Identification of Natural Molecular Determinants of Ross River Virus Type I Interferon Modulation.

Ross River virus (RRV) belongs to the genus Alphavirus and is prevalent in Australia. RRV infection can cause arthritic symptoms in patients and may include rash, fever, arthralgia, and myalgia. Type I interferons (IFN) are the primary antiviral cytokines and trigger activation of the host innate immune system to suppress the replication of invading viruses. Alphaviruses are able to subvert the type I IFN system, but the mechanisms used are ill defined. In this study, seven RRV field strains were analyzed for induction of and sensitivity to type I IFN. The sensitivities of these strains to human IFN-ß varied significantly and were highest for the RRV 2548 strain. Compared to prototype laboratory strain RRV-T48, RRV 2548 also induced higher type I IFN levels both in vitro and in vivo and caused milder disease. To identify the determinants involved in type I IFN modulation, the region encoding the nonstructural proteins (nsPs) of RRV 2548 was sequenced, and 42 amino acid differences from RRV-T48 were identified. Using fragment swapping and site-directed mutagenesis, we discovered that substitutions E402A and R522Q in nsP1 as well as Q619R in nsP2 were responsible for increased sensitivity of RRV 2548 to type I IFN. In contrast, substitutions A31T, N219T, S580L, and Q619R in nsP2 led to induction of higher levels of type I IFN. With exception of E402A, all these variations are common for naturally occurring RRV strains. However, they are different from all known determinants of type I IFN modulation reported previously in nsPs of alphaviruses.IMPORTANCE By identifying natural Ross River virus (RRV) amino acid determinants for type I interferon (IFN) modulation, this study gives further insight into the mechanism of type I IFN modulation by alphaviruses. Here, the crucial role of type I IFN in the early stages of RRV disease pathogenesis is further demonstrated. This study also provides a comparison of the roles of different parts of the RRV nonstructural region in type I IFN modulation, highlighting the importance of nonstructural protein 1 (nsP1) and nsP2 in this process. Three substitutions in nsP1 and nsP2 were found to be independently associated with enhanced type I IFN sensitivity, and four independent substitutions in nsP2 were important in elevated type I IFN induction. Such evidence has clear implications for RRV immunobiology, persistence, and pathology. The identification of viral proteins that modulate type I IFN may also have importance for the pathogenesis of other alphaviruses.

Prophylactic Antiheparanase Activity by PG545 Is Antiviral In Vitro and Protects against Ross River Virus Disease in Mice.

Recently we reported on the efficacy of pentosan polysulfate (PPS), a heparan sulfate mimetic, to reduce the recruitment of inflammatory infiltrates and protect the cartilage matrix from degradation in Ross River virus (RRV)-infected PPS-treated mice. Here, we describe both prophylactic and therapeutic treatment with PG545, a low-molecular-weight heparan sulfate mimetic, for arthritogenic alphaviral infection. We first assessed antiviral activity in vitro through a 50% plaque reduction assay. Increasing concentrations of PG545 inhibited plaque formation prior to viral adsorption in viral strains RRV T48, Barmah Forest virus 2193, East/Central/South African chikungunya virus (CHIKV), and Asian CHIKV, suggesting a strong antiviral mode of action. The viral particle-compound dissociation constant was then evaluated through isothermal titration calorimetry. Furthermore, prophylactic RRV-infected PG545-treated mice had reduced viral titers in target organs corresponding to lower clinical scores of limb weakness and immune infiltrate recruitment. At peak disease, PG545-treated RRV-infected mice had lower concentrations of the matrix-degrading enzyme heparanase in conjunction with a protective effect on tissue morphology, as seen in the histopathology of skeletal muscle. Enzyme-linked immunosorbent assay quantification of cartilage oligomeric matrix protein and cross-linked C-telopeptides of type II collagen as well as knee histopathology showed increased matrix protein degradation and cartilage erosion in RRV-infected phosphate-buffered saline-treated mice compared to their PG545-treated RRV-infected counterparts. Taken together, these findings suggest that PG545 has a direct antiviral effect on arthritogenic alphaviral infection and curtails RRV-induced inflammatory disease when administered as a prophylaxis.

How myeloid cells contribute to the pathogenesis of prominent emerging zoonotic diseases.

Up to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014-2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host's innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus.

UNLABELLED: The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

Role of pentraxin 3 in shaping arthritogenic alphaviral disease: from enhanced viral replication to immunomodulation.

The rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (CHIKV) and Ross River virus (RRV), and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. The immune responses underlying alphavirus virulence remain enigmatic. We found that pentraxin 3 (PTX3) was highly expressed in CHIKV and RRV patients during acute disease. Overt expression of PTX3 in CHIKV patients was associated with increased viral load and disease severity. PTX3-deficient (PTX3(-/-)) mice acutely infected with RRV exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. Furthermore, binding of the N-terminal domain of PTX3 to RRV facilitated viral entry and replication. Thus, our study demonstrates the pivotal role of PTX3 in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis.

Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss.

Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (µCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.

Role of envelope N-linked glycosylation in Ross River virus virulence and transmission.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αß) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

Pentosan Polysulfate: a Novel Glycosaminoglycan-Like Molecule for Effective Treatment of Alphavirus-Induced Cartilage Destruction and Inflammatory Disease.

UNLABELLED: Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) cause large-scale epidemics of severe musculoskeletal disease and have been progressively expanding their global distribution. Since its introduction in July 2014, CHIKV now circulates in the United States. The hallmark of alphavirus disease is crippling pain and inflammation of the joints, a similar immunopathology to rheumatoid arthritis. The use of glycans as novel therapeutics is an area of research that has increased in recent years. Here, we describe the promising therapeutic potential of the glycosaminoglycan (GAG)-like molecule pentosan polysulfate (PPS) to alleviate virus-induced arthritis. Mouse models of RRV and CHIKV disease were used to characterize the extent of cartilage damage in infection and investigate the potential of PPS to treat disease. This was assessed using histological analysis, real-time PCR, and fluorescence-activated cell sorting (FACS). Alphaviral infection resulted in cartilage destruction, the severity of which was alleviated by PPS therapy during RRV and CHIKV clinical disease. The reduction in cartilage damage corresponded with a significant reduction in immune infiltrates. Using multiplex bead arrays, PPS treatment was found to have significantly increased the anti-inflammatory cytokine interleukin-10 and reduced proinflammatory cytokines, typically correlated with disease severity. Furthermore, we reveal that the severe RRV-induced joint pathology, including thinning of articular cartilage and loss of proteoglycans in the cartilage matrix, was diminished with treatment. PPS is a promising new therapy for alphavirus-induced arthritis, acting to preserve the cartilage matrix, which is damaged during alphavirus infection. Overall, the data demonstrate the potential of glycotherapeutics as a new class of treatment for infectious arthritis. IMPORTANCE: The hallmark of alphavirus disease is crippling pain and joint arthritis, which often has an extended duration. In the past year, CHIKV has expanded into the Americas, with approximately 1 million cases reported to date, whereas RRV continues to circulate in the South Pacific. Currently, there is no licensed specific treatment for alphavirus disease, and the increasing spread of infection highlights an urgent need for therapeutic intervention strategies. Pentosan polysulfate (PPS) is a glycan derivative that is orally bioavailable, has few toxic side effects, and is currently licensed under the name Elmiron for the treatment of cystitis in the United States. Our findings show that RRV infection damages the articular cartilage, including a loss of proteoglycans within the joint. Furthermore, treatment with PPS reduced the severity of both RRV- and CHIKV-induced musculoskeletal disease, including a reduction in inflammation and joint swelling, suggesting that PPS is a promising candidate for drug repurposing for the treatment of alphavirus-induced arthritis.

Bindarit, an inhibitor of monocyte chemotactic protein synthesis, protects against bone loss induced by chikungunya virus infection.

UNLABELLED: The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used microcomputed tomographic (µCT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in the proximal tibial epiphysis of CHIKV-infected mice compared to the results for mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. The expression levels of the monocyte chemoattractant proteins (MCPs), including MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCP inhibitor bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphavirus-induced bone loss. IMPORTANCE: Arthritogenic alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host proinflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) is important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicates in bone and triggers bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints, and bone loss can be ameliorated by treatment with an MCP-inhibiting drug, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favoring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans.

Dual proinflammatory and antiviral properties of pulmonary eosinophils in respiratory syncytial virus vaccine-enhanced disease.

UNLABELLED: Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCE: This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.

Mouse models of alphavirus-induced inflammatory disease.

Part of the Togaviridae family, alphaviruses are arthropod-borne viruses that are widely distributed throughout the globe. Alphaviruses are able to infect a variety of vertebrate hosts, but in humans, infection can result in extensive morbidity and mortality. Symptomatic infection can manifest as fever, an erythematous rash and/or significant inflammatory pathologies such as arthritis and encephalitis. Recent overwhelming outbreaks of alphaviral disease have highlighted the void in our understanding of alphavirus pathogenesis and the re-emergence of alphaviruses has given new impetus to anti-alphaviral drug design. In this review, the development of viable mouse models of Old Word and New World alphaviruses is examined. How mouse models that best replicate human disease have been used to elucidate the immunopathology of alphavirus pathogenesis and trial novel therapeutic discoveries is also discussed.

