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Mosquito-borne viruses, or arboviruses, have been part of the infectious disease landscape for centuries, and are often, but not exclusively, endemic to equatorial and subtropical regions of the world. The past two decades saw the re-emergence of arthritogenic alphaviruses, a genus of arboviruses that includes several members that cause severe arthritic disease. Recent outbreaks further highlight the substantial public health burden caused by these viruses. Arthritogenic alphaviruses are often reported in the context of focused outbreaks in specific regions (eg, Caribbean, southeast Asia, and Indian Ocean) and cause debilitating acute disease that can extend to chronic manifestations for years after infection. These viruses are classified among several antigenic complexes, span a range of hosts and mosquito vectors, and can be distributed along specific geographical locations. In this Review, we highlight key features of alphaviruses that are known to cause arthritic disease in humans and outline the present findings pertaining to classification, immunogenicity, pathogenesis, and experimental approaches aimed at limiting disease manifestations. Although the most prominent alphavirus outbreaks in the past 15 years featured chikungunya virus, and a large body of work has been dedicated to understanding chikungunya disease mechanisms, this Review will instead focus on other arthritogenic alphaviruses that have been identified globally and provide a comprehensive appraisal of present and future research directions.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain–Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml−1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus associated with a robust systemic infection and an acute inflammatory rheumatic disease. A high fiber diet has been widely promoted for its ability to ameliorate inflammatory diseases. Fiber is fermented in the gut into short chain fatty acids such as acetate, propionate, and butyrate, which enter the circulation providing systemic anti-inflammatory activities. Herein we show that mice fed a high fiber diet show a clear exacerbation of CHIKV arthropathy, with increased edema and neutrophil infiltrates. RNA-Seq analyses illustrated that a high fiber diet, in this setting, promoted a range of pro-neutrophil responses including Th17/IL-17. Gene Set Enrichment Analyses demonstrated significant similarities with mouse models of inflammatory psoriasis and significant depression of macrophage resolution phase signatures in the CHIKV arthritic lesions from mice fed a high fiber diet. Supplementation of the drinking water with butyrate also increased edema after CHIKV infection. However, the mechanisms involved were different, with modulation of AP-1 and NF-κB responses identified, potentially implicating deoptimization of endothelial barrier repair. Thus, neither fiber nor short chain fatty acids provided benefits in this acute infectious disease setting, which is characterized by widespread viral cytopathic effects and a need for tissue repair.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/-and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/-mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. Trial Registration: ClinicalTrials.gov NCT00281294 © 2017 Wilson et al.

Chikungunya virus (CHIKV) is a mosquito-borne, single-stranded, positive-sense RNA virus (genus alphavirus) that has caused sporadic outbreaks of predominantly rheumatic disease every 2–50 years, primarily in Africa and Asia (1). Most recently, during 2004–2011, CHIKV produced the largest epidemic ever recorded for an alphavirus, affecting an estimated 1.4–6 million patients. Imported cases were also reported in nearly 40 countries, including Europe, Japan, and the US. The word “chikungunya” is derived from the Makonde language (Tanzania) and means “that which bends up,” referring to the severe joint pain–induced posture of afflicted individuals (1,2). CHIKV belongs to a group of mosquito-borne arthritogenic alphaviruses, which include the Australasian Ross River virus (RRV) and Barmah Forest virus, the African o’nyong-nyong virus, and the European Ockelbo and Pogosta viruses(1). Although many viruses can cause arthralgia/arthritis, none do so with the reliability of these alphaviruses. In adults, symptomatic infections are nearly always associated with arthropathy (1,3). The disease is characterized by acute and chronic polyarthritis/polyarthralgia, which is usually symmetric and often incapacitating. Other symptoms, such as fever, rash, myalgia, and/or fatigue, are often also present during the acute phase (1). The joints most commonly affected are multiple peripheral small joints (interphalangeal joints, wrists, and ankles) and large joints such as the shoulders, knees, and spine. The arthropathy usually resolves progressively over several weeks to months, usually without long-term sequelae, although CHIKV can sometimes produce