Characterization of bioengineered pestiviruses The aim of this project will be to develop new bioengineered pestiviruses and characterize these in terms of genetic stability, replication kinetics, virus growth etc. Infectious cDNA clones are a prerequisite for directed genetic manipulation of pestivirus RNA genomes. At DTU Vet Lindholm, we have developed a novel strategy to facilitate manipulation and rescue of modified pestiviruses from infectious cDNA clones based on bacterial artificial chromo-somes 50 (BACs). The strategy involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E. coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome and hence is not limited to the use of internal restriction sites. Rescue of modi-fied pestiviruses can be obtained by electropora-tion of cell cultures with full-length RNA tran-scripts in vitro transcribed from the recombined BAC clones. We have used this approach to gen-erate a series of new pestivirus BACs modified within different genomic regions and infectious pestiviruses have been rescued from several of these new constructs, demonstrating that recombination-mediated mutagenesis of pesti-virus BACs provides a useful tool for expediting Fluorescence microscopy of bioengineered pestivirus the construction of recombinant pestiviruses. Significance Bioengineered pestiviruses can be utilized for a variety of applications such as vaccine develop-ment, screening for antiviral drugs and for identification of essential components in the pestivirus lifecycle. Techniques • Standard molecular biology techniques including nucleotide sequencing • Recombination-mediated mutagenesis • Rescue of modified viruses by electroporation • Characterization of virus replication by quantitative PCR and fluorescence microscopy ECTS: 15-50 Place: DTU Vet, Lindholm, Kalvehave Contact senior scientist Thomas Bruun Rasmussen, tbru@vet.dtu.dk, DTU Vet, Lindholm, Kalvehave
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