Unintended spread of a biosafety level 2 recombinant retrovirus

Abstract

BackgroundContamination of vertebrate cell lines with animal retroviruses has been documented repeatedly before. Although such viral contaminants can be easily identified with high sensitivity by PCR, it is impossible to screen for all potential contaminants. Therefore, we explored two novel methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant.ResultsThe first hint for the presence of contaminating retroviruses in one of our cell lines was obtained by electron microscopy of exosome-like vesicles released from the supernatants of transfected 293T cells. Random amplification of particle associated RNAs (PAN-PCR) from supernatant of contaminated 293T cells and sequencing of the amplicons revealed several nucleotide sequences showing highest similarity to either murine leukemia virus (MuLV) or squirrel monkey retrovirus (SMRV). Subsequent mass spectrometry analysis confirmed our findings, since we could identify several peptide sequences originating from monkey and murine retroviral proteins. Quantitative PCRs were established for both viruses to test currently cultured cell lines as well as liquid nitrogen frozen cell stocks. Gene fragments for both viruses could be detected in a broad range of permissive cell lines from multiple species. Furthermore, experimental infections of cells negative for these viruses showed that both viruses replicate rapidly to high loads. We decided to further analyze the genomic sequence of the MuLV-like contaminant virus. Surprisingly it was neither identical to MuLV nor to the novel xenotropic MuLV related retrovirus (XMRV) but showed 99% identity to a synthetic retrovirus which was engineered in the 1980s.ConclusionThe high degree of nucleotide identity suggests unintended spread of a biosafety level 2 recombinant virus, which could also affect the risk assessment of gene-modified organisms released from contaminated cell cultures. The study further indicates that both mass spectrometry and PAN-PCR are powerful methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant. Both methods might be useful tools for testing cell lines before using them for critical purposes.

The human retrovirus XMRV in prostate cancer and chronic fatigue syndrome

“…With regard to laboratory events, common cell lines have been shown to harbor XMRV and other retroviruses. 31,73 Human candidates for xenotransplantation of porcine tissue and organs might benefit from testing for XMRV infection to avoid possible recombination with porcine endogenous retroviruses. 74 Conclusions XMRV is the first human retrovirus to be discovered in many years and has generated considerable interest among both researchers and patients.…”

Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts

“…While we did not report on the viral findings, others have done so 14,15 and we refer to this XMLV strain as N417 although it has also been referred to as VB3.2. 21 Recent reports of squirrel monkey retroviruses (SMRV) infecting human cultures (by unknown routes or sources) and their possible large scale horizontal spread have also been published, 35,36 and several human cell lines for human immunodeficiency virus research were found to release infectious XMLV. 22,37 However, there are virtually no reports of contamination of xenograft cultures published during the past 15 y and most scientists appear unaware of the contamination problem (authors' observations).…”

From where did the 2009 'swine-origin' influenza A virus (H1N1) emerge?

“…Gene sequences of the 1977 isolate and others collected in the 1950s were almost identical, indicating that the virus had not replicated and evolved in the interim, and had probably been held in a laboratory freezer between 1950 and 1977 and 'escaped' during passaging. The suggestion that persistently infected cells might be involved is also not outlandish; influenza virus can persistently and latently infect MDCK cells [27], and viruses do travel between laboratories in cells [28]. …”