Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA's physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA's bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

A high-dose inoculum size results in persistent viral infection and arthritis in mice infected with chikungunya virus.

Chikungunya virus (CHIKV) is an emerging mosquito-transmitted alphavirus that leads to acute fever and chronic debilitating polyarthralgia. To date, the mechanism underlying chronic recurrent arthralgia is unknown. In the present study, newborn wild-type C57BL/6 mice were infected with CHIKV, and the virological and pathological features of CHIKV infection were analyzed over a period of 50 days. Acute viral infection was readily established by footpad inoculation of CHIKV at doses ranging from 10 plaque forming unit (PFU) to 106 PFU, during which inoculation dose-dependent viral RNA and skeletal muscle damage were detected in the foot tissues. However, persistent CHIKV was observed only when the mice were infected with a high dose of 106 PFU of CHIKV, in which low copy numbers (103-104) of viral positive strand RNA were continuously detectable in the feet from 29 to 50 dpi, along with a low level and progressive reduction in virus-specific CD8+ T cell responses. In contrast, viral negative strand RNA was detected at 50 dpi but not at 29 dpi and was accompanied by significant local skeletal muscle damage at 50 dpi when mild synovial hyperplasia appeared in the foot joints, although the damage was briefly repaired at 29 dpi. These results demonstrated that a high viral inoculation dose leads to viral persistence and progression to chronic tissue damage after recovery from acute infection. Taken together, these results provide a useful tool for elucidating the pathogenesis of persistent CHIKV infection and viral relapse-associated chronic arthritis.

Tender and swollen joint counts are poorly associated with disability in chikungunya arthritis compared to rheumatoid arthritis.

Chronic rheumatological manifestations similar to those of rheumatoid arthritis (RA) are described after chikungunya virus infection. We aimed to compare the relevance of joint counts and symptoms to clinical outcomes in RA and chronic chikungunya disease. Forty patients with chronic chikungunya arthralgia and 40 patients with RA were enrolled in a cross-sectional study. The association of tenderness and swelling, clinically assessed in 28 joints, and patient evaluations of pain and musculoskeletal stiffness with modified Health Assessment Questionnaire (HAQ) and quality of life (QoL) assessments were investigated. Tender and swollen joint counts, pain and stiffness scores were all associated with the HAQ disability index in RA (all r > 0.55, p ≤ 0.0002), but only stiffness was significantly associated with disability in chikungunya (r = 0.38, p = 0.02). Joint counts, pain and stiffness were also associated with most QoL domains in RA patients. In contrast, in chikungunya disease, tender joint counts were associated only with one QoL domain and swollen joints for none, while pain and stiffness were associated with several domains. Our results confirm the relevance of joint counts in RA, but suggest that in chronic chikungunya disease, joint counts have more limited value. Stiffness and pain score may be more important to quantify chikungunya arthritis impact.

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Persistent Joint Pain Following Arthropod Virus Infections.

PURPOSE OF REVIEW: Persistent joint pain is a common manifestation of arthropod-borne viral infections and can cause long-term disability. We review the epidemiology, pathophysiology, diagnosis, and management of arthritogenic alphavirus infection. RECENT FINDINGS: The global re-emergence of alphaviral outbreaks has led to an increase in virus-induced arthralgia and arthritis. Alphaviruses, including Chikungunya, O'nyong'nyong, Sindbis, Barmah Forest, Ross River, and Mayaro viruses, are associated with acute and/or chronic rheumatic symptoms. Identification of Mxra8 as a viral entry receptor in the alphaviral replication pathway creates opportunities for treatment and prevention. Recent evidence suggesting virus does not persist in synovial fluid during chronic chikungunya infection indicates that immunomodulators may be given safely. The etiology of persistent joint pain after alphavirus infection is still poorly understood. New diagnostic tools along and evidence-based treatment could significantly improve morbidity and long-term disability.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

Robust COX-2-mediated prostaglandin response may drive arthralgia and bone destruction in patients with chronic inflammation post-chikungunya.

