Aim Complete genome amplification of viral RNA provides a new tool for generation of modified pestiviruses. We have recently reported a full genome amplification strategy for direct recovery of infectious pestivirus (Rasmussen et al., 2008). This comprised rescue of BDV strain “Gifhorn” from a full-length RT-PCR amplicon demonstrating that long RT-PCR can be used for direct generation of an infectious pestivirus. The strategy is not limited to amplification of BDV “Gifhorn”, but can be further utilized for amplification of a diverse selection of pestivirus strains and for the generation of modified pestiviruses. Methods Pestivirus genomes were amplified from either total RNA preparations using long RT-PCR or from infectious cDNA clones using long PCR. Viral RNA was extracted from cell cultures inoculated with pestivirus (e.g. BDV “Gifhorn” or BVDV “CP7”) using a combined Trizol/RNeasy protocol. Total RNA was reverse transcribed to cDNA at 50C for 90 minutes using SuperScript III reverse transcriptase (Invitrogen). Full-length PCR amplification was performed using primers specific for the extreme 5’- and 3’-ends of the viral genomes. A T7 promoter was incorporated in the 5’-primers for direct in vitro transcription of the amplicons. Long (RT)-PCR was performed using Accuprime High Fidelity or Elongase enzyme mix (Invitrogen), which consists of mixtures of Taq and proofreading Pyrococcus GB-D DNA polymerases. Reactions containing 2 l cDNA were amplified using 94C for 30 seconds followed by 35 cycles of 94°C for 15 seconds, 65°C for 30 seconds and 68°C for 12 minutes. Results Complete genomes from a selection of pestivirus strains and infectious cDNA clones were amplified using long (RT)-PCR. Amplicons of approximately 12.5 kb was obtained for the amplified pestivirus strains. Specific primers for complete genome amplification of the different pestivirus strains were obtained from nucleotide sequences of the extreme genomic ends. Less than 50 nucleotides of the genomic ends of a given strain were needed for amplification of the whole genome. By incorporation of unique restriction sites within the primers the amplicons were prepared for cloning into low-copy vectors to produce new infectious cDNA clones. Conclusions Using this full genome amplification strategy the efforts in producing new viral variants can be expedited and focused on a variety of other viral strains and hence is not limited to the availability of an existing infectious clone. The long RT-PCR strategy significantly simplifies and streamlines the workflow and facilitates generation of new modified pestiviruses and also allows direct full-length sequence analysis. References Rasmussen et al., J. Virol. Methods 149(2), 330 (2008).
|Publication status||Published - 2008|
|Event||7th ESVV Pestivirus Symposium - Uppsala, Sweden|
Duration: 16 Sept 2008 → 19 Sept 2008
Conference number: 7
|Conference||7th ESVV Pestivirus Symposium|
|Period||16/09/2008 → 19/09/2008|