This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.
Bibliographical noteFunding Information:
We thank Dr. Karl-Klaus Conzelmann for kindly providing pTIT plasmid and the BSR-T7/5 cells. Also we thank Dr David F. Stojdl for kindly providing the LC-Kan Maraba plasmid. We acknowledge the excellent technical work of the PIADC Animal Care Unit personnel during the animal experiments. This work was performed under USDA Research Service CRIS project No. 8064-3200-059-00D.
- Site-specific recombination
- Vesicular stomatitis virus
- cDNA clone