A gammaretrovirus, called the xenotropic murine leukemia virus (MLV)-related virus (XMRV), has been reported in persons with prostate cancer (PC), chronic fatigue syndrome (CFS), and blood donors with PCR prevalences up to 67% 
. A related MLV was also reported in about 86% of CFS persons in a separate study 
. The finding of XMRV and MLV in humans is controversial as subsequent studies have failed to confirm the initial reports 
. Recent data showing that XMRV was generated in the laboratory during the passage of a human prostate cancer xenograft in nude mice during the generation of the XMRV-infected prostate cancer cell line 22Rv1 raises further doubts about the association of XMRV with human disease 
. The origin of MLV in humans has also been questioned by the finding of reagents, human cell lines and specimens that are contaminated with MLV sequences 
. However, additional studies aimed at defining the prevalence of XMRV and related viruses in humans and their association with diseases using an array of diagnostic tests are currently in progress 
MLVs are endogenous gammaretroviruses that constitute about 8–10% of the mouse genome and can cause leukemia, lymphoma, and neurological disorders in mice 
. XMRV shares about 96% nucleotide identity with MLVs classified as xenotropic and which replicate only in non-mouse cells 
. Thus, while mice are the likely species origin of MLV-related viruses, exposures that may have led to possible cross-species infections may be diverse, ranging from natural exposure to mice to possible exposures of biologicals, such as vaccines 
. Mice and other rodents have been, or are currently used, in the production of vaccines. For example, the first live polio vaccine was grown in mice and tested on humans in 1950, smallpox, yellow fever, and rabies viruses were cultured in the brains of mice for vaccine production and several live, attenuated vaccines are produced on mammalian cell lines from mice, pigs, chickens, and cats 
. Thus, MLV may have been introduced into vaccines during attenuation of the master seed stock during successive passage in rodents or during vaccine production from contaminated reagents or growth in mouse or other rodent cell lines.
The Japanese encephalitis virus (JEV) vaccine (SA14-14-2, Rongsheng, China) is an example of a live vaccine that was attenuated via passage in rodents, including mouse brain, primary hamster kidney (PHK) cells, mouse spleen and skin, Syrian hamster (Mesocricetus auratus
) spleens, and suckling mice skin 
. Production of the SA-14-14-2 vaccine is done in the hamster PHK cell line. According to the manufacturer, the master seed virus of the SA-14-14-2 JEV vaccine and the PHK cell lines used for vaccine production were shown to be free of adventitious agents and pathogens. The SA-14-14-2 JEV vaccine has been used for over 20 years and administered to over 300 million children in China, South Korea, Nepal, and India.
Although the master seeds and cell substrates used for vaccine production are prescreened for adventitious agents, newer technologies, including sequence-independent amplification, followed by ultra-deep DNA sequencing, and microarrays have shown that some cell substrates and live-attenuated vaccines still contain adventitious viruses, including endogenous retroviruses like avian leukosis virus (ALV), and porcine circovirus 
. Previous studies have also documented the presence of endogenous avian retroviruses in currently used avian cell-derived vaccines 
. Endogenous retroviruses exist in all mammals as proviral DNA integrated in the germ line of the host and are passed from parent to offspring. Thus, endogenous retroviruses cannot be eliminated from cell lines or live animals by pre-screening. While most endogenous retroviruses are replication defective, some exist as intact genomes that are capable of expressing infectious virus.
We screened eight live attenuated human vaccines for XMRV and related MLV and adventitious agents using PCR and metagenomics. The eight vaccines included JEV (SA-14-14-2), varicella (Varivax), measles, mumps, and rubella (MMR-II), measles (Attenuvax), rubella (Meruvax-II), rotavirus (Rotateq and Rotarix), and yellow fever virus. All eight vaccines were negative for XMRV and closely related MLV sequences using these two approaches. We found novel hamster genomic and retrovirus sequences in the JEV vaccine mostly likely originating from vaccine production in Syrian hamster cells. Our findings do not support the hypothesis that vaccines are a possible source of XMRV or MLV introduction into humans and are consistent with accumulating evidence on the absence of these viruses in humans.