title: Host-pathogen interactions during alphavirus infection
Creators: name: Lim, Elisa X
Publisher: name: Griffith University
Year: 2021
Subjects: subject: alphavirus infection
subject: joint-associated
subject: murine joints
subject: human primary chondrocytes

description: Arthritogenic alphavirus infection causes debilitating pain in the joints and muscles with many patients experiencing such symptoms chronically. However, there is insufficient evidence to explain the underlying causes behind symptoms of persistent arthralgia and myalgia. Joint-associated tissues are the main site of inflammation during alphavirus infection, and it has been shown that alphaviruses induce damage to the cartilage and synovium. Therefore, the cell types present in these tissues play critical roles in disease pathogenesis. The findings described in this thesis contribute to the general understanding of host-pathogen interactions during alphavirus infection of joint-associated cell types. Here, the analysis of murine joints revealed chondrocytes as a target of RRV infection (Chapter 1). Further evaluation of human primary chondrocytes and skeletal muscle cells through short-term in vitro cell culture showed that these cell types could support productive RRV infection. Our study presents the first evidence of the role of chondrocytes in alphavirus disease pathogenesis. Currently, there are gaps in our understanding of chronic alphavirus disease, especially in the absence of detectable viraemia after recovery from infection. Here, we have investigated the r sponses of several cell types in joint-associated tissues during chronic infection. Human primary cells and their corresponding cell line counterparts for chondrocytes, muscle cells and fibroblast-like synoviocytes (FLS) were infected with four alphaviruses of clinical importance, namely Ross River virus (RRV), Barmah Forest virus (BFV), chikungunya virus (CHIKV) and o’nyong’nyong virus (ONNV). We found that all cell types studied were able to retain residual alphaviral nucleic acids after recovery from infection despite several passages in culture (up to 10 weeks), indicating the potential of these cell types as reservoirs for the virus and/or viral RNA (Chapter 2). Regretfully, we were unable to determine the roles of the lingering viral nucleic acids though we hypothesise that they may play roles in causing chronic inflammation. During this study, we also established persistent alphavirus infection in chondrocyte C28/I2 and muscle RD cell lines (Chapter 2) and hypothesise that these two cell types could act as potential harbours for virus evasion from the immune system. The characterisation of genetic variants present in samples from persistent infections led to the identification of several mutations which could potentially be important for alphavirus persistence. We speculate that C28/I2 and RD cell lines are suitable candidates for exploring alphavirus evolution through selective pressures applied by in vitro serial passaging of infected cells. Our findings indicate that infected chondrocytes, muscle cells and FLS contribute to alphavirus disease pathogenesis through increased expression of pro-inflammatory cytokines associated with clinical disease such as IL-6, MCP-1 and IL-8 (Chapter 1 and 2). However, further studies are required to determine if the presence of residual alphaviral nucleic acids serves as PAMPs that are responsible for eliciting chronic inflammatory responses. While we have shown that RRV-infected chondrocytes play a role in causing alphavirus-induced inflammation, we also observed that these cells cause cartilage damage through disruption of ECM homeostasis. As the main cell type of the cartilage, chondrocytes are responsible for the regulation of ECM synthesis and degradation. During RRV-infection of chondrocytes, we observed reduced gene expression of key ECM constituents COL1A1, COL2A1 and ACAN and elevated gene expression of ECM breakdown enzymes like HPSE, ADAMTS4 and MMP9 (Chapter 1, 2 and 3). We also observed evidence of this through our transcriptomic analysis of RRVinfected and uninfected bystander chondrocytes. This is also the first study that investigates the direct and indirect responses to alphavirus infection of chondrocyte (Chapter 3). As an avascular tissue type, chondrocytes are not easily accessible to virus infection. However, we found evidence of RRV RNA in the chondrocytes of infected mice (Chapter 1) and have shown that these cells are susceptible to alphavirus infection (Chapters 1-4). Therefore, we can only speculate on the possible routes of alphavirus infection of chondrocytes. The use of in vitro chondrocyte models with complex ECM architecture allows for greater physiological relevance in the study of cartilage and their responses to alphavirus infection. The synovium is a neighbouring tissue with access to the blood supply network and provides nutrients to the cartilage. Therefore, it is possible that chondrocytes can acquire alphavirus infection via the synovium. Fibroblast-like synoviocytes (FLS) are the main resident cell type of the synovium and maintains the synovial fluid through the expression of ECM components and breakdown enzymes like MMP3. We found that interactions between chondrocytes and FLS result in increased viral infectivity profiles (Chapter 4). Our study also demonstrates that the ECM surrounding the chondrocytes acts as a physical barrier that prevents access to virus particles. Treatment of cells with MMP3 was able to loosen the interactions of the ECM and expose the chondrocytes to virus infection, resulting in greater virus attachment and infectivity compared to non-treated cells. Taken together, this thesis presents key findings on the possible mechanisms involved in alphavirus disease pathogenesis and the roles of cell types of joint-associated tissues in causing the chronic symptoms of joint and muscle pain felt by the majority of infected patients.