Virus propagation and culture

Most experiments in virology have required virus replication in cultured cells, although today many experiments rely entirely on the cloning of individual genes and the expression of proteins in cell cultures without full virus infection. Historically, those viruses that replicate well in cell culture have been studied in the greatest detail. Lack of in vitro propagation has seriously curtailed progress in research, vaccine production, and development of antiviral drugs for many viruses.

Viruses are amplified within cultures to create virus stocks that are then usually stored at -70^.C, although some viruses remain stable at -20^.C or even 4^.C. It is important to record when multiple virus stocks are made from each other and to exercise caution when continuously propagating new virus stocks. The high number of genomic generations achieved during each amplification means that mutations can accumulate rapidly, potentially altering the virus over subsequent generations. It is preferable to return to an earlier stock of the virus for the production of new stocks, rather than using the next generation each time.  Minimizing the level of mutation during virus stock preparation can be achieved by infecting only a low proportion (1–10%) of the cells in the culture. This is known as using a low multiplicity of infection (m.o.i.) whereby virus is added to a level equivalent to only 0.01–0.1 infectious virions per cell.

The virions infect a low number of cells, leaving the remainder to continue mitosis for 24–48 hours. Following replication in the subset of infected cells, newly formed virions infect the remaining cells, resulting in significant amplification of the virus. Once replication has been achieved in the majority of cells, virus is harvested in one of two ways. Extracellular virus that has been released from cells by budding or exocytosis can be harvested directly from the culture medium. Intracellular virus that remains cell-associated must be released by cell lysis either by repeated freezing and thawing cycles or by using an ultrasonic bath. Infected cells yield various numbers of new (progeny) virus particles, ranging from 10 to 10 000 particles per cell, and this is usually quantified accurately by an infectivity assay.