Growth and Assay of Viruses - Viral Multiplication

GROWTH AND ASSAY OF VIRUSES

Viruses are generally propagated in the laboratory by mixing the virus and susceptible cells together and incubating the infected cells until lysis occurs. After lysis, the cells and cell debris are removed by a brief centrifugation and the resulting supernatant is called a lysate.

     The growth of animal viruses requires that the host cells be cultivated in the labora-tory. To prepare cells for growth in vitro, a tissue is removed from an animal and the cells are disaggregated using the proteolytic enzyme trypsin. The cell suspension is seeded into a plastic petri dish in a medium containing a complex mixture of amino acids, vitamins, minerals, and sugars. In addition to these nutritional factors, the growth of animal cells requires components present in animal serum. This method of growing cells is referred to as tissue culture, and the initial cell population is called a primary culture. The cells at-tach to the bottom of the plastic dish and remain attached as they divide and eventually cover the surface of the dish. When the culture becomes crowded, the cells generally cease dividing and enter a resting state. Propagation can be continued by removing the cells from the primary culture plate using trypsin and reseeding a new plate.

Cells taken from a normal (as opposed to cancerous) tissue cannot usually be propa-gated in this manner indefinitely. Eventually most of the cells die; a few may survive, and these survivors often develop into a permanent cell line. Such cell lines are very useful as host cells for isolating and assaying viruses in the laboratory, but they rarely bear much resemblance to the tissue from which they originated. When cells are taken from a tumor and cultivated in vitro, they display a very different set of growth properties, including long-term survival, reflecting their tumor phenotype .

When a virus is propagated in tissue culture cells, the cellular changes induced by the virus, which usually culminate in cell death, are often characteristic of a particular virus and are referred to as the cytopathic effect of the virus .

     Viruses are quantitated by a method called the plaque assay (see Plaque Assay under Quantitation of Viruses for a detailed description of the method). Briefly, viruses are mixed with cells on a petri plate such that each infectious particle gives rise to a zone of lysed or dead cells called a plaque. From the number of plaques on the plate, the titer of infectious particles in the lysate is calculated. Virus titers are expressed as the number of plaque-forming units per milliliter (pfu/mL).