Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
http://www.cyto.purdue.edu/hmarchiv/index.htm
Eric Massicotte
Responsable, Service de cytométrie en flux
Institut de Recherches Cliniques de Montréal (IRCM)
110 ave. des Pins Ouest
Montréal, Québec
Canada, H2W 1R7
Téléphone: (514) 987-5724
Hello everybody,
Here is a summary of the answers I got so far regarding the safety of sorting a cell line (COS) transduced with a retroviral vector derived from HIV and producing viral-like particles that aren't supposed to form the enveloppe.
The names of all respondents have been removed in case anyone wants to protect their privacy.
Responses:
1) Hi Eric Since all cells potentially contain pathogens, whether detected/characterized or not, I would think that BSL-2 level or higher should be required for all sorting of live human or human-derived cells. I would not sort these without containment of the instrument, or of me, especially given the nature of the original virus! I've seen several MoFlo's with customized containment apparatus'.
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2) I personally wouldn't sort anything connected to the HIV virus no matter what the investigator says. I think for this work a dedicated machine in a restricted area should be the minimum requirement. This is for your safety as well as others. Better safe than sorry.....
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3) I have worked with VLPs before. In brief, I created VLPs for the SARS virus but I never ran any on a flow cytometer. The principle of a VLP is that it is non-infectious, i.e. it contains only structural proteins from a virus or from more than one virus. An example of this for an HIV VLP would be to use the gag protein to create a 'scaffolding' and the env protein to be expressed on the surface. No genetic material would be present (otherwise to my understanding it would be termed a psuedovirion). More information on what the VLPs consist of, i.e. which proteins he is using, would be required to determine if the VLP would be infectious or not. As I said, if no genetic material is present, and only viral proteins are present, then the VLP would be non-infectious. As he is wanting to sort cells producing VLPS, then you would need to find out what DNA is present within the cell (i.e. genetic material encoding HIV proteins). Another thought would be whether the genetic material he has introduced into the cell could form psuedovirions from endogenous retroviruses, or if the particular cell line is known to be infected with other viruses (e.g. HTLV positive). If so, then it would be theoretically possible for an infectious pseudovirion to be produced. Once again, more information about what he has put into the cell line, and about the status of the cell line with regards to other viral infections, would be required to determine how biohazardous the cells would be.
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4) If the cells are producing only VLPs, which I assume, then no additional precautions are necessary as VLPs are indeed non-infectious.
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5) I am responding to your question regarding the feasibility of sorting cell lines with HIV-based lentivirus LTR defective, Vpr- , tat- , Env- , Nef- , Gag +. The likely hood of such construct reverting to viable HIV virus is slim. However as long as there is no clear proof that such construct cannot relocate/rearange on some other frame and become viable, I would suggest that such live cell sorting experiments should be performed only if the instrument is installed in accordance with class three biohazard containment. Unfortunately I do not have any specify literature regarding this issue. There are several publications that indicated that HIV VLP's with natural and experimental Gag deletion can revert to viable form with relocation of mutant fragments.
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6) I don't think that it's a major problem. Remember that we always process samples in the clinical labs that may be infected and therefore take precautions. What is so different here?
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7) The number of mutations does minimize the chance of reversion to virulence. The gag-GLP fusion also adds another mutation, further minimizing the risks. In a risk managment mindset, this is unlikely to be infectious in humans. However, I would like to see the data showing no risk of infecting human cells(ie take particle/culture supernatant and try to infect human cell lines and look for lack of virus production or proviral itegration), before being prepared to lower the biocontainment level to a level 2 risk. As the manager of your Flow facility you have a requirement under due diligence to provide this data to your staff (Workplace health and safety wise) and ultimately will be asked by the Public Health Agency of Canada, Office of Lab security to prove why you lowered the risk level to a level 2 from a level 3 (http://www.phac-aspc.gc.ca/ols-bsl/)
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8) Dear Eric,
I am including the proposed wording for the new standard for cell sorting we are currently finalizing for sorting established cell lines which is based on information contained in an interpretation of the Blood Borne Pathogen Standard. The interpretation can be found under the Osha web site at www.osha.gov <http://www.osha.gov/> under laws and regulations, interpretations, search for "establish human cell lines". In your case also concerns about recombinationn of the replication incompetent virus to form an infectious one makes it necessary to treat the experiment as biohazardous.
Handling of all unfixed human specimens and primary cell cultures as if infectious is recommended. This practice also applies to established cell lines that are in vitro or animal-passaged human explanted tissues transformed by spontaneous mutation or a natural or laboratory infection with an immortalization agent, e.g., Epstein Barr virus. In fact, cell lines from the American Type Culture Collection (ATCC) and other sources bear warnings that they may contain bloodborne pathogens, and ATCC recommends they be accorded the same biosafety level as the ones known to be infected with HIV. Likewise specimens from non-human primates and animal tissues, explants, or cell cultures known to be deliberately infected with human pathogens are subject to safety procedures as outlined in the Bloodborne Pathogen Standard.
Only rigorously characterized human cell lines that have been proven by stringent techniques such as PCR, sensitive antigen detection, stimulation and co-culture assays, enzyme analysis etc. to be completely devoid of bloodborne pathogens could be excluded. However, as most laboratories are not able to provide reliable confirmation that the samples are pathogen free before they are subjected to cell sorting using biosafety precautions as outlined in the standard is strongly advised.
Thank you all for your support. This gives me good documentation to start a real conversation with the users and the institute's biohazard comittee.
Regards
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