Methods for Testing Vectors for
Replication Competent Virus (RCV)

Most viral vectors used today are disabled such that replication competent viruses are not readily formed by any biological process that might occur in normal hosts. Biosafety encourages the use of such vectors in all relevant applications. In particularly sensitive applications, demonstrating that the viral stock used has no apparent contamination with replication competent vectors is essential for biosafety. Of course, assays for replication competence will never be perfect or absolute, so the Institutional Biosafety Committee (IBC) asks that one use a current procedure of demonstrated sensitivity and specificity. Below is a summary guide of the current IBC recommendations for common classes of vectors. If another procedure or reference method is used to accomplish the same conclusion, researchers are asked to submit that procedure and published article with their IBC registration.

In general, the IBC will require use of such an assay whenever viruses or virus-infected cells are used in whole animals. Even more rigorous testing may be required in some instances, such as a vector bearing a pathogenic gene or in human gene therapy, or in any materials that could be released in the environment. Viral testing is not generally required if experiments are conducted entirely in tissue culture.

Virus	Method	Reference
Adenovirus	Test for RCV by PCR for E1a prior to use. Confirmation of absence of RCV must be documented by researcher prior to use in animals.	Reference 1 1
Adeno-associated virus (with adenovirus helper)	Test for RCV by PCR prior to use. Confirmation of absence of RCV must be documented by researcher prior to use in animals.	Reference 2
Adeno-associated virus (Adenovirus-free)	Testing not required
Lentivirus	Test for RCV by ELISA assay for p24 antigen in the media of a cell culture inoculated with viral stock solution. Confirmation of absence of RCV must be documented by researcher prior to use in animals. RCV testing is not required for third-generation, commercially available Lentiviral vector systems.	Reference 3
Retrovirus (ecotrophic and amphotrophic)	Test for RCV by amplification in a permissive cell line followed by screening by appropriate detection assay. Confirmation of absence of RCV must be documented by researcher prior to use in animals.	Reference 4 1 Reference 5 1
Vaccinia	Not applicable due to use as a replicating vector.