Inhibitory Effect of a Nucleotide Analog on Infectious Salmon Anemia Virus Infection

Abstract

The infectious salmon anemia virus (ISAV), which belongs to the Orthomyxoviridae family, has been responsible for major losses in the salmon industry, with mortalities close to 100% in areas where Atlantic salmon (Salmo salar) is grown. This work studied the effect of ribavirin (1-␤-D-ribofuranosyl-1,2,3-triazole-3-carbaxaide), a broad-spectrum antiviral compound with proven ability to inhibit the replicative cycle of the DNA and RNA viruses. The results show that ribavirin was able to inhibit the infectivity of ISAV in in vitro assays. In these assays, a significant inhibition of the replicative viral cycle was observed with a 50% inhibitory concentration (IC 50 ) of 0.02 g/ml and an IC 90 of 0.4 g/ml of ribavirin. After ribavirin treatment, viral proteins were not detectable and a reduction of viral mRNA association with ribosomes was observed. Ribavirin does not affect the levels of EF1a, nor its association with polysomes, suggesting that the inhibition of RNA synthesis occurs specifically for the virus mRNAs and not for cellular mRNAs. Moreover, ribavirin caused a significant reduction in genomic and viral RNA messenger levels. The study of the inhibitory mechanism showed that it was not reversed by the addition of guanosine. Furthermore, in vivo assays showed a reduction in the mortality of Salmo salar by more than 90% in fish infected with ISAV and treated with ribavirin without adverse effects. In fact, these results show that ribavirin is an antiviral that could be used to prevent ISAV replication either in vitro or in vivo.

Bona Fide Evidence for Natural Vertical Transmission of Infectious Salmon Anemia Virus in Freshwater Brood Stocks of Farmed Atlantic Salmon (Salmo salar) in Southern Chile

“…The cells were fixed, and cell sections were blocked with 3% bovine serum albumin (BSA) containing 0.5% Tween 20 for 30 min at 37°C (25). A monoclonal antibody against the ISAV nucleoprotein (clone 2C2/H4; Grupo Bios, BiosChile) diluted 1:500 in 1ϫ PBS and 3% BSA (26) was incubated overnight at 4°C, detected by using a goat anti-mouse antibody conjugated with Alexa Fluor 568 (Life Technologies, USA) diluted 1:1,000 in 1ϫ PBS and 3% BSA, and incubated for 1 h at room temperature. For wheat germ agglutinin (WGA) depiction, samples were stained with 5 g/ml Alexa Fluor 488-conjugated WGA (Invitrogen, USA) for 1 h at room temperature.…”

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

“…At day 7 postinfection, ASK cells infected with rISAV S6-EGFP-HPR from each passage of the recombinant virus were analyzed by confocal microscopy using the procedures described by RivasAravena et al (46). The fixed cells were observed with an LSM 510 (Zeiss) confocal microscope with the LSM image browser program to detect rISAV S6-EGFP-HPR by EGFP fluorescence.…”

“…The fixed cells were observed with an LSM 510 (Zeiss) confocal microscope with the LSM image browser program to detect rISAV S6-EGFP-HPR by EGFP fluorescence. In addition, rISAV was detected by monoclonal anti-HE (BiosChile) as previously described (46).…”

Ribavirin stimulates the immune response of Atlantic salmon

“…Samples of fish were taken at this point to evaluate the initial levels of transcripts (day 0). Then, 168 fish were treated with 6.5 mol of ribavirin/kg of fish for 10 days in the feed, in duplicate farming tanks as described (Rivas-Aravena et al, 2011). After that, the fish were fed without ribavirin.…”

“…In fish, it has been reported that some viral infections are inhibited by ribavirin. In particular, infections with viral hemorrhagic septicemia virus (VHSV) (Marroqui et al, 2007), chum salmon reovirus (CSV) (DeWitte-Orr and Bols, 2007), infectious pancreatic necrosis virus (IPNV) (Jashés et al, 1996;Migus and Dobos, 1980), infectious hematopoietic necrosis virus (IHNV) (Hudson et al, 1988) and infectious salmon anemia virus (ISAV) (Rivas-Aravena et al, 2011) have been shown to be dramatically reduced by ribavirin.…”