Chikungunya virus persists in joint-associated macrophages and promotes chronic disease in mice

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), Mayaro virus and Ross River virus, cause long-lasting musculoskeletal pain and inflammation. However, the mechanisms driving chronic disease remain unclear. Here, we used single-cell RNA sequencing, spatial transcriptomics and flow cytometry to investigate joint-associated tissues in alphavirus-infected mice at a late stage of infection. We identified an accumulation of inflammatory macrophages in joint-associated tissues with elevated expression of inflammatory markers. These cells harbour CHIKV RNA, suggesting ongoing viral replication during chronic disease. We also identified an accumulation of CD4⁺ T cells in these tissues expressing Ifng, and found that depletion of CD4⁺ T cells diminished major histocompatibility complex class II expression on joint macrophages, highlighting their potential role in inflammation. Treatment with a small molecule inhibitor of CHIKV replication during chronic disease reduced viral RNA and joint inflammation. Our data suggest that macrophages harbour replicating viral RNA and contribute to the sustained joint inflammation associated with chronic alphavirus disease.