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Revision as of 09:19, 27 August 2012

Contents 1 Safety 1.1 Virus-related issues 1.1.1 Cowpea Chlorotic Mottle Virus (CCMV) 1.1.2 Hepatitis B (HBV) 1.1.3 Turnip Yellows Virus (TuYV) 1.1.4 Potato Leafroll Virus (PLRV)

Safety

Virus-related issues

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). [Safety Procedures]