Unintended spread of a biosafety level 2 recombinant retrovirus ( MuLV-like)

[Emily]

http://www.retrovirology.com/content/6/1/86
Retrovirology 2009, 6:86doi:10.1186/1742-4690-6-86
Unintended spread of a biosafety level 2 recombinant retrovirus

Published: 22 September 2009

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.
[snip]
The present report extents these studies by identifying for the first time a presumably synthetic chimeric retrovirus as a contaminant. This gene-modified organism seems to have replicated and spread intensely in a broad set of cell lines for several years without being noticed. This hybrid amphotropic/Moloney murine leukemia virus was engineered in the 1980s [7,8] and neither the virus itself nor the plasmid (pAMS) containing its proviral genome were ever used in our laboratory. Although the precise source for the contamination could not be traced back, sharing cell lines with other laboratories seems the most likely explanation. A frozen aliquot of 293T cells (HEK 293tsA201), which we obtained from ECACC, was not contaminated. While SMRV contaminations were detected in different laboratories, testing of three other laboratories? cell lines did not reveal contaminations with the hybrid amphotropic/Moloney murine leukemia virus ....

 

[AlaskaDenise]

Re: Unintended spread of a biosafety level 2 recombinant retrovirus ( MuLV-like)

Comments about article, as linked on original page:
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How did these retroviruses spread so widely?

A Dusty Miller (29 September 2009) Fred Hutchinson Cancer Research Center

Speaking as an investigator who does much work with retroviruses, and as the creator of the pAMS hybrid retrovirus mentioned in this report as having spread promiscuously in multiple cell lines, I find it incredible to see such widespread contamination by retroviruses in the authors' laboratories. Provided the PCR data in Table 3 are correct and do not represent amplification of endogenous retroviral sequences, and because I work with many of the listed cells lines and know them to be free of replication-competent retroviruses, I conclude that most of the spread has occurred in the authors' laboratories. I also know that it is relatively easy to prevent spread of retroviruses in cultured cells by using good cell culture techniques. Presumably one or a few infected cell lines were imported into the lab and provided the source of the subsequent widespread contamination.

First, I would ask whether many of the authors' cell lines are also contaminated with mycoplasma. This would be another sign of poor cell culture technique where organisms secreted by one cell type are transferred to another, probably by transfer of culture medium containing the organisms. This can occur by using the same pipette to feed multiple cell types, or by feeding a contaminated cell type and then using that pipette to get more medium from a bottle that is later used to feed other cells.

Beyond the use of poor cell culture practices, the other worry is transfer of viruses during storage of cells in liquid nitrogen. Liquid nitrogen can enter and leave standard freezing vials, and I have always wondered if it would be possible that frozen virus might be transmitted between cell lines by this route, although I have never detected this. Storage of cells in the vapor phase above the liquid nitrogen should prevent this provided the liquid nitrogen level never rises above the stored vials. Which type of frozen cell storage was used in this case? In my lab we usually freeze virus-producing cells in sealed glass vials to prevent any possibility of virus transmission, especially when dealing with pathogenic retroviruses. I believe many of the central cell line repositories store cell lines in glass vials as well.

I'd be interested in the authors' thoughts.



Response to “How did these retroviruses spread so widely?”

Thomas Grunwald (07 October 2009) Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany

As mentioned in our paper “Unintended spread of a biosafety level 2 recombinant retrovirus” contamination of cell lines with replication competent retroviruses has been repeatedly observed, even in laboratories of experienced retrovirologists. Systematic investigations on the frequency of such contaminations are, to the best of our knowledge, missing. In response to a recommendation of the German Central Commission for Biosafety (ZKBS), that laboratories working with gene-modified organisms should test their cell lines for the presence of squirrel monkey retrovirus (SMRV), 128 of 4279 tested aliquots of cell lines were reported to be positive. Approximately 70 different cell lines from at least 13 different laboratories throughout Germany were positive for SMRV. Since not all aliquots of the same cell line were positive, this indicates that contaminations do occur under commonly employed cell culture techniques.

We agree with Dr. Miller´s assumption, that we have imported one or a few infected cell lines and that the contaminants subsequently spread in our laboratory. However, we reject his insinuation that this has been due to poor cell culture techniques. We are using sterile, disposable tissue culture plastic ware and receive media, fetal calf serum and all other cell culture solutions from common suppliers. Cell cultures are regularly checked for mycoplasma and in the rare case of positivity the cell lines are either disposed off or treated with antibiotics until mycoplasma can no longer be detected. All lab members working with tissue cultures are of course trained not to use the same pipette to split different cell lines or to re-fill a pipette that has been previously used to feed cell lines. At least since 2001, all cell lines are stored in the vapor phase above the liquid nitrogen in plastic cryo tubes with a screw cap. Virus stocks are stored at – 80°C, and not as virus infected cultures in liquid nitrogen tanks.

Thus, it remains unclear, as in most previous reports on retroviral contaminants, how these contaminants spread. Formation of aerosols during the splitting of a contaminated culture and subsequent contamination of cultures splitted in the same tissue culture hood via aerosols should also be considered. Even if this is a rare event, the virus might accumulate over time in several cell lines, since different cell lines are maintained simultaneously in the same laboratory for years, particularly if a contamination is not suspected due to lack of cytopathic effects.

While one can only speculate, how the retroviral contaminants spread so widely, we do know that by regular testing for the contaminants and systematic elimination of all contaminated cultures we were able to maintain our cell cultures free of SMRV and the hybrid retrovirus without other major changes in cell culture techniques and storage.

http://www.retrovirology.com/content/6/1/86/comments

 

[tropical]

Re: Unintended spread of a biosafety level 2 recombinant retrovirus ( MuLV-like)

#1:
" 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.
[snip]
The present report extents these studies by identifying for the first time a presumably synthetic chimeric retrovirus as a contaminant. This hybrid amphotropic/Moloney murine leukemia virus was engineered in the 1980s [7,8] and neither the virus itself nor the plasmid (pAMS) containing its proviral genome were ever used in our laboratory. Although the precise source for the contamination could not be traced back, sharing cell lines with other laboratories seems the most likely explanation." This gene-modified organism seems to have replicated and spread intensely in a broad set of cell lines for several years without being noticed.



Who said it is not artifitial ...


Thus, must we mantain further confidence in cell-lines ...

Those "shared" cell lines must be sterilized 100% prior further "business",
or there is a lack off techno possibility to do that ...