Location: Location not imported yet.Title: Validation of a site-specific recombination cloning technique for the rapid development of an infectious cDNA clone of a virulent field strain of vesicular stomatitis New Jersey virus
|VELAZQUEZ-SALINAS, LAURO - The National Autonomous University Of Mexico|
|BARRERA, JOSE - Leidos|
|CLARK, BENJAMIN - Orise Fellow|
|VERDUGO-RODRIGUEZ, ANTONIO - The National Autonomous University Of Mexico|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2019
Publication Date: 1/9/2019
Citation: Velazquez-Salinas, L., Pauszek, S.J., Barrera, J., Clark, B., Borca, M.V., Verdugo-Rodriguez, A., Arzt, J., Rodriguez, L.L. 2019. Validation of a site-specific recombination cloning technique for the rapid development of an infectious cDNA clone of a virulent field strain of vesicular stomatitis New Jersey virus. Journal of Virological Methods. Volume 265, March 2019, Pages 113-116. https://doi.org/10.1016/j.jviromet.2019.01.003.
Interpretive Summary: Vesicular stomatitis New Jersey virus (VSNJV) is an important pathogen of horses, cattle and pigs that causes sporadic outbreaks in the United States. In cattle and pigs, the clnical sings resemble those of foot-and-mouth disease, a devastating foreign animal disease eradicated since 1929 in the United States. In order to study the disease it is important to have the abilty to derive VSV using recombinant DNA techniques (i.e. reverse genetics). Here we describe a methology that allowed the creation of a VSV field strain affecting the United States in 2012 derived from genetic sequences. We showed that the virus had similar growth charateristics to the field virus both in cell culture and in experimentally inoculated pigs. This methodology will allow future studies to understand disease transmission and control methods.
Technical Abstract: In this study, we report the use of a site-specific recombination cloning technique for rapid development of cDNA infectious clones of 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 BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization revealed similar growth kinetics and plaque morphologies between recombinant and parental viruses. Furthermore, experimental infection of pigs with recombinant virus resulted in severe vesicular stomatitis clinical signs similar to those previously reported for the parental field strain. Our results validate the use of a site-directed specific recombination cloning as a useful alternative method for the rapid construction of stable cDNA infectious clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of infectious clones of this relevant virus.