I have to transfect human and primate cells with an HIV-1 full sequence plasmid. I would like to know at which biosafety level my cell culture should be. Some sources state BSL2, some state BSL3, and even more confusing, there is a source that states this should be done in a BSL2 lab with BSL3 practices.
BSL-3. The cells will be producing HIV-1. There are many kinds of HIV plasmids. Some are missing key proteins to prevent propagation. Some are chimeric, such as SHIV. So what is an appropriate safety level can vary. In this case, you are dealing with fully infectious HIV-1. Full precautions are appropriate.
Be aware also that your plasmid is potentially quite problematic to handle. If it is injected into your body, and the plasmid enters a cell, it will produce virus, and that cell does not need to be a T-cell or other leukocyte that would be normally expected. The ratio of transfection for free plasmid is low, but it is definitely not zero, and all it takes is one cell transfected. Your chances are near 100% for infection if you get an accidental plasmid injection. For virus contamination, it is very low. So be careful of needles and sharp objects that could be contaminated. Irradiate (UV), autoclave, and use chlorox of some other sterilizing agent of equal effectiveness on all materials associated with the plasmid.
That said, in research, corners are cut sometimes. The cost of maintaining a certified BSL-3 is high just due to regulations. So, I have, myself, handled BSL-3 viruses in a BSL-2.5. This is a BSL-2 facility that, as you say, observes BSL-3 protocols. It had a dressing room. antechamber with an airlock. Full hoods and negative pressure to contain organisms. Everything that a BSL-3 would have except the certification. Double glove, face shields, and a fresh clean lab coat with elastic cuffs to protect the arms were always done. Step in a chlorox bath going in, and going out. Discard all lab gear on exit, including for bathroom breaks. No exceptions. (The bathroom break is how, for instance someone got a genital infection with vaccinia that was not fun at all. Didn't want to go through the bother of gown and glove, etc. "I'll just be a minute." or , "It won't matter this once.")
Practically speaking, how it is done depends on what is available, the regulations of your nation, the decisions of your institution, and your own understanding and practice. It used to be that smallpox was handled on open benches using classical flame and chlorox wipe methods. This worked for a long time. But, inevitably mistakes were made. A lab had cockroaches, perhaps because staff were eating in the lab. Cockroaches tracked virus into wall. Mouse tracked it up to higher floor and onto a telephone. Outbreak.
In the end, for all lab researchers, what they do is up to them, and the facilities around them. With the best of facilities, not taking things seriously or not being aware of and thinking about contamination, can lead to problems. With lesser facilities, and careful attention, actual lab safety can be higher.
it should be under biosafety level 2 as it is non-rep- lication-competent viruses or pseudoviruse.
The biosafety level for working with HIV plasmid depends on the type of plasmid and the type of experiment. Generally, HIV plasmid is considered as a BSL-2 agent, as it is derived from HIV, which is a human pathogen that can cause serious or lethal disease. However, some HIV plasmids may be handled at BSL-1 if they are replication incompetent, non-infectious, and do not encode any viral proteins. Additionally, some experiments may require a higher level of biosafety, such as BSL-2+ or BSL-3, if they involve the generation of infectious viral particles, the use of human or animal cells or tissues, or the manipulation of large quantities (high concentrations) of HIV plasmid.
• Therefore, the biosafety level for transfecting the HIV plasmid may vary depending on the specific characteristics and risks of the plasmid & the experiment.
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