culture was standard.
- Plaque assay: Agar overlay on a monolayer produces discrete plaques, each arising from one infectious particle, and the count gives titre in plaque-forming units.
A stepwise approach to cultivating an unknown virus
Cultivation is a sequence of decisions, and each step narrows what the virus might be. Work through the steps in order, because a missed step can waste weeks of effort.
- Collect the right specimen: Use vesicle fluid, throat swab or stool in viral transport medium, kept cold, since viability falls quickly at room temperature.
- Remove bacteria: Filtration or antibiotics clear bacterial contamination, so that only the virus is inoculated onto the cells or host.
- Choose the first host: Start with the cell line most likely to support the suspected virus, such as HEp-2 cells for respiratory viruses or Vero cells for poliovirus.
- Watch for cytopathic effect: Examine the monolayer daily under the inverted microscope and note the time taken for changes to appear.
- Confirm the identity: Use haemadsorption, immunofluorescence or nucleic acid detection, because cytopathic effect alone is not specific.
- Escalate to eggs or animals: If cell culture fails, inoculate embryonated eggs or suckling mice, which remain useful for viruses that grow poorly in cell lines.
Modern diagnostic laboratories detect most viruses directly by PCR, so cultivation now serves mainly to isolate strains for vaccine production and research. Its theoretical value in exams is still high, because the methods explain how viruses were first identified.
Where the exam tries to catch you
Cultivation questions reward precise matching of host system to virus, and most wrong answers pair a virus with the wrong host.
- Trap: Taking cytopathic effect as proof of one particular virus Correct: Many viruses cause similar cell changes, so identification needs immunofluorescence, haemadsorption or nucleic acid detection.
- Trap: Using the allantoic cavity to recognise pox viruses Correct: Pox viruses are identified on the chorioallantoic membrane through pock lesions, whereas the allantoic cavity suits influenza.
- Trap: Assuming every vaccine virus grows in cell culture Correct: The yellow fever 17D vaccine seed is grown in embryonated eggs, and rabies vaccine was historically made from animal nerve tissue.
Frequently asked questions
Which viruses are grown in the allantoic cavity of embryonated eggs?
Influenza viruses are the classic examples grown in the allantoic cavity of embryonated hen's eggs. This route has long been the traditional method for producing influenza vaccine.
Why are pox viruses detected on the chorioallantoic membrane?
Pox viruses such as variola and vaccinia produce visible pock lesions on the chorioallantoic membrane of embryonated eggs. This pock pattern helped laboratories identify poxviruses before molecular methods became available.
What does haemadsorption show in virus cultivation?
Haemadsorption shows that infected cells express viral haemagglutinin on their surface, so red blood cells stick to the monolayer. It is a classic test for influenza and parainfluenza viruses and is read directly from the culture.
How does a plaque assay measure a virus?
A plaque assay overlays a cell monolayer with agar so that each infectious particle forms one discrete area of cell death. Counting the plaques gives the titre as plaque-forming units per millilitre.
Why are suckling mice used to isolate coxsackieviruses?
Suckling mice are highly susceptible to coxsackieviruses and develop illness or death after inoculation, which allows the virus to be isolated. Coxsackie A and B viruses were classically identified this way before cell culture became standard.