Retrovirology. 2009 Sep 22;6:86.
Unintended spread of a biosafety level 2 recombinant retrovirus.
_Stang A_ (mip://04de0f28/pubmed?term="Stang%20A"[Author]) ,
_Petrasch-Parwez E_ (mip://04de0f28/pubmed?term="Petrasch-Parwez%20E"[Author]) , _Brandt
S_ (mip://04de0f28/pubmed?term="Brandt%20S"[Author]) , _Dermietzel R_
(mip://04de0f28/pubmed?term="Dermietzel%20R"[Author]) , _Meyer HE_
(mip://04de0f28/pubmed?term="Meyer%20HE"[Author]) , _Stühler K_
(mip://04de0f28/pubmed?term="Stühler%20K"[Author]) , _Liffers ST_
(mip://04de0f28/pubmed?term="Liffers%20ST"[Author]) , _Uberla K_
(mip://04de0f28/pubmed?term="Uberla%20K"[Author]) , _Grunwald T_ (mip://04de0f28/pubmed?term="Grunwald%20T"[Author]) .
Department of Molecular and Medical Virology, Ruhr-University Bochum,
D-44780 Bochum, Germany. alexander.stang@rub.de
Abstract
BACKGROUND: Contamination 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.
RESULTS: The 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.
CONCLUSION: The 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.
_http://www.ncbi.nlm.nih.gov/pubmed/19772602_
(http://www.ncbi.nlm.nih.gov/pubmed/19772602)
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