An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp(1) to Rp(5) were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp(1) to Rp(5) and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp(1) to RP5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp(5)-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 mu M and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutacrenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.