Murine leukaemia viruses (MLVs) are simple γ-retroviruses of mice that are endogenous to the mouse genome, with several being replication-competent. In 2006, a research group using virus arrays, identified a novel MLV recombinant, XMRV, in human prostate cancer (PCa) tissue and cell lines. The virus appeared to infect and replicate efficiently in human cells. Disease associations with PCa and chronic fatigue syndrome caused a worldwide concern over the presence of XMRV in the human blood supply and patient populations. The final conclusions settled in 2011, and XMRV was widely accepted as a result of contamination in patient samples while its rescue was proven directly due to the propagation of the human cancer cell lines in nude mice in vivo.

The re-emerged perception of the ability of MLVs to recombine and form novel recombinants that can infect human cells propelled the need of investigation in all cases where mouse and human cells are placed in contact for biotechnology applications in vitro, such as for the expansion of human epidermal primary stem cells prior human transplantation.

We first sought to screen a variety of mouse embryonic feeder (MEF) cells that are used in human stem cell culture, for the presence of replication-competent retroviruses. To this aim, we developed a tool case of molecular assays to detect MLVs and their products and then tested various MEFs with known endogenous virus activators. We identified one stably expressed ecotropic MLV retrovirus in a CF1-MEF cell line and characterised it with molecular and infectivity assays, as well as next generation sequencing (NGS). Following, we co-cultivated human corneal stem cells with the CF1-MEF cells to test the ability of the CF1-related virus to cause human cell infection and used integration splinkerette PCR and NGS to detect possible integration site sequences in the human stem cells.

In all, this study provides with a comprehensive up to date assessment over the risk of transmission of MLV viruses onto human stem cells for conditions that are comparable to the protocols used in human eye cell transplantation. We conclude that the infection of stem cells is confirmed possible by xenotropic MLVs as demonstrated for limbal eye stem cells and XMRV, but unlikely as expected for the ecotropic CF1 cell-derived virus; the possibility of recombination in mouse feeders however, cannot be excluded during prolonged co-cultivations.