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ABSTRACT: Human metapneumovirus (hMPV) is a significant viral respiratory pathogen of infants and children, the elderly and immunocompromised individuals. Disease associated with hMPV infection resembles that of human respiratory syncytial virus (RSV) and includes bronchiolitis and pneumonia. The glycosylated G attachment protein of hMPV is required for viral entry in vivo and has also been identified as an inhibitor of innate immune responses.
We designed and validated two siRNA molecules against the G gene using A549 cells and demonstrated consistent 88-92% knock-down for one siRNA molecule, which was used in subsequent experiments. Significant reduction of G mRNA in A549 cells infected with hMPV did not result in a reduction in viral growth, nor did it significantly increase the production of type I interferon (α/β) in response to infection. However, there was a moderate increase in IFN-β mRNA expression in response to infection in siG-transfected cells compared to untransfected and si-mismatch-transfected cells. Expression of G by recombinant adenovirus did not affect type I IFN expression.
G has been previously described as a type I interferon antagonist, although our findings suggest it may not be a significant antagonist.
Virology Journal 06/2012; 9:105. · 2.34 Impact Factor
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The Journal of Infectious Diseases 05/2012; 206(3):455-7: 457-9. · 6.41 Impact Factor
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Bronwyn M Gunn,
Thomas E Morrison,
Alan C Whitmore,
Lance K Blevins,
Linda Hueston,
Robert J Fraser,
Lara J Herrero,
Ruben Ramirez,
Paul N Smith, Suresh Mahalingam,
Mark T Heise
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ABSTRACT: 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.
PLoS Pathogens 03/2012; 8(3):e1002586. · 9.13 Impact Factor
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ABSTRACT: In the past decade, chikungunya--a virus transmitted by Aedes spp mosquitoes--has re-emerged in Africa, southern and southeastern Asia, and the Indian Ocean Islands as the cause of large outbreaks of human disease. The disease is characterised by fever, headache, myalgia, rash, and both acute and persistent arthralgia. The disease can cause severe morbidity and, since 2005, fatality. The virus is endemic to tropical regions, but the spread of Aedes albopictus into Europe and the Americas coupled with high viraemia in infected travellers returning from endemic areas increases the risk that this virus could establish itself in new endemic regions. This Seminar focuses on the re-emergence of this disease, the clinical manifestations, pathogenesis of virus-induced arthralgia, diagnostic techniques, and various treatment modalities.
The Lancet 11/2011; 379(9816):662-71. · 38.28 Impact Factor
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ABSTRACT: Chikungunya virus (CHIKV) is associated with outbreaks of infectious rheumatic disease in humans. Using a mouse model of CHIKV arthritis and myositis, we show that tumor necrosis factor-α, interferon-γ, and monocyte chemotactic protein 1 (MCP-1) were dramatically induced in tissues from infected mice. The same factors were detected in the serum of patients with CHIKV-induced polyarthralgia and polyarthritis, with MCP-1 levels being particularly elevated. Bindarit (MCP inhibitor) treatment ameliorated CHIKV disease in mice. Histological analysis of muscle and joint tissues showed a reduction in inflammatory infiltrate in infected mice treated with bindarit. These results suggest that bindarit may be useful in treating CHIKV-induced arthritides in humans.
The Journal of Infectious Diseases 10/2011; 204(7):1026-30. · 6.41 Impact Factor
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09/2011; , ISBN: 978-953-307-694-2
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The Journal of Infectious Diseases 08/2011; 204(3):489-91. · 6.41 Impact Factor
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 07/2011; 108(29):12048-53. · 9.68 Impact Factor
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ABSTRACT: Animal models, which mimic human disease, are invaluable tools for understanding the mechanisms of disease pathogenesis and development of treatment strategies. In particular, animal models play important roles in the area of infectious arthritis. Alphaviruses, including Ross River virus (RRV), o'nyong-nyong virus, chikungunya virus (CHIKV), mayaro virus, Semliki Forest virus and sindbis virus, are globally distributed and cause transient illness characterized by fever, rash, myalgia, arthralgia and arthritis in humans. Severe forms of the disease result in chronic incapacitating arthralgia and arthritis. The mechanisms of how these viruses cause musculoskeletal disease are ill defined. In recent years, the use of a mouse model for RRV-induced disease has assisted in unraveling the pathobiology of infection and in discovering novel drugs to ameliorate disease. RRV as an infection model has the potential to provide key insights into such disease processes, particularly as many viruses, other than alphaviruses, are known to cause infectious arthritides. The emergence and outbreak of CHIKV in many parts of the world has necessitated the need to develop animal models of CHIKV disease. The development of non-human primate models of CHIKV disease has given insights into viral tropism and disease pathogenesis and facilitated the development of new treatment strategies. This review highlights the application of animal models of alphaviral diseases in the fundamental understanding of the mechanisms that contribute to disease and for defining the role that the immune response may have on disease pathogenesis, with the view of providing the foundation for new treatments.
