Effects of ribavirin on the replication and genetic stability of porcine reproductive and respiratory syndrome virus

Abstract

BackgroundAlthough modified live virus (MLV) vaccines are commonly used for porcine reproductive and respiratory syndrome virus (PRRSV) control, there have been safety concerns due to the quick reversion of MLV to virulence during replication in pigs. Previous studies have demonstrated that mutant viruses emerged from lethal mutagenesis driven by antiviral mutagens and that those viruses had higher genetic stability compared to their parental strains because they acquired resistance to random mutation. Thus, this strategy was explored to stabilize the PRRSV genome in the current study.ResultsFour antiviral mutagens (ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride) were evaluated for their antiviral effects against VR2332, a prototype of type 2 PRRSV. Among the mutagens, ribavirin and 5-fluorouracil had significant antiviral effects against VR2332. Consequently, VR2332 was serially passaged in MARC-145 cells in the presence of ribavirin at several concentrations to facilitate the emergence of ribavirin-resistant mutants. Two ribavirin-resistant mutants, RVRp13 and RVRp22, emerged from serial passages in the presence of 0.1 and 0.2 mM ribavirin, respectively. The genetic stability of these resistant mutants was evaluated in MARC-145 cells and compared with VR2332. As expected, the ribavirin-resistant mutants exhibited higher genetic stability compared to their parental virus.ConclusionsIn summary, ribavirin and 5-fluorouracil effectively suppressed PRRSV replication in MARC-145 cells. However, ribavirin-resistant mutants emerged when treated with low concentrations (≤0.2 mM) of ribavirin, and those mutants were genetically more stable during serial passages in cell culture.

The Attenuation Phenotype of a Ribavirin-Resistant Porcine Reproductive and Respiratory Syndrome Virus Is Maintained during Sequential Passages in Pigs

“…Two ribavirin-resistant mutant viruses (RVRp13 and RVRp22) were rescued through the serial passaging of VR2332 (a prototype strain of North American PRRSV) in the presence of 0.1 or 0.2 mM ribavirin, respectively, as characterized in a previous study (61). In the present study, both mutants, RVRp13 and RVRp22, were evaluated in terms of their genetic stability during replication in pigs in comparison to MLV (Ingelvac PRRS MLV; Boehringer Ingelheim, USA), a vaccine virus that was attenuated by sequential passages of VR2332 in MARC-145 cells (1,(64)(65)(66).…”

“…In the present study, both mutants, RVRp13 and RVRp22, were evaluated in terms of their genetic stability during replication in pigs in comparison to MLV (Ingelvac PRRS MLV; Boehringer Ingelheim, USA), a vaccine virus that was attenuated by sequential passages of VR2332 in MARC-145 cells (1,(64)(65)(66). For this purpose, each virus (RVRp13, RVRp22, and MLV) was purified by three rounds of plaque purification in MARC-145 cells to prepare a highly homologous virus inoculum, similar to a previously described method (61,67,68). The plaque-purified viruses were propagated again, and virus titers were measured in MARC-145 cells and stored at Ϫ80°C until use.…”

“…Virus titers were measured in PAMs and MARC-145 cells at the end of each passage using a microtitration infectivity assay (70) on rescued viruses in each virus-challenged group and were compared to the titers of inoculated viruses from the previous passage. The detailed procedure of virus titration has been described in a previous study (61). At 5 to 6 days postinoculation (dpi) of the virus assay, the virus titers were measured, and the virus antigen was detected by immunofluorescence microscopy.…”

“…However, mutagen-resistant mutants reemerged by escaping lethal mutagenesis when RNA viruses were sequentially passed in the sublethal concentrations of those antiviral mutagens, and those mutants were found to be genetically more stable than their parental viruses (56,(58)(59)(60). Therefore, in a previous study (61), this strategy was applied to select ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22), which are genetically and phenotypically stable through acquired resistance to random mutation. Consequently, in the present study, both of these ribavirin-resistant mutant viruses were evaluated in terms of their genetic and phenotypic stability during replication in three pig-to-pig passages in comparison with a commercial MLV vaccine (Ingelvac PRRS MLV).…”

Hematological adverse effects and pharmacokinetics of ribavirin in pigs following intramuscular administration

“…Ribavirin was demonstrated to have an antiviral effect in in vitro studies on porcine disease caused by viruses such as foot-and-mouth disease (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine epidemic diarrhea virus (PEDV) (Airaksinen et al, 2003;Kim & Lee, 2013;Khatun et al, 2015). However, still there is limited information available regarding in vivo application of ribavirin in food animals.…”