Characterization of Barmah Forest virus pathogenesis in a mouse model.

Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in human BFV infection are very similar to those of RRV. Although RRV disease has been studied extensively, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis, and to identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Here, to the best of our knowledge, we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to that of RRV. In comparison with RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-α, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge, this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understand disease pathogenesis. With an increasing number of BFV cases occurring annually, a better understanding of the disease mechanisms is essential for future therapeutic development.

Mannose binding lectin is required for alphavirus-induced arthritis/myositis.

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3(-/-) mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.

Macrophage migration inhibitory factor receptor CD74 mediates alphavirus-induced arthritis and myositis in murine models of alphavirus infection.

OBJECTIVE: Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses that are characterized by arthritis, arthralgia, and myalgia. In previous studies, we identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral diseases. The present study was undertaken to characterize the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides. METHODS: Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histologic analysis, real-time polymerase chain reaction, flow cytometry, and plaque assay. RESULTS: In comparison to wild-type mice, CD74-/- mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterized predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in the infected tissue of CD74-/- mice, but production of proinflammatory cytokines and chemokines was not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells, and serum MIF levels were significantly elevated in patients with RRV or CHIKV infection. CONCLUSION: CD74 appears to regulate immune responses to alphaviral infection through its effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease, and blocking these factors with novel therapeutic agents could substantially ameliorate the pathologic manifestations.

Critical role for macrophage migration inhibitory factor (MIF) in Ross River virus-induced arthritis and myositis.

Arthrogenic alphaviruses, such as Ross River virus (RRV), chikungunya, Sindbis, mayaro and o'nyong-nyong viruses circulate endemically worldwide, frequently causing outbreaks of polyarthritis. The exact mechanisms of how alphaviruses induce polyarthritis remain ill defined, although macrophages are known to play a key role. Macrophage migration inhibitory factor (MIF) is an important cytokine involved in rheumatoid arthritis pathogenesis. Here, we characterize the role of MIF in alphavirus-induced arthritides using a mouse model of RRV-induced arthritis, which has many characteristics of RRV disease in humans. RRV-infected WT mice developed severe disease associated with up-regulated MIF expression in serum and tissues, which corresponded to severe inflammation and tissue damage. MIF-deficient (MIF(-/-)) mice developed mild disease accompanied by a reduction in inflammatory infiltrates and muscle destruction in the tissues, despite having viral titers similar to WT mice. In addition, reconstitution of MIF into MIF(-/-) mice exacerbated RRV disease and treatment of mice with MIF antagonist ameliorated disease in WT mice. Collectively, these findings suggest that MIF plays a critical role in determining the clinical severity of alphavirus-induced musculoskeletal disease and may provide a target for the development of antiviral pharmaceuticals. The prospect being that early treatment with MIF-blocking pharmaceuticals may curtail the debilitating arthritis associated with alphaviral infections.

Synoviocytes assist in modulating the effect of Ross River virus infection in micromass-cultured primary human chondrocytes.

Introduction. Ross River virus (RRV) is a mosquito-borne virus prevalent in Australia and the islands of the South Pacific, where it causes an arthritogenic illness with a hallmark feature of severe joint pain. The joint space is a unique microenvironment that contains cartilage and synovial fluid. Chondrocytes and synoviocytes are crucial components of the joint space and are known targets of RRV infection.Hypothesis/Gap statement. Understanding the relationship between synoviocytes and chondrocytes during RRV infection will provide further insights into RRV-induced joint pathology.Methodology. To better understand the unique dynamics of these cells during RRV infection, we used primary chondrocytes cultured in physiologically relevant micromasses. We then directly infected micromass chondrocytes or infected primary fibroblast-like synoviocytes (FLS), co-cultured with micromass chondrocytes. Micromass cultures and supernatants were collected and analysed for viral load with a PCR array of target genes known to play a role in arthritis.Results. We show that RRV through direct or secondary infection in micromass chondrocytes modulates the expression of cellular factors that likely contribute to joint inflammation and disease pathology, as well as symptoms such as pain. More importantly, while we show that RRV can infect micromass-cultured chondrocytes via FLS infection, FLS themselves affect the regulation of cellular genes known to contribute to arthritis.Conclusion. Single-cell culture systems lack the complexity of in vivo systems, and understanding the interaction between cell populations is crucial for deciphering disease pathology, including for the development of effective therapeutic strategies.

