Feline coronavirus is a single-stranded, positive-sense RNA virus with a membrane envelope. The FCoV genome contains 11 putative ORFs. These include 2 large ORFs that encode viral nonstructural replicase proteins; 4 structural ORFs that encode spike, envelope, membrane, and nucleocapsid proteins; and 5 accessory ORFs of unknown function. Similar to other coronaviruses, FCoV uses a discontinuous transcription mechanism to synthesize both full-length and sub-genomic negative-strand RNAs, which then function as templates for synthesis of full-length genomic mRNAs and sgRNAs.1 Both viral genome and sgRNAs share a common 5′ leader sequence and a common 3′ UTR.2
Feline coronavirus is a virus that commonly infects domestic cats, typically resulting in subclinical infection or mild enteritis. However, some FCoV-infected cats develop FIP, a progressive and fatal disease that accounts for most infectious disease–related deaths in pet cats.3 Feline infectious peritonitis may have an acute onset with peritoneal or pleural effusion (or both) or may have a more protracted course with pyogranulomatous infiltration of multiple tissues. Both clinical scenarios invariably end in death. The pathogenesis of FIP involves mutant FCoV that can replicate efficiently in monocytes and macrophages, which results in dysregulation of host cell-mediated immunity and allows the virus to replicate unchecked to a high titer. Contributing to the pathogenesis of FIP are cytokines and inflammatory mediators released from infected macrophages and other inflammatory cells that infiltrate the tissue as well as antigen-antibody complexes and complement activation.4 Thus, efficient systemic viral replication appears to have a critical role in FIP pathogenesis.
To date, there is no specific treatment for this fatal disease. The use of siRNAs is a novel antiviral strategy that specifically targets viral mRNA and genomic RNA for degradation by endogenous cellular enzymes.5 This technology has been used successfully for viral diseases such as viral hepatitis and severe acute respiratory syndrome both in vitro and in ex vivo studies.6–8 Recently, 2 individual siRNAs that target the FCoV leader region and nucleocapsid gene were shown to inhibit FCoV replication in vitro.9 The purpose of the study reported here was to evaluate the ability of siRNAs (singly and in combination) to inhibit in vitro replication and gene expression of FCoV. To this end, FCoV-specific siRNAs that hybridize to viral coding or noncoding regulatory regions of the FCoV genome were designed and evaluated in various single or combination siRNA test conditions. Assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, RT-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.
Crandell-Rees feline kidney
Dulbecco modified Eagle medium
Feline enteric coronavirus
Feline infectious peritonitis
Feline infectious peritonitis virus
Multiplicity of infection
Open reading frame
Small interfering RNA
Stealth siRNA, Invitrogen, Carlsbad, Calif.
Block-iT RNAi designer, Invitrogen, Carlsbad, Calif.
Crandell-Rees feline kidney cell line, American Type Culture Collection, Manassas, Va.
DMEM:F-12, Lonza, Walkersville, Md.
Fetal Bovine serum, Atlanta Biologicals, Lawrenceville, Ga.
Lipofectamine 2000, Invitrogen, Carlsbad, Calif.
FIPV WSU 79-1146, American Type Culture Collection, Manassas, Va.
FECV WSU 79-1683, American Type Culture Collection, Manassas, Va.
Fluorescently labeled siRNA, Invitrogen, Carlsbad, Calif.
IntraPrep permeabilization reagent, Immunotech, Marseille, France.
Polyclonal Ab, VMRD, Pullman, Wash.
Flow cytometr, Epics XL, Beckman Coulter Inc, Fullerton, Calif.
RNeasy Plus Mini Kit, Qiagen, Valencia, Calif.
Superscript III Platinum One-Step qRT-PCR Kit, Invitrogen, Carlsbad, Calif.
SmartCycler II, Cepheid, Sunnyvale, Calif.
CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega Corp, Madison, Wis.
ELx800 Universal Microplate Reader, Bio-Tek Inc, Winooski, Vt.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
4. de Groot-Mijnes JDF, Van Dun JM, van der Most RG, et al. Natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease. J Virol 2005; 79: 1036–1044.
5. Spurgers KB, Sharkey CM, Warfield KL, et al. Oligonucleotide antiviral therapeutics: antisense and RNA interference for highly pathogenic RNA viruses. Antiviral Res 2008; 78: 26–36.
6. Chandra PK, Kundu AK, Hazari S, et al. Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes. Mol Ther 2012; 20: 1724–1736.
7. Li T, Zhang Y, Fu L, et al. siRNA targeting the leader sequence of SARS-CoV inhibits virus replication. Gene Ther 2005; 12: 751–761.
8. Chang Z, Babiuk LA, Hu J. Therapeutic and prophylactic potential of small interfering RNAs against severe acute respiratory syndrome. BioDrugs 2007; 21: 9–15.
9. McDonagh P, Sheehy PA, Norris JM. In vitro inhibition of feline coronavirus replication by small interfering RNAs. Vet Microbiol 2011; 150: 220–229.
10. Li S, Crothers J, Haqq CM, et al. Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA. J Biol Chem 2005; 280: 23709–23717.
11. Gut M, Leutenegger CM, Huder JB, et al. One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses. J Virol Methods 1999; 77: 37–46.
12. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008; 3: 1101–1108.
14. Hartmann K, Ritz S. Treatment of cats with feline infectious peritonitis. Vet Immunol Immunopathol 2008; 123: 172–175.
15. Legendre AM, Bartges JW. Effect of polyprenyl immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis. J Feline Med Surg 2009; 11: 624–626.
16. He ML, Zheng BJ, Chen Y, et al. Kinetics and synergistic effects of siRNAs targeting structural and replicase genes of SARS-associated coronavirus. FEBS Lett 2006; 580: 2414–2420.
Sequences of siRNAs (and their position within the FIPV WSU 79-1146 genome) used in experiments to evaluate the ability of siRNAs to inhibit in vitro viral replication and gene expression of FIPV WSU 79-1146 and FECV WSU 79-1683.
|siRNA||Nucleotide sequence*||Position in the genome (bp range)|
Nucleotide sequences are based on GenBank accession No. DQ010921.