| XMRV is a gammaretrovirus initially reported in prostate cancer and chronic fatigue syndrome human patient samples. Recent data has provided strong evidence that the virus evolved through rare retroviral recombination events in human tumor cell lines established through murine xenograft experiments, and that its human association was due to laboratory contamination of patient samples. Although XMRV is most likely not present in the general population, it has the potential to infect humans. The subject of this thesis was to characterize XMRV viral replication in rats, in vitro and in vivo, as a basis for an animal model.;The permissibility of rat cells to XMRV infection was tested in vitro by infection of NRK rat cells with XMRV (obtained from the molecular clone Vp62). NRK cells support productive infection of XMRV, characterized by cellular changes that resulted in the production of viral progeny that seemed to replicate more efficiently in rat cells compared to transiently produced XMRV (Vp62). Genetic analysis of this viral replication further suggests that non-genetic mechanisms may be involved in the observed changes.;To determine the permissibility of rats to XMRV infection in vivo, NRK-grown XMRV was used to infect neonatal Sprague-Dawley rats. Using an XMRV specific nested-PCR targeting three proviral regions (5'LTR and 5' non-coding region, Env and 3`LTR), we assayed harvested rat tissues for presence of XMRV viral sequences at time points 4 weeks to 1 year post-infection. Although XMRV was not detected at early time points post-infection, we were able to detect one animal that had limited but prolonged replication in the spleen after 1 yr of infection, as shown by the presence of XMRV viral sequences from the primary tissue and virus isolation from the infected splenocytes by co-culturing with a permissive cell line.;We explored the viral dissemination and target potential of XMRV in rats by infection of a second cohort of Sprague-Dawley rats at higher input inocula with virus from human 22rv1 tumor cells. We identified XMRV infection in 4/11 animals both at early and late time points p.i. (3, 6, 26 and 27 wks. p.i.). The greatest viral dissemination was detected in an animal 27 week infection, based on the amplification of XMRV viral DNA from tissues including hematopoetic cells, prostate, and testis. Sequencing analysis of PCR-amplified proviral sequences revealed that the viral sequences in the 27wk animal were characterized with the introduction of a unique fixed mutation in the 5' non-coding leader gag region (nt. 375). The sequencing also confirmed that the PCR assay was detecting XMRV and not a contaminating mERV. Based on this data we conclude that the XMRV sequences detected here reflect in vivo infection in multiple rat tissues, but the level of infection is low. Although our data suggests that XMRV is not pathogenic in rats, its biohazard potential in humans needs to be further addressed. |
