Heterogeneity of clinical isolates of chikungunya virus and its impact on the responses of primary human fibroblast-like synoviocytes.

Low-passage clinical isolates of chikungunya virus (CHIKV) were found to be a mixture of large- and small-plaque viruses, with small-plaque viruses being the predominant species. To investigate the contribution of plaque variants to the pathology of the joint, primary human fibroblast-like synoviocytes (HFLS) were used. Large- and small-plaque viruses were purified from two clinical isolates, CHIKV-031C and CHIKV-033C, and were designated CHIKV-031L and CHIKV-031S and CHIKV-033L and CHIKV-033S, respectively. The replication efficiencies of these viruses in HFLSs were compared and it was found that CHIKV-031S and CHIKV-033S replicated with the highest efficiency, while the parental clinical isolates had the lowest efficiency. Interestingly, the cytopathic effects (CPE) induced by these viruses correlated with neither the efficiency of replication nor the plaque size. The small-plaque viruses and the clinical isolates induced cell death rapidly, while large-plaque viruses induced slow CPE in which only 50 % of the cells in infected cultures were rounded up and detached on day 5 of infection. The production of proinflammatory cytokines and chemokines from infected HFLSs was evaluated. The results showed that the large-plaque viruses and the clinical isolates, but not small-plaque variants, were potent inducers of IL-6, IL-8 and MCP-1, and were able to migrate monocytes/macrophages efficiently. Sequencing data revealed a number of differences in amino acid sequences between the small- and large-plaque viruses. The results suggest that it is common for clinical isolates of CHIKV to be heterogeneous, while the variants may have distinct roles in the pathology of the joint.

Differences in response of primary human myoblasts to infection with recent epidemic strains of Chikungunya virus isolated from patients with and without myalgia.

In addition to fever, rash, and arthralgia/arthritis, myalgia is another dominant symptom in Chikungunya virus (CHIKV) infection. How CHIKV induces myalgia is unclear. To better understand the viral factors involved in CHIKV-induced myalgia, CHIKVs were isolated from patients with and without myalgia designated myalgia-CHIKV and mild-CHIKV, respectively. The response of myoblasts to infection by the two groups of clinical isolates of CHIKV was investigated. Both groups of CHIKV replicated well in primary human myoblasts. However, the myalgia-CHIKVs replicated to a higher titer and caused the death of infected myoblast more rapidly than the mild-CHIKVs. CHIKV-infected myoblasts increased production of four out of five inflammatory cytokines examined (MCP-1, IP-10, MIP-1α, and IL-8) in comparison to mock-infected cells. Comparison between the myoblast inflammatory cytokine responses showed that myalgia-CHIKVs were stronger activators of cytokines than mild-CHIKVs. This means that recent epidemic strains of CHIKV exhibited different degrees of myoblast permissiveness as evidenced by differences in the ability to replicate and to stimulate inflammatory responses in myoblasts. This data suggest that the myopathic syndrome in recent epidemics is dependent upon the strain of CHIKV.

Osteoclastogenesis induced by CHIKV-infected fibroblast-like synoviocytes: a possible interplay between synoviocytes and monocytes/macrophages in CHIKV-induced arthralgia/arthritis.

Fibroblast-like synoviocytes are known to migrate from joint to joint and are proposed to be one of the key players in the inflammatory cascade amplification in rheumatoid arthritis patients. In the recent CHIKV epidemic, patients developed arthritis-like syndrome and the synoviocyte is one of the suspected players in CHIKV-induced polyarthritis. Thus, to learn more on this syndrome, the responses of fibroblast-like synoviocytes to chikungunya virus (CHIKV) infection, and the interaction between CHIKV-infected synoviocytes and phagocytes, were investigated. Primary human fibroblast-like synoviocyte (HFLS) cultures were infected with clinical isolates of CHIKV at an MOI of 0.001pfu/cell. Data indicated that HFLS are permissive to CHIKV replication, generating peak titers of 10(5)-10(6)pfu/ml. Interestingly, CHIKV-infected HFLS cultures secreted mainly the mediators that are responsible for phagocytes recruitment and differentiation (RANKL, IL-6, IL-8 and MCP-1) but not arthritogenic mediators (TNF-α, IL-1ß, MMP-1, MMP-2 or MMP-13). The interaction between CHIKV-infected synoviocytes and phagocytes was studied using UV-irradiated, CHIKV-infected HFLS supernatant. Data revealed that supernatants from CHIKV-infected HFLS cultures not only induced migration of primary human monocytes, but also drove monocytes/macrophages into osteoclast-like cells. These differentiated osteoclast-like cells produced high levels of TNF-α and IL-6, principal mediators of arthritis. This data suggests a potential interplay between infected HFLS and recruiting phagocytes which may responsible for the arthralgia/arthritis in CHIKV-infected patients.