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Published on: August 9, 2013
Effect of small interfering RNAs on in vitro replication and gene expression of feline coronavirus
Eman A Anis1, Rebecca P Wilkes, Stephen A Kania
1Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996., Department of Virology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Beheira, Egypt 32511.
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 TCID₅₀ 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.
Insights
Small interfering RNAs (siRNAs) effectively inhibited feline coronavirus (FCoV) replication in cell cultures. Combinations of siRNAs targeting multiple FCoV genome regions showed significant antiviral activity, suggesting potential therapeutic applications.
Area of Science:
- Virology
- Molecular Biology
- RNA Interference
Background:
- Feline coronavirus (FCoV) causes significant disease in cats, including feline infectious peritonitis.
- Current treatment options for FCoV infections are limited, necessitating the exploration of novel therapeutic strategies.
Purpose of the Study:
- To assess the efficacy of small interfering RNAs (siRNAs) in inhibiting in vitro replication and gene expression of feline coronavirus (FCoV).
Main Methods:
- Utilized Crandell-Rees feline kidney cell cultures infected with FCoV.
- Tested five synthetic siRNAs targeting different FCoV genome regions individually and in combination.
- Assessed viral inhibition through real-time RT-PCR for genomic RNA, flow cytometry for protein expression, and TCID₅₀ assays for virus titration.
Main Results:
- Individual siRNAs exhibited variable inhibitory effects on FCoV.
- Combinations of siRNAs targeting diverse viral genome regions demonstrated superior antiviral activity compared to single-target siRNAs.
- siRNA combinations achieved approximately 95% reduction in viral replication compared to controls.
Conclusions:
- siRNAs targeting both coding and noncoding regions of the FCoV genome effectively inhibited viral replication in vitro.
- RNA interference holds promise as a potential therapeutic strategy for treating feline infectious peritonitis.
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