Lentiviral Vectors- Dangerous?

[ennui]

Lots of labs use lentivirus vectors for transfecting cells with certain genes. But are these safe to use?

 

I know that they're genetically modified, and not competent for replication, so what would happen if you became infected accidentally? Would the virus just infect a limited number of white cells, and then be destroyed?

 

Are there any cases of lab workers getting infected by them and developing disease?

[CharonY]

Two major risk factors are the potential of generating viruses that become competent again in vivo and possibly oncogenesis.

While the vector as it is usually used should not be really harmful, there is always the possibility that changes might occur post-infection.

This will also make it difficult to find out if they are causative agents in diseases or cancer that may occur later on. At least I have not heard of any infection that could have been traced back to a lab accident with a modified vector yet. The actual risk really depends on the origin of the vector, its safety features and what insert is used for transfection.

Edited November 11, 2009 by CharonY

[brainscattered]

hi,

 

i recently had a stupid accident and am not quite sure what to do now or if the accident could have any aftermath at all?

 

i was handling a primary cell line culture which has previously been transduced with a lentivirus (derived from HIV - 3 vector system (envelope from vsv) + the vector carrying the desired construct). the construct is not oncogenic in nature. after the cells were infected, i changed medium to normal virus-free DMEM and a day later took off the fresh medium, washed the cells with pbs and split them to a 10 cm plate. 2 days later, cells were washed again ad split to 2 new plates containing coverslips. immediately before i took out the coverslips i washed the cells with pbs and thereafter lifted the coverslips using a needle.

 

unfortunately, i pricked myself by accident with that very same needle after scraping around in the cell culture dish in order to lift the coverslips. now i am afraid that i might have introduced virus or virus containing cells into my bloodstream. the cells should be eliminated rapidy and the virus itself should not be capapble of replication, though.

 

 

 

 

is it possible that after so many washing steps there might still have been virus present and if so do i need to worry about infection? after all, the primary cells i was working with should not be able to produce new virus...

 

 

 

 

thank you for your answers.

 

 

[CharonY]

All accidents of this nature have to be put on record. I.e. talk to your labmanager/PI and also the safety manager. Also, I would take a close look at procedures and get rid of sharp implements whenever not necessary. The coverslips can also be lifted by tweezers that can be sterilized, for instance.

[brainscattered]

thank you for your reply. the accient has been recorded and i have made some inquiries into the nature of the 3rd generation lentiviruses. of course, the accident was stupid and should never happen again, but the probability of serious health hazards seems to be (at least so i can tell in my case) very low. presence of any viral particles after washing and trypsinizing the cells twice already decreases viral titers greatly. the half-life of lentiviruses in cell culture (on acceptor cells, not the producer cell line) is about a day or so and i cultured for several days after removing the viral supernatant. however, half life seems to be increased in cell cultures harbouring macrophages or dedritic cells (internalization of viral particles and subsequent release)

 

moreover, recombination events which could give rise to virus competent of reproduction have not yet been observed (at least 4 recombinations in non-homologous regions would have to take place ...). last but not least, essential genes responsible for pathogenicity of HIV have been deleted - thus, it is impossible to generate wild type HIV just by uncontrolled recombination of the lentiviral vector genome.

 

therefore, the greatest risk seems to be insertional mutagenesis and incorporation of the transgene into the researchers genome.

 

Nonetheless, lifting coverslides with pincers alone is a torture - the coverslides appear to 'stick' to the bottom of the cell culture dish and you have to get underneath somehow (that is what i use the thin, sharp needle for...) - does anyone know a technique (simple as it may be) in order to lift coverslides without help of sharp object such as needles?

 

thank you and cheers

 

 

[CharonY]

Excellent. I just did not want to write an answer here, because as it is always better to go through the official lines rather than listening to what someone may tell you on an anonymous board. I am happy that you took care of that. Regarding the coverslip it depends on the precise setup and what you need to do. In some cases lifting the coverslip with a sticky tape may work. I also have very fine tweezers that work fine, however you can also prick yourself with them (though not quite as easily as with needles).

Edited October 27, 2011 by CharonY