Identification and characterization of a Ross River virus variant that grows persistently in macrophages, shows altered disease kinetics in a mouse model, and exhibits resistance to type I interferon

Alphaviruses, such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV), cause outbreaks of human rheumatic disease worldwide. RRV is a positive-sense single-stranded RNA virus endemic to Australia and Papua New Guinea. In this study, we sought to establish an in vitro model of RRV evolution in response to cellular antiviral defense mechanisms. RRV was able to establish persistent infection in activated macrophages, and a small-plaque variant (RRV _PERS) was isolated after several weeks of culture. Nucleotide sequence analysis of RRV _PERS found several nucleotide differences in the nonstructural protein (nsP) region of the RRV _PERS genome. A point mutation was also detected in the E2 gene. Compared to the parent virus (RRV-T48), RRV _PERS showed significantly enhanced resistance to beta interferon (IFN-β)-stimulated antiviral activity. RRV _PERS infection of RAW 264.7 macrophages induced lower levels of IFN-β expression and production than infection with RRV-T48. RRV _PERS was also able to inhibit type I IFN signaling. Mice infected with RRV _PERS exhibited significantly enhanced disease severity and mortality compared to mice infected with RRV-T48. These results provide strong evidence that the cellular antiviral response can direct selective pressure for viral sequence evolution that impacts on virus fitness and sensitivity to alpha/beta IFN (IFN-α/β).