severe disease manifestations and mortality (1,2). Chronic alpha-viral rheumatic disease is likely due to the persistence of viral replication in the joint tissue (1,4–6), with no evidence that autoimmune responses are responsible (3). Alphaviral arthritides are generally treated with simple analgesics, such as paracetamol and/or nonsteroidal antiinflammatory drugs (NSAIDs), which can provide relief, although symptom reduction is often inadequate (1,2). The small market size for alphaviral arthritides generally, and the rapid, sporadic, and unpredictable nature of outbreaks caused by certain alpha-viruses like CHIKV (1), present major hurdles for the development and deployment of virus-specific interventions such as vaccines (7) or antibodies (8). Thus, therapeutic drug treatment will likely remain the only option for most patients. A number of new treatments for CHIKV disease have been investigated. Unfortunately, chloroquine treatment was ineffective in human trials (9). Findings from murine studies suggest that interferon-α (IFNα) treatment is only effective if given before infection (10), and that anti–tumor necrosis factor (anti-TNF) agents may, if administered during the acute phase, exacerbate disease (11). Steroid treatment (12) and steroid treatment combined with NSAIDs (13) appeared to provide clinical benefit in patients with RRV disease and in those with CHIKV disease, respectively; however, steroid treatment may not be appropriate for these self-limiting diseases, given the potential side effects (12). The most active area of antirheumatic drug development is for RA (14–16). The market size is large, with an estimated RA prevalence of ∼1% among adult white populations in Europe and the US. Importantly, there is also a growing awareness that ideal new therapeutic agents for RA should not compromise antipathogen immunity (16), a consideration that is also clearly important in treatments of viral arthritides (3). Although there are some similarities between alphaviral arthritides and RA (3), there are also key differences. RA is generally a progressive disease that results in bone and cartilage erosion, has a female:male prevalence ratio of ∼2.5:1, is more common in people older than age 65 years, is associated with specific HLA–DRB1 alleles (17), involves neutrophils, and is believed to be driven by autoimmune T cells (primarily Th17) and B cells specific for citrullinated proteins (18). In contrast, alphaviral arthritides are generally self-limiting, do not normally show erosive changes, have a female:male prevalence ratio of ∼1.2:1, are rare in children, are characterized by mononuclear infiltrates (2,4,5,10), have no established HLA association, and have T and B cell responses that are generally directed at CHIKV antigens (5,10). Results from murine studies also suggest that the contribution of T and B cells to alphaviral rheumatic disease may be limited (19–21). We recently developed an experimental model of CHIKV arthritis in adult wild-type mice that mimics many of the features of human CHIKV disease, including a 4–5-day viremia that is followed by arthritic disease (10). In the present study, we report data from a microarray analysis of foot tissues from mice following infection with 2 CHIKV isolates, an Asian isolate from the 1960s and an isolate from the recent epidemic on Reunion Island (10). These data were used to identify a consensus CHIKV arthritis gene signature, which was compared with the signatures previously reported for patients with RA (22) and for collagen-induced arthritis (CIA), a mouse model of RA (23). There was a surprisingly significant overlap between the CHIKV arthritis and the RA gene signatures, and the CHIKV arthritis and the CIA gene signatures. These results provide hope that at least some of the antiinflammatory drugs and biologic agents being developed for RA might also be effective in the treatment of alphaviral arthritides.

Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities., Author summary Chikungunya virus (CHIKV) and Ross River virus are mosquito-borne alphaviruses that cause epidemics of human arthritic disease that usually last from weeks to months. Arthropathy predominantly manifests in the joints of limbs and the joints at the extremities (e.g. hand and feet). Herein we show a surprisingly large reduction in viral loads and foot arthropathy in mice when animals were housed at 30°C rather than the conventional 22°C, with the feet of the former mice being ≈3–4°C warmer. Using RNA-Seq analyses of mice feet, we illustrate that this small increase in temperature results in a significant increase in both the unfolded protein response and anti-viral type I interferon responses. Taken together these results suggest that the predominantly peripheral alphaviral arthropathy is due to the usually slightly lower temperature of limbs and extremities, which results in less effective type I interferon responses and a subsequent increase in viral loads and ensuing arthritic disease.

