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Team:Wageningen UR/Virus-related issues
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Safety
Any project related to viruses sounds very dangerous and is therefore in need of an extra biosafety emphasis. However, our group isn’t working with viruses but instead with “Virus-Like Particles” (VLP’s). VLP’s consist of viral Coat Proteins (CP’s) which, in the right conditions, will spontaneously assemble into a shell that very much resembles the original virus in shape and size. However, since VLP’s consist of only the Coat Proteins, they lack both the external sites that are usually required for the infection of cells and the internal machinery needed for viral replication. Moreover, they lack the viral DNA/RNA to be transcribed and replicated. Consequently, these particles can safely be used without risk of any unintended viral activity (Roy and Noad 2008).
Nevertheless, an overview of which virus components we used and how we got them is included below.
Cowpea Chlorotic Mottle Virus (CCMV)
CCMV is a Risk Group I virus because it poses no threat to humans or animals, but it might nonetheless be harmful for the flora in our direct environment should it ever be accidentally released. However, the CCMV itself or its genome were never in our possession and thus posed no viral threats to our environment.
The viral Coat Protein encoding gene we used was obtained from a plasmid stock provided to us by Dr. Kormelink of the faculty of virology at Wageningen UR. The plasmid itself was a standard pET-28a(+) ‘’E. coli’’ vector harboring the CCMV Coat Protein encoding sequence (GenBank: NC_003542.1 (1360..1932)) behind an IPTG inducible promotor. No other viral genes were present. This was verified by DNA sequencing.
Hepatitis B (HBV)
HBV is a DNA-based Risk Group III virus because it is considered to pose a high risk for individual researchers. We have no legal classifications nor the experience to work with such a virus. However, the Hepatitis B Virus itself or its genome were never in our possession and thus posed no viral threats to ourselves, other people or the environment. The viral core protein encoding gene that we used was obtained from a plasmid stock supplied to us by [Londen through Kormelink in vector] (GenBank: NC_003977.1 (1814..2452))
No other viral genes were present. This was verified by DNA sequencing.
Turnip Yellows Virus (TuYV)
Like CCMV, TuYV is a Risk Group I virus because it poses no threat to humans or animals, but it might nonetheless be harmful to the flora in our direct environment should it ever be accidentally released. The viral Coat Protein encoding gene we introduced for Turnip Yellows Virus (TuYV) (GenBank: NC_003743.1 (3483..5495)) was obtained from a plasmid encoding the entire viral genome (GenBank: X13063.1). This plasmid was provided to us, via Dr. Kormelink of Wageningen UR’s Virology faculty, by the [French group, Veronique]
With the entire virus genome on one plasmid, it is in theory possible that complete viruses could be formed from it. However, this would require both transcription and translation to take place in significant amounts, which is only possible if a competent cell took it up. This would provide the cell with no foreseeable selective advantage. Still, to prevent this from happening the eppendorf tube containing the plasmids was always handled with Good Microbiological Techniques (GMT) and extra care was taken to keep it sterile. To restrict any risk to a minimum the eppendorf tube containing the plasmid was opened only thrice, to serve as a template for PCR. After completion of our project, this DNA will be destroyed by addition of an excess of hydrochloric acid.
We think that with GMT, even in the unlikely case that a virus of TuYV would have formed, the likelihood of it leaving the samples and then the lab, ultimately infecting aphids or plants in our environment, is rather low.
Potato Leafroll Virus (PLRV)
Like CCMV and TuYV, PLRV is a Risk Group I virus because it poses no threat to humans or animals. This virus is native to our direct environment, but accidental release could still mean serious harm to the flora in our environment and in the worst case scenario even an economic disaster for local farmers.
The viral Coat Protein encoding genes for Potato Leafroll Virus (PLRV) were isolated from infected potato plant leaves. The leaves were provided by the ‘Dutch General Inspection Service for Agricultural seed and seed potatoes’ (www.nak.nl). Part of the leaves was used for RNA isolation, the remainder was frozen immediately at -80°C. This absolutely minimized the chance of spreading of PLRV. The virus can be spread by several aphid species, most efficiently by Myzus persicae. The insect is absolutely necessary for spreading of the virus because mechanical transmission (rubbing infected leaves on healthy ones) does not work. After acquiring the virus, by feeding on infected leaves, the aphid is only infectious after 24 hours or more. We can almost exclude this event as a possibility to happen in our lab. After RNA isolation, cDNA was synthesized using PLRV coat protein specific primers as well as random primers. Subsequent PCR reactions were performed using PLRV coat protein primers. Products of subsequent cloning steps were sequenced and contain only the PLRV coat protein gene. All other (intermediate) products will, after completion of our project, be destroyed.
[Safety Procedures]
