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

Abstract

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 ...

Attenuating Mutations in nsP1 Reveal Tissue-Specific Mechanisms for Control of Ross River Virus Infection

“…However, in vitro type I IFN sensitivity assays demonstrated that both RRV-T48-nsP1 6M and RRV-T48-nsP1 S79C; L224I were more sensitive than RRV T48 to type I IFN. Recently, Lidbury et al described a mutant RRV (RRV PERS ) isolated from persistently infected murine macrophages (63). In agreement with our findings, in which sensitivity to type I IFN in vitro correlated with pathogenicity in vivo, RRV PERS displayed increased virulence in mice and increased resistance to type I IFN in vitro.…”

“…In agreement with our findings, in which sensitivity to type I IFN in vitro correlated with pathogenicity in vivo, RRV PERS displayed increased virulence in mice and increased resistance to type I IFN in vitro. Five of the 12 amino acid differences between RRV T48 and RRV PERS were located in nsP1 (63). In addition, the mutations in nsP1 that arose during SINV passage in the presence of decreased levels of GTP also conferred enhanced sensitivity to interferon (49,64).…”

The growth of arthralgic Ross River virus is restricted in human monocytic cells

“…compared to a progeny production upper than 8 log PFU.mL -1 in HEK-293 and VERO cells (Lidbury et al 2011) ( Fig. 1B).…”

Understanding immunobiology of chikungunya virus disease using mouse models

“…RRV is able to establish persistent infections in many cell lines including mouse muscle cell (Eaton and Hapel, 1976), human synovial fibroblasts (Journeaux et al, 1987) and macrophages cell lines (Linn et al, 1996, Way et al, 2002, Lidbury et al, 2011. CHIKV was found to have persisted in spleen macrophages in macaques months after CHIKV infection (Labadie et al, 2010, Messaoudi et al, 2013.…”

“…The ability of alphaviruses to acquire mutations and better evade the antiviral effects of IFNα/β have been reported (Lidbury et al, 2011, Stoermer Burrack et al, 2014 with CHIKV and other alphaviruses having evolved strategies to counter the host's type I and II interferon responses Klimstra, 2008, Fros et al, 2010). Virus isolated from Rag-1 -/-mice on 100 and 429 days post-infection behaved no differently from parental virus (with respect to viraemia and foot swelling) when isolated from blood, expanded in C6/36 cells, and used to infect WT mice (data not shown).…”