Leveraging Synthetic Virology for the Rapid Engineering of Vesicular Stomatitis Virus

Vesicular stomatitis virus, a negative-sense, non-segmented RNA virus of Rhabdoviridae family is a prototype virus that is widely studied for advancing molecular virology. Replicative cycle of VSV is relatively quick, within 4-6 h and favorable for research in vaccines developments. However, there are several limitations among previous traditional technologies for viruses engineering (homologous recombination in plasmids) is taken long time periods and need significant research expertise, also restriction enzyme-based cloning methods are limited to the location of restriction sites. Thus, the study aimed to introduce a new methodology in a framework that resulted a quick design-construct and test the synthetic viruses. A full-length Indiana strain of VSV plasmid was used in this study to rescue the wildtype virus. In brief, the fragment sequence was designed (by rearrange the P and M gene order) to be inserted in plasmid vector for DNA synthesis and gene assembly. Then, it was transformed into selective medium and screened for successful cloning and plasmid construction. The successful cloning of synthesized VSV was transfected into the wild type of VSV and passaged. Last, the rescued products were quantified for gene copy number (by qPCR) and the cytopathic effect was observed under microscope.  As a result, 1.1 kilobases synthetic VSV showed no significant differences compared to the natural viruses on phenotypic analysis after rescue and titration. In conclusion, rapid engineering on synthetic VSV construction provides a novel development on vaccines and drug therapies that lead to improve human health.
(INV)