Single-Round Infectious Particle Production by DNA-Launched Infectious Clones of Bungowannah Pestivirus.

Abstract

Reverse genetics systems are powerful tools for functional studies of viral genes or for vaccine development. Here, we established DNA-launched reverse genetics for the pestivirus Bungowannah virus (BuPV), where cDNA flanked by a hammerhead ribozyme sequence at the 5' end and the hepatitis delta ribozyme at the 3' end was placed under the control of the CMV RNA polymerase II promoter. Infectious recombinant BuPV could be rescued from pBuPV-DNA-transfected SK-6 cells and it had very similar growth characteristics to BuPV generated by conventional RNA-based reverse genetics and wild type BuPV. Subsequently, DNA-based ERNS deleted BuPV split genomes (pBuPV∆ERNS/ERNS)-co-expressing the ERNS protein from a separate synthetic CAG promoter-were constructed and characterized in vitro. Overall, DNA-launched BuPV genomes enable a rapid and cost-effective generation of recombinant BuPV and virus mutants, however, the protein expression efficiency of the DNA-launched systems after transfection is very low and needs further optimization in the future to allow the use e.g., as vaccine platform.

• Authors: Anja Dalmann, Kerstin Wernike, Eric J Snijder, Nadia Oreshkova, Ilona Reimann, Martin Beer
• Journal: Viruses (Viruses) Vol. 12 Issue 8 (08 04 2020) ISSN: 1999-4915 [Electronic] Switzerland
• PMID: 32759644 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Complementary
• RNA, Catalytic
• RNA Polymerase II
• bacteriophage T7 induced DNA polymerase
• DNA-Directed DNA Polymerase

Topics

• Animals
• Cell Line
• Cloning, Molecular
• Cytomegalovirus (genetics)
• DNA, Complementary (genetics)
• DNA-Directed DNA Polymerase (genetics)
• Genome, Viral
• Pestivirus (genetics, pathogenicity)
• Promoter Regions, Genetic
• RNA Polymerase II (genetics)
• RNA, Catalytic (genetics)
• Reverse Genetics (methods)
• Swine