Retrovirus Assays
The majority of cells contain endogenous retrovirus or retrovirus-like sequences, and some cells (e.g., murine cells) may produce infectious retrovirus. Regulatory guidelines generally require retrovirus testing by infectivity assay and electron microscopy, on master cell banks and cells cultured up to or beyond the limit of in vitro cell age, as well as unprocessed bulk material (ICH Q5A, FDA PTC 1993, 1997).

Infectivity assays performed will depend on the anticipated retrovirus contaminant, but may involve, for example, the use of XC assays to detect infectious ecotropic MLV, S+L- assays using feline or mink cell lines, to detect amphotropic or xenotropic MLV, or Mus dunni cells, with either immunofluorescence end-point or a PG4 cell end-point, for detection of ecotropic, amphotropic, xenotropic and mink cell focus-forming viruses.

For non-murine retroviruses, infectivity testing on appropriate indicator cells (selected for their susceptibility to different retrovirus types) is recommended. Such testing strategies would be determined case-by-case and should be discussed with Vitrology and regulatory authorities before implementation. Often, infectivity tests may be augmented with sensitive PERT assays. Under some circumstances, for example when tumorigenic cell substrates are proposed for use, it could be appropriate to pre-treat cells with chemical agents known to induce reactivation or replication of endogenous or latent viruses. Subsequent co-cultivation or transmissibility assays with cells susceptible to a wide range of retroviruses, combined with a relevant detection system (e.g., PERT assay and electron microscopy) may be used to confirm the absence of detectable virus, or to amplify viruses with a specific host cell tropism (e.g., cocultivation with HEK 293 cells, to detect retroviruses capable of infecting human cells).

Validated retrovirus infectivity assays for the detection of ecotropic, amphotropic, xenotropic and mink-cell focus forming viruses will be released early in 2008. Co-cultivation assays to amplify viruses with a tropism for human cells will also become available over 2008, and may be available on request.

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