Assessment of the inhibitory effect of ribavirin on the rainbow trout rhabdovirus VHSV by real-time reverse-transcription PCR.
Viral hemorrhagic septicemia virus (VHSV) is one of the most ubiquitous viruses in salmonid aquaculture in Europe. This infectious disease results in significant losses in the farming industry and therefore effective therapeutic agents are needed to control outbreaks caused by this pathogen. Thus, accurate methods to test new antiviral compounds need to be developed. Our goal was to establish a model system for testing novel antivirals with potential applications to aquaculture. In a previous study, a TaqMan real-time RT-PCR assay was designed to detect and quantitate VHSV in rainbow trout tissues [Chico, V., Gomez, N., Estepa, A., Perez, L., 2006. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J. Virol. Methods 132, 154-159]. In this report, we applied the real-time RT-PCR assay to the evaluation of the inhibitory effect of ribavirin, a well-known broad spectrum antiviral drug, in a cell culture system. When added from the beginning of the infection, ribavirin caused a dose-dependent reduction of VHSV RNA accumulation. Real-time RT-PCR measurements showed 99.8% inhibition at 25 microg/ml ribavirin, with an IC50 of 0.43 microg/ml. Ribavirin maintained its inhibitory activity against VHSV when added at 6 h post-infection. Quantitation of N protein messenger RNA and plus-stranded RNA showed a substantial decrease of viral transcription in ribavirin-treated cells. Partial reversion of the effect of ribavirin by addition of GTP was observed, confirming that ribavirin targets the synthesis of guanidine nucleotides in the cells. This is the first report of a real-time PCR-based assay for addressing the efficacy and mechanism of action of an antiviral agent for rainbow trout.
Marroquí
Laura
L
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avda. Ferrocarril s/n, 03202 Elche, Spain.
Estepa
Amparo
A
Perez
Luis
L
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002347:Carps; D002460:Cell Line; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D006160:Guanosine Triphosphate; D029141:Novirhabdovirus / Q000187:drug effects*; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012254:Ribavirin / Q000494:pharmacology*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D000998:Antiviral Agents; D019590:Nucleocapsid Proteins; D012254:Ribavirin; D006160:Guanosine Triphosphate