Patients following infection by chikungunya virus (CHIKV) can suffer for months to years from arthralgia and arthritis. Interestingly, methotrexate (MTX) a major immune-regulatory drug has proved to be of clinical benefit. We have previously shown that CHIKV can persist in the joint of one patient 18 months post-infection and plausibly driving chronic joint inflammation but through ill-characterized mechanisms. We have pursued our investigations and report novel histological and in vitro data arguing for a plausible role of a COX-2-mediated inflammatory response post-CHIKV. In the joint, we found a robust COX-2 staining on endothelial cells, synovial fibroblasts and more prominently on multinucleated giant cells identified as CD11c+ osteoclasts known to be involved in bone destruction. The joint tissue was also strongly stained for CD3, CD8, CD45, CD14, CD68, CD31, CD34, MMP2, and VEGF (but not for NO synthase and two B cell markers). Dendritic cells were rarely detected. Primary human synovial fibroblasts were infected with CHIKV or stimulated either by the synthetic molecule polyriboinosinic:polyribocytidylic acid (PIC) to mimic chronic viral infection or cytokines. First, we found that PIC and CHIKV enhanced mRNA expression of COX-2. We further found that PIC but not CHIKV increased the mRNA levels of cPLA2α and of mPGES-1, two other central enzymes in PGE2 production. IFNß upregulated cPLA2α and COX-2 transcription levels but failed to modulated mPGES-1 mRNA expression. Moreover, PIC, CHIKV and IFNß decreased mRNA expression of the PGE2 degrading enzyme 15-PGDH. Interestingly, MTX failed to control the expression of all these enzymes. In sharp contrast, dexamethasone was able to control the capacity of pro-inflammatory cytokines, IL-1ß as well as TNFα, to stimulate mRNA levels of cPLA2α, COX-2 and mPGES-1. These original data argue for a concerted action of CHIKV (including viral RNA) and cytokines plausibly released from recruited leukocytes to drive a major COX-2-mediated PGE2 proinflammatory responses to induce viral arthritis.

Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity.

While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)-a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase-deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.

A Critical Role for STING Signaling in Limiting Pathogenesis of Chikungunya Virus.

The stimulator of interferon gene (STING) pathway controls both DNA and RNA virus infection. STING is essential for induction of innate immune responses during DNA virus infection, while its mechanism against RNA virus remains largely elusive. We show that STING signaling is crucial for restricting chikungunya virus infection and arthritis pathogenesis. Sting-deficient mice (Stinggt/gt) had elevated viremia throughout the viremic stage and viral burden in feet transiently, with a normal type I IFN response. Stinggt/gt mice presented much greater foot swelling, joint damage, and immune cell infiltration than wild-type mice. Intriguingly, expression of interferon-γ and Cxcl10 was continuously upregulated by approximately 7 to 10-fold and further elevated in Stinggt/gt mice synchronously with arthritis progression. However, expression of chemoattractants for and activators of neutrophils, Cxcl5, Cxcl7, and Cxcr2 was suppressed in Stinggt/gt joints. These results demonstrate that STING deficiency leads to an aberrant chemokine response that promotes pathogenesis of CHIKV arthritis.

Zika Virus and Arthritis/Arthralgia: A Systematic Review and Meta-Analysis.

Dengue, chikungunya and Zika viruses share similar disease features, rendering them difficult to distinguish clinically. Incapacitating arthralgia/arthritis is a specific manifestation associated with chikungunya virus infection. However, the profile of arthralgia/arthritis in Zika virus (ZIKV) cases has not been well characterized. Articles were extracted from PubMed and Scopus databases reporting original data from patients with arthralgia/arthritis, according to the Cochrane Collaboration. Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, 137 articles reporting ZIKV-associated joint symptoms were reviewed. Arthralgia was more frequently reported (n = 124 from case studies, n = 1779 from population-based studies) than arthritis (n = 7 and n = 121, respectively). Arthralgia was resolved in <1 week in 54%, and within 1-2 weeks in 40% of cases. The meta-analysis of cases in population-based studies identified a pooled prevalence of 53.55% for arthralgia. The pooled prevalence of arthralgia/arthritis during outbreaks depended on the geographic location, with a higher joint symptom burden observed in the Americas compared to South East Asia (Brazil: 60.79%; Puerto Rico: 68.89% and South East Asia: 26.46%). We conclude that non-specific constitutional arthralgia is the most common joint manifestation during ZIKV infection, being present in nearly half of cases but resolving by two weeks in >90% of these. We found no evidence of chronic rheumatic manifestations following ZIKV infection.

