Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (μCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.Interleukin-6 | Ross River virus disease | viral arthritis | osteoclastogenesis A rthritogenic alphaviruses including Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), o'nyongnyong virus (ONNV), and Barmah Forest virus (BFV) are classified under the genus Alphavirus ("Old World" alphaviruses) of the Togaviridae family (1). RRV is a small, enveloped, positivesense single-stranded RNA virus transmitted by mosquitoes (2, 3). RRV disease (RRVD) in humans commonly affects the ankles, knees, and peripheral joints. The hallmarks of RRVD include incapacitating joint pain and polyarthralgias, with a level of disability comparable to rheumatoid arthritis (RA) (4, 5). Similar to RA, the onset of RRVD can be sudden and debilitating, and the prolonged manifestations of RRVD in some patients have been proposed to be due to the actions of proinflammatory mediators including interleukin-6 (IL-6), interleukin-1 (IL-1), and chemokine (C-C motif) ligand 2; monocyte chemotactic protein-1 (CCL2; MCP-1) (6-8).Recently, bone lesions in joints of CHIKV-infected patients have been reported (9), providing evidence that alphavirusinduced disease can result in bone pathologies (10, 11). In physiological conditions, osteoblasts (OBs) form bone, and this cell lineage also expresses both receptor activator of nuclear factorkappaB ligand (RANKL) and its soluble decoy receptor, osteoprotegerin (OPG). The expression of RANKL by the OB lineage is stimulated by IL-6 and IL-1β among other proinflammatory cytokines (12, 13), whereas CCL2 is thought to be an important chemoattractant for monocytic precursors during inflammatory processes (14,15). Together with an elevation in RANKL/OPG...