Date of Award
Doctor of Philosophy (PhD)
Chikungunya virus (CHIKV) is an arthritogenic alphavirus that during acute disease causes fever as well as severe joint and muscle pain. Chronic joint and muscle pain persists in a significant subset of patients, yet we still have a poor understanding of what drives this chronic disease. While replicating virus has not been detected in the joints of patients with chronic arthritis or in various animal models at chronic time points, persistent viral RNA can be detected for months after acute infection.
To identify the cells that could be contributing to chronic CHIKV pathogenesis, we developed recombinant viruses that express Cre recombinase (CHIKV-3ʹ-Cre and CHIKV-5ʹ-Cre). These viruses replicated in cell types targeted by CHIKV, including myoblasts and fibroblasts, and they induced acute arthritis in a murine model of CHIKV arthritis. Importantly, they also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type (WT) virus. CHIKV-3ʹ-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. The majority of these cells localized to the dermis and muscle, and immunofluorescence profiling revealed that these tdTomato+ cells were dermal and muscle fibroblasts and myofibers. Treatment with an antibody against Mxra8, a host receptor for CHIKV, reduced the levels of viral RNA and the total number of tdTomato+ cells in the chronic phase, with a preferential reduction in fibroblasts. Anti-Mxra8 treatment demonstrated a correlation between chronic viral RNA levels and the number of surviving tdTomato+ cells, thus suggesting that viral RNA can be found within these persistent tdTomato+ cells. This CHIKV-3ʹ-Cre and tdTomato reporter mouse system demonstrates that cells can survive CHIKV infection in vivo and represents a powerful tool to study the chronic pathogenesis of CHIKV infection.
In the process of optimizing this reporter system, we observed noticeable effects on pathogenesis depending on viral inoculum. Increasing the dose of CHIKV (WT, 5ʹ-Cre, and 3ʹ-Cre) was associated with an earlier peak of swelling and infectious titers in the ipsilateral ankle, earlier resolution of swelling, and faster clearance of infectious virus in the ipsilateral ankle and distal sites. This accelerated clearance phenotype could explain why levels of persistent viral RNA tended to be lower in the ipsilateral ankle with increasing initial inoculum.
Ultimately, our CHIKV-Cre system could be applied to many aspects of studying chronic CHIKV pathogenesis. For example, single cell or nucleus RNA-seq could help determine whether persistent viral RNA is concentrated in tdTomato+ cells, the nature of this persistent viral RNA, and whether the host transcriptome of tdTomato+ cells is altered. Electron microscopy could help determine the subcellular location of persistent viral RNA within tdTomato+ cells. CHIKV-Cre infection of Diphtheria toxin receptor (DTR) mice could allow for depletion of DTR+ cells and exploration of how these cells contribute to chronic pathogenesis. Overall, our CHIKV-Cre and tdTomato reporter mouse system marks myofibers and fibroblasts as cells that may harbor viral components and likely contribute to chronic CHIKV pathogenesis. Further characterization of these cells will no doubt aid in our understanding and development of potential treatments for this debilitating infection.
Chair and Committee
Deborah J. Lenschow
Adrianus C. Boon, Michael S. Diamond, Robyn S. Klein, Thaddeus S. Stappenbeck,
Young, Alissa Roxanne, "A Recombinant Virus and Reporter Mouse System to Study Chronic Chikungunya Virus Pathogenesis" (2018). Arts & Sciences Electronic Theses and Dissertations. 1705.