Chikungunya virus (CHIKV) is a mosquito borne alphavirus, whose primary disease manifestation in humans is acute and chronic, often debilitating polyarthritis/polyathralgia. Current treatments are often inadequate. This thesis aims to elucidate the roles of several immune components in CHIKV disease, using a mouse model of CHIKV infection and disease, which recapitulates many aspects of human disease. An improved understanding of the immunobiology of CHIKV is required for future development of new interventions for this recently re-emergent virus. CHIKV polyarthritis/polyathralgia is associated with a prolific monocytes/macrophage infiltration of musculoskeletal tissue in humans, monkeys and mice. The infection in all species is associated with high levels of chemokine (C-C motif) ligand 2 (CCL2), a chemokine that binds chemokine (C-C motif) receptor 2 (CCR2) and is involved in recruitment of monocytes to the sites of infection. Targeting CCL2 or CCR2 signalling has thus been proposed as a potential treatment option for CHIKV disease. Results herein show that CHIKV infection of mice deficient in CCR2 led to a significantly more pronounced and prolonged arthritis that was associated with cartilage damage. The prominent monocytes/macrophages infiltrate seen in wild-type mice was replaced by a prolific neutrophil infiltrate in CCR2-/- mice. The switch in the arthritic infiltrate was associated with changes in the expression of multiple inflammatory mediators, with the loss of anti- inflammatory macrophages and their activities (e.g. efferocytosis) also implicated in the exacerbated inflammatory response. These results illustrated that the recruited monocytes/macrophages in wild-type mice (although associated with acute disease) also have a protective role in CHIKV arthritis. These results suggest caution may be warranted when considering therapeutic targeting of CCR2 for treatment of CHIKV arthritis (Chapter 3). To study the role of different components of the immune system in CHIKV viraemia and disease, mice deficient in B cells, T cells, CD4+ T cells, natural killer cells and IFN-g were infected with CHIKV. Results herein confirmed that B cells are crucial for controlling CHIKV viraemia, but showed that T cells also play a role, albeit secondary to antibodies. B cell deficient mice experienced a life long viraemia with little overt pathology evident, supporting the view that CHIKV disease is largely an immunopathology. In T and B cell deficient mice, viraemia was likely controlled by chronic up-regulation of tumour necrosis factor and interferon gamma, but not interferon alpha/beta. Importantly, CD4+ T cells and interferon gamma were shown to play important roles in acute CHIKV arthritis, with T cells previously thought not to play a significant role (Chapter 4). CHIKV disease in humans is often associated with chronic arthritic symptoms that persist for months, occasionally over a year, after CHIKV infection. Persistence of CHIKV has been postulated to be responsible for the chronic arthritis. To further investigate this issue, chronic infection and disease parameters were analysed in the established wild-type mouse model of acute CHIKV arthritis. Viraemia is cleared day 4-5 post-infection and neutralising antibodies are detected as early as day 4 post-infection, and rose significantly thereafter. Despite the high levels of neutralising antibodies, replication competent CHIKV could be recovered from feet of infected mice up to day 14 post-infection. Importantly, significant levels of CHIKV RNA (including negative strand RNA) could be detected by quantitative real-time polymerase chain reaction in feet up to day 100 post-infection. Interferon stimulated gene 54, a factor that is up-regulated in response to viral infection and double stranded RNA, was found also to be persistently up-regulated in these feet, suggesting ongoing replication of viral RNA. Microarray analysis of these feet revealed that (i) many of the pathways up-regulated during acute arthritis were maintained during chronic infection, (ii) on-going local inflammatory responses were dominated by type I IFN responses, and (iii) many of the inflammatory pathways identified in chronically infected mouse feet were also found in chronic CHIKV patients. These observations suggest that chronic disease is due to ongoing inflammation stimulated by persistently replicating viral RNA and viral proteins encoded by that RNA (Chapter 5). In conclusion, this thesis has shown, that inflammatory CCR2+ monocyte can be critical for preventing excessive pathology and resolving inflammation, that distinct immune factors control CHIKV viraemia and arthritis, and that long-term persistence of replicating CHIKV RNA might be the cause of protracted arthritic manifestations in CHIKV patients. These studies will hopefully inform future development of intervention for CHIKV.

Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2 −/− mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2 −/− mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2 −/− mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2 −/− mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2 + monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. IMPORTANCE Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.