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-␤)-stimulated antiviral activity. RRV PERS infection of RAW 264.7 macrophages induced lower levels of IFN-␤ 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-␣/␤).Arthritogenic alphaviruses are distributed globally and are maintained in nature by cycles of transmission between hematophagous arthropods (for example, mosquitoes) and enzootic vertebrate hosts (mammals, marsupials, or birds) (33, 53). Nearly all symptomatic infections with these alphaviruses manifest with joint symptoms (arthritis and arthralgia), with myalgia, rash, and lethargy also being common. Ross River virus (RRV) is an Australian alphavirus associated with chronic polyarthritis and causes up to 8,000 cases annually in Australia, with the number of cases reported in new localities increasing (42,55). During the late 1970s and early 1980s, a number of South Pacific island nations experienced a major outbreak of RRV disease (RRVD) affecting more than 50,000 people (17). More recently, a related virus, chikungunya virus (CHIKV), caused similar rheumatic disease in one-third of the population of Réunion Island in the Western Indian Ocean and an estimated 1.39 million cases in India (22,23,37,52). This outbreak was also associated for the first time with some severe clinical manifestation and mortality (37).In RRV-infected patients, the disease is usually severe at onset with a gradual resolution over 3 to 6 months (40). Alphavirus-induced rheumatic disease is thought to have a substantial immunopathological component. For example, in animal models tissue damage results from the induction of proinflammatory cytokines and chemokines and recruitment of macrophages in response to infection (29,43,55). RRV infects and replicates in human and mouse macrophages, and infection of the mouse macrophage cell line RAW 264.7 results in the establishment of ...

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