Principles
of Molecular Virology CELL CULTURE METHODS
Cell
culture began early in the twentieth century with whole-organ cultures, then
progressed to methods involving individual cells, either primary cell cultures
(somatic cells from an experimental animal or taken from a human patient which
can be maintained for a short period in culture) or immortalized cell lines,
which, given appropriate conditions, continue to grow in culture indefinitely.
In
1949, John Enders and his colleagues were able to propagate poliovirus in
primary human cell cultures.This achievement ushered in what many regard as the
‘Golden Age of Virology’ and led to the identification and isolation
during the 1950s and 1960s of many viruses and their association with human
diseases—for example, many enteroviruses and
respiratory viruses, such as adenoviruses. Widespread virus isolation led to
the realization that subclinical virus infections were very common; for
example, even in epidemics of the most virulent strains of poliovirus there are
approximately 100 subclinical infections for each paralytic case of
poliomyelitis.
Renato
Dulbecco in 1952 was the first to quantify accurately animal viruses using a
plaque assay. In this technique, dilutions of the virus are used to infect a
cultured cell monolayer, which is then covered with soft agar to restrict
diffusion of the virus, resulting in localized cell killing and the appearance
of plaques after the monolayer is stained (Figure 1.1). Counting the number of
plaques directly determines the number of infectious virus particles applied to
the plate. The same technique can also be used biologically to clone a virus
(i.e., isolate a pure form from a mixture of types). This technique had been in
use for some time to quantify the number of infectious virus particles in
bacteriophage suspensions applied to confluent ‘lawns’ of bacterial cells on agar plates,
but its application to viruses of eukaryotes enabled rapid advances in the
study of virus replication to be made. Plaque assays largely replaced earlier
endpoint dilution techniques, such as the tissue culture infectious dose
(TCID50) assay, which are statistical means of measuring virus populations in
culture; however, endpoint techniques may still be used in certain
circumstances—for example, for viruses that do not
replicate in culture or are not cytopathic and do not produce plaques, (e.g.,
human immunodeficiency virus).
Figure 1.1 Plaque assays are performed by applying a
suitable dilution of a virus preparation to a confluent or semiconfluent
adherent monolayer of susceptible cells. After allowing time for virus
attachment to and infection of the cells, liquid medium is replaced by a
semisolid culture medium containing a polymer such as agarose or carboxymethyl
cellulose, which restricts diffusion of virus particles from infected cells.
Only direct cell-to-cell spread can occur, resulting in localized destruction of
the monolayer. After a suitable period, the medium is usually removed and the
cells stained to make the holes in the monolayer (plaques) more easily visible.
Each plaque therefore results from infection by a single plaque-forming unit
(p.f.u.).
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