Application of versatile reverse genetics system for feline coronavirus

TL;DR

Researchers developed a new reverse genetics system for feline coronavirus to study its genes and disease mechanisms more effectively.

Contribution

A rapid PCR-based reverse genetics system for generating and modifying feline coronavirus with high efficiency and accuracy.

Findings

Recombinant feline coronavirus was successfully generated with high sequence accuracy and comparable growth to parental viruses.

The system enabled stable expression of reporter genes and evaluation of antiviral inhibitors using luciferase assays.

Chimeric viruses with spike gene modifications showed similar characteristics to parental viruses.

Abstract

Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). Although multiple gene mutations in FCoV likely account for FIP pathogenesis, molecular studies for FCoV have been limited due to the lack of a suitable reverse genetics system. In the present study, we established a rapid PCR-based system to generate recombinant FCoV using the circular polymerase extension reaction (CPER) method for both serotype 1 and 2 viruses. Recombinant FCoV was successfully rescued at sufficient titers to propagate the progeny viruses with high sequence accuracy. The growth kinetics of recombinant FCoV were comparable to those of the parental viruses. We successfully generated recombinants harboring the spike gene from a different FCoV strain or a reporter HiBiT tag using the CPER method. The chimeric virus demonstrated similar characteristics with the parental virus of the spike gene. The reporter tag stably expressed after five serial passages in the susceptible cells, and the reporter virus could be applied to evaluate the sensitivity of antiviral inhibitors using the luciferase assay system to detect HiBiT tag. Taken together, our versatile reverse genetics system for FCoV shown herein is a robust tool to characterize viral genes even without virus isolation and to investigate the molecular mechanisms of the proliferation and pathogenicity of FCoV.

Feline infectious peritonitis is a highly fatal disease in cats caused by feline coronavirus variants that can infect systemically. Due to the lack of a versatile toolbox for manipulating the feline coronavirus genome, an efficient method is urgently needed to study the virus proteins responsible for the severe disease. Herein, we established a rapid reverse genetics system for the virus and demonstrated the capability of the recombinant viruses to be introduced with desired modifications or reporter genes without any negative impacts on virus characteristics in cell culture. Recombinant viruses are also useful to evaluate antiviral efficacy. Overall, our system can be a promising tool to reveal the molecular mechanisms of the viral life cycle of feline coronavirus and disease progression of feline infectious peritonitis.

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