Current drug targets 03/2011; 12(7):1024-36. · 3.93 Impact Factor
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ABSTRACT: Alphaviruses, such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV), cause outbreaks of human rheumatic disease worldwide. RRV is a positive-sense single-stranded RNA virus endemic to Australia and Papua New Guinea. In this study, we sought to establish an in vitro model of RRV evolution in response to cellular antiviral defense mechanisms. RRV was able to establish persistent infection in activated macrophages, and a small-plaque variant (RRV(PERS)) was isolated after several weeks of culture. Nucleotide sequence analysis of RRV(PERS) found several nucleotide differences in the nonstructural protein (nsP) region of the RRV(PERS) genome. A point mutation was also detected in the E2 gene. Compared to the parent virus (RRV-T48), RRV(PERS) showed significantly enhanced resistance to beta interferon (IFN-β)-stimulated antiviral activity. RRV(PERS) infection of RAW 264.7 macrophages induced lower levels of IFN-β expression and production than infection with RRV-T48. RRV(PERS) was also able to inhibit type I IFN signaling. Mice infected with RRV(PERS) exhibited significantly enhanced disease severity and mortality compared to mice infected with RRV-T48. These results provide strong evidence that the cellular antiviral response can direct selective pressure for viral sequence evolution that impacts on virus fitness and sensitivity to alpha/beta IFN (IFN-α/β).
Journal of Virology 03/2011; 85(11):5651-63. · 5.40 Impact Factor
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ABSTRACT: The viral determinants of alphavirus-induced rheumatic disease have not been elucidated. We identified an RRV strain (DC5692) which, in contrast to the T48 strain, does not induce musculoskeletal inflammation in a mouse model of RRV disease. Substitution of the RRV T48 strain nonstructural protein 1 (nsP1) coding sequence with that from strain DC5692 generated a virus that was attenuated in vivo despite similar viral loads in tissues. In contrast, substitution of the T48 PE2 coding region with the PE2 coding region from DC5692 resulted in attenuation in vivo and reduced viral loads in tissues. In gain of virulence experiments, substitution of the DC5692 strain nsP1 and PE2 coding regions with those from the T48 strain was sufficient to restore full virulence to the DC5692 strain. These findings indicate that determinants in both nsP1 and PE2 have critical and distinct roles in the pathogenesis of RRV-induced musculoskeletal inflammatory disease in mice.
Virology 02/2011; 410(1):216-27. · 3.35 Impact Factor
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ABSTRACT: Mosquito-borne alphaviruses such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV) cause sporadic, sometimes large, outbreaks of rheumatic disease worldwide. This study was designed to test the effect of treating RRV-induced arthritis using the anti-tumor necrosis factor (anti-TNF) drug etanercept in a mouse model of rheumatic disease.
Mice were infected with RRV and treated with etanercept. Weight gain was measured, tissue viral titers were determined, and histologic changes in muscle and joint tissues were assessed.
RRV-infected mice treated with etanercept showed decreased weight gain, higher viral titers in muscle, joints, and blood, and more tissue damage and inflammatory cell recruitment than RRV-infected mice without treatment.
Anti-TNF therapy is unlikely to be useful in treating alphaviral arthritides. During alphaviral epidemics, careful monitoring of patients being treated with anti-TNF agents may be warranted.
Arthritis & Rheumatism 02/2011; 63(2):488-91. · 7.87 Impact Factor
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ABSTRACT: OBJECTIVES.: Mosquito-borne alphaviruses such as chikungunya virus, O'nyong-nyong virus and Ross River virus (RRV) cause sporadic, sometimes large, outbreaks of rheumatic disease worldwide. Here we test the effect of treating RRV-induced arthritis using the anti-TNF drug, etanercept, in a mouse model of rheumatic disease. METHODS.: Mice were infected with RRV and treated with etanercept. Weight loss, tissue viral titers and histological examination of muscle and joint tissues were undertaken. RESULTS.: RRV-infected mice treated with etanercept showed increased weight loss, higher viral titres in muscle, joints and blood, more tissue damage and inflammatory cell recruitment than RRV-infected mice without treatment. CONCLUSION.: Anti-TNF therapy is unlikely to have utility in treating alphaviral arthritides. During alphaviral epidemics careful monitoring of patients being treated with anti-TNF drugs may be warranted.
Arthritis & Rheumatism 10/2010; