Effect of small interfering RNAs on in vitro replication and gene expression of feline coronavirus.

Anis, Eman A; Wilkes, Rebecca P; Kania, Stephen A; Legendre, Alfred M; Kennedy, Melissa A

OBJECTIVE To evaluate the ability of small interfering RNAs (siRNAs) to inhibit in vitro viral replication and gene expression of feline coronavirus (FCoV). SAMPLE Cell cultures of Crandell-Rees feline kidney cells. PROCEDURES 5 synthetic siRNAs that each targeted a different region of the FCoV genome were tested individually and in various combinations for their antiviral effects against 2 strains of FCoV (feline infectious peritonitis virus WSU 79-1146 and feline enteric coronavirus WSU 79-1683) in cell cultures. Tested combinations targeted the FCoV leader and 3' untranslated region, FCoV leader region and nucleocapsid gene, and FCoV leader region, 3' untranslated region, and nucleocapsid gene. For each test condition, assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, reverse-transcription PCR analysis; flow cytometric evaluation of the reduction of viral protein expression in infected cells; and assessment of virus replication inhibition via titration of extracellular virus with a TCIDâ‚…â‚€ infectivity assay. RESULTS The 5 siRNAs had variable inhibitory effects on FCoV when used singly. Combinations of siRNAs that targeted different regions of the viral genome resulted in more effective viral inhibition than did individual siRNAs that targeted a single gene. The tested siRNA combinations resulted in approximately 95% reduction in viral replication (based on virus titration results), compared with findings in negative control, nontargeting siRNA-treated, FCoV-infected cells. CONCLUSIONS AND CLINICAL RELEVANCE In vitro replication of FCoV was specifically inhibited by siRNAs that targeted coding and noncoding regions of the viral genome, suggesting a potential therapeutic application of RNA interference in treatment of feline infectious peritonitis.