Pathogenicity and virulence of chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted, RNA virus that causes an often-severe musculoskeletal illness characterized by fever, joint pain, and a range of debilitating symptoms. The virus has re-emerged as a global health threat in recent decades, spreading from its origin in Africa across Asia and the Americas, leading to widespread outbreaks impacting millions of people. Despite more than 50 years of research into the pathogenesis of CHIKV, there is still no curative treatment available. Current management of CHIKV infections primarily involves providing supportive care to alleviate symptoms and improve the patient's quality of life. Given the ongoing threat of CHIKV, there is an urgent need to better understand its pathogenesis. This understanding is crucial for deciphering the mechanisms underlying the disease and for developing effective strategies for both prevention and management. This review aims to provide a comprehensive overview of CHIKV and its pathogenesis, shedding light on the complex interactions of viral genetics, host factors, immune responses, and vector-related factors. By exploring these intricate connections, the review seeks to contribute to the knowledge base surrounding CHIKV, offering insights that may ultimately lead to more effective prevention and management strategies for this re-emerging global health threat.

Independent repeated mutations within the alphaviruses Ross River virus and Barmah Forest virus indicates convergent evolution and past positive selection in ancestral populations despite ongoing purifying selection.

Ross River virus (RRV) and Barmah Forest virus (BFV) are arthritogenic arthropod-borne viruses (arboviruses) that exhibit generalist host associations and share distributions in Australia and Papua New Guinea (PNG). Using stochastic mapping and discrete-trait phylogenetic analyses, we profiled the independent evolution of RRV and BFV signature mutations. Analysis of 186 RRV and 88 BFV genomes demonstrated their viral evolution trajectories have involved repeated selection of mutations, particularly in the nonstructural protein 1 (nsP1) and envelope 3 (E3) genes suggesting convergent evolution. Convergent mutations in the nsP1 genes of RRV (residues 248 and 441) and BFV (residues 297 and 447) may be involved with catalytic enzyme mechanisms and host membrane interactions during viral RNA replication and capping. Convergent E3 mutations (RRV site 59 and BFV site 57) may be associated with enzymatic furin activity and cleavage of E3 from protein precursors assisting viral maturation and infectivity. Given their requirement to replicate in disparate insect and vertebrate hosts, convergent evolution in RRV and BFV may represent a dynamic link between their requirement to selectively 'fine-tune' intracellular host interactions and viral replicative enzymatic processes. Despite evidence of evolutionary convergence, selection pressure analyses did not reveal any RRV or BFV amino acid sites under strong positive selection and only weak positive selection for nonstructural protein sites. These findings may indicate that their alphavirus ancestors were subject to positive selection events which predisposed ongoing pervasive convergent evolution, and this largely supports continued purifying selection in RRV and BFV populations during their replication in mosquito and vertebrate hosts.

Approaches to the treatment of disease induced by chikungunya virus.

Chikungunya virus, a re-emerging mosquito-borne alphavirus, causes fever, rash and persistent arthralgia/arthritis in humans. Severe outbreaks have occurred resulting in infections of millions of people in Southeast Asia and Africa. Currently there are no antiviral drugs or vaccines for prevention and treatment of chikungunya infections. Herein we report the current status of research on antiviral drugs and vaccines for chikungunya virus infections.

Detection of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) and heterologous Leishmania species antibodies among blood donors in a region of Australia with marsupial Leishmania endemicity.

OBJECTIVES: The Australian Leishmania (Mundinia) macropodum parasite causes cutaneous leishmaniasis among marsupial species. Although cutaneous leishmaniasis is a major public health burden worldwide, it is not clear if humans are naturally exposed to the unique L. macropodum. To assess whether humans have an immunoglobulin (Ig) G response to L. macropodum, we examined anti-Leishmania antibodies among humans residing in a region of marsupial Leishmania endemicity in Australia. METHODS: Using a serological enzyme-linked immunosorbent assay, we characterized Leishmania-specific IgG and IgG subclass responses to soluble Leishmania antigen from L. macropodum, and other Leishmania species (L. donovani, L. major, and L. mexicana) in 282 blood donor samples. RESULTS: We found that 20.57% of individuals demonstrated a positive total IgG response to L. macropodum. For individuals with antibodies to soluble Leishmania antigen from one Leishmania species, there was no increased likelihood of recognition to other Leishmania species. For samples with detectable L. macropodum IgG, IgG1 and IgG2 were the prevalent subclasses detected. CONCLUSION: It is not yet clear whether the IgG antibody detection in this study reflects exposure to Leishmania parasites or a cross-reactive immune response that was induced against an unrelated immunogen. Future studies should investigate whether L. macropodum can result in a viable infection in humans.