Establishment of a reverse genetics system for virulent systemic feline calicivirus using circular polymerase extension reaction

Abstract

Summary

Feline caliciviruses can cause oral and upper respiratory tract infections in cats. However, a virulent and systemic feline calicivirus (VS-FCV) variant implicated in multisystem lesions and death in cats has emerged recently. To date, the mechanism underlying virulence variations in VS-FCV remains unclear. The aim of the present study was to provide a tool for exploring genetic variation in VS-FCV, by constructing an infectious clone of VS-FCV SH/2014. First, a full-length cDNA molecular clone of VS-FCV SH/2014 strain, which contains an Xba I recognition site generated by mutating one base (A→T) as a genetic marker, was constructed using the circular polymerase extension reaction (CPER) method. Second, the full-length cDNA clone was introduced into Crandell-Rees feline kidney cells using liposomes to rescue recombinant VS-FCV SH/2014 (rVS-FCV SH/2014). Third, the rescued viruses were identified by real-time PCR, immunofluorescence assay, western blotting, and electron microscopy. The full-length cDNA molecular clone of the VS-FCV SH/2014 strain was successfully constructed and that rVS-FCV SH/2014 could be rescued efficiently. rVS-FCV SH/2014 had the expected genetic markers and morphology and growth characteristics similar to those of the parental virus. The reverse genetics system provides a research platform for future studies on VS-FCV genetic variation and pathogenesis.