Sequencing and phylogenetic analysis of partial S1 genes of avian orthoreovirus isolates in Shandong province during 2015-2017.

Outbreaks of avian orthoreovirus (ARV) infection with primary symptoms of arthritis/tenosynovitis syndrome have been occurring more frequently in broiler flocks in China in recent years. This study aimed to investigate the genetic characteristics of ARV field strains in broiler flocks exhibiting arthritis/tenosynovitis syndrome from 9 cities in Shandong province during 2015 to 2017. A total of 64 synovial and tendon samples were obtained from broilers with significant arthritis/tenosynovitis syndrome, and 21 ARV field strains were obtained. Phylogenetic analysis of the σC nt/aa sequences revealed that only 4 isolates were clustered in genotype I, including vaccine strains S1133, 1733, and most of the ARV field strains identified previously in China. Eleven and 6 ARV field isolates were identified in genotypes II and V, sharing 70.9 to 76.0% and 53.0 to 55.2% nt identities with the vaccine strains, respectively. Previous studies in China have not reported these 2 serotypes of field strains, and prevalence of these ARV variants may be increasing in Chinese broiler flocks. Results of this study suggest that large-scale investigation of epidemic ARV should be conducted to explore the genetic diversity of ARV field isolates in China.

[Chronic chikungunya arthritis]. / Chronische Chikungunya-Arthritis.

This article describes the case of a 48-year-old female traveller returning from Bangladesh who presented with persisting pain and joint swelling due to chikungunya virus infection, which had persisted for months. Typical symptoms are a high fever and musculoskeletal disorders, which can last for months up to years. The chronic inflammatory form is treated similarly to those recommended for other chronic inflammatory joint diseases. Due to the increasing prevalence of the virus and its vectors as well as the unbroken travel activity, an increase in imported cases in Europe and establishment of the pathogen in southern Europe are to be expected.

Chikungunya Virus Evades Antiviral CD8+ T Cell Responses To Establish Persistent Infection in Joint-Associated Tissues.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes explosive epidemics of a febrile illness characterized by debilitating arthralgia and arthritis that can endure for months to years following infection. In mouse models, CHIKV persists in joint tissues for weeks to months and is associated with chronic synovitis. Using a recombinant CHIKV strain encoding a CD8+ T cell receptor epitope from ovalbumin, as well as a viral peptide-specific major histocompatibility complex class I tetramer, we interrogated CD8+ T cell responses during CHIKV infection. Epitope-specific CD8+ T cells, which were reduced in Batf3-/- and Wdfy4-/- mice with known defects in antigen cross-presentation, accumulated in joint tissue and the spleen. Antigen-specific ex vivo restimulation assays and in vivo killing assays demonstrated that CD8+ T cells produce cytokine and have cytolytic activity. Despite the induction of a virus-specific CD8+ T cell response, the CHIKV burden in joint-associated tissues and the spleen were equivalent in wild-type (WT) and CD8α-/- mice during both the acute and the chronic phases of infection. In comparison, CD8+ T cells were essential for the control of acute and chronic lymphocytic choriomeningitis virus infection in the joint and spleen. Moreover, adoptive transfer of virus-specific effector CD8+ T cells or immunization with a vaccine that induces virus-specific effector CD8+ T cells prior to infection enhanced the clearance of CHIKV infection in the spleen but had a minimal impact on CHIKV infection in the joint. Collectively, these data suggest that CHIKV establishes and maintains a persistent infection in joint-associated tissue in part by evading CD8+ T cell immunity.IMPORTANCE CHIKV is a reemerging mosquito-transmitted virus that in the last decade has spread into Europe, Asia, the Pacific Region, and the Americas. Joint pain, swelling, and stiffness can endure for months to years after CHIKV infection, and epidemics have a severe economic impact. Elucidating the mechanisms by which CHIKV subverts antiviral immunity to establish and maintain a persistent infection may lead to the development of new therapeutic strategies against chronic CHIKV disease. In this study, we found that CHIKV establishes and maintains a persistent infection in joint-associated tissue in part by evading antiviral CD8+ T cell immunity. Thus, immunomodulatory therapies that improve CD8+ T cell immune surveillance and clearance of CHIKV infection could be a strategy for mitigating chronic CHIKV disease.

A possible role for autoimmunity through molecular mimicry in alphavirus mediated arthritis.

Alphaviral infections are foremost in causing debilitating clinical outcomes in humans characterized by rheumatic arthritis like conditions. Though the presence of virus in joints and associated inflammation has been implicated as one of the reasons for the acute and chronic polyarthritis post alphaviral infections, the basis for rheumatic like outcomes is not clear. Through an in silico analysis, we have investigated the possibility of an autoimmune process mediated through molecular mimicry in alphaviral infection induced pathogenicity. Interestingly, sequence alignment of the structural polyproteins belonging to arthritogenic alphaviruses revealed conserved regions which share homology with human proteins implicated in rheumatoid arthritis (RA). These conserved regions were predicted to exhibit binding to HLA class II alleles, showcasing their potential to incite T cell help. Molecular docking of the viral peptide and the corresponding homologous region in the human protein onto HLA-DRB1 revealed strong similarities in their binding patterns. Linear and conformational B cell epitope prediction analyses showed that these potential mimics have high propensity to elicit an efficient B cell response. We thus propose that the origin of polyarthritis post-arthritogenic alphaviral infections may also be mediated through a hitherto unknown autoimmune response due to the presence of cross-reactive epitopes between viral and human proteins.

Long-Term Persistence of Chikungunya Virus-Associated Manifestations and Anti-Chikungunya Virus Antibody in Southern Thailand: 5 Years After an Outbreak in 2008-2009.

Chikungunya fever, a disease caused by chikungunya virus (CHIKV), reemerged and affected over 52,000 people in southern Thailand in 2008 and 2009. The CHIKV strain involved in this outbreak was the East Central South African (ECSA) strain with the E1-A226V mutation. The prevalence of CHIKV-associated chronic discomfort varied by virus lineage. This retrospective cohort study aims to describe the CHIKV-related symptoms persisting in CHIKV-affected patients, related factors, and the presence of anti-CHIKV immunoglobulin G (IgG) antibodies 5 years after the onset of disease. From 5,344 of the study population screened, a total of 89 affected patients reported persistent arthralgia 5 years after the disease onset (nonrecovery rate = 1.7%). Of the 141 affected patients enrolled, 122 cases (86.5%; 77 cases with persistent arthralgia and 45 cases of fully recovered) still had detectable levels of anti-CHIKV IgG antibodies. Long-term persistence of chronic joint symptoms is associated with the severity of the disease during the initial phase of the infection, but not gender, age, or comorbidities. The common manifestations were arthralgia (75.3%), morning joint stiffness (39.0%), muscle pain (19.5%), and occasional joint swelling (16.9%). Chronic joint symptoms could occur in either a fluctuating or a persistent manner and usually caused moderate pain. The joints affected were mainly fingers (71.4%), wrists (51.9%), and knees (50.6%). Most patients had polyarthralgia with symmetrical joint involvement. In some cases with persistent arthralgia, atypical manifestations, including severe depression with suicide attempts, severe weight loss, and severe hair loss, were found, and some patients still experienced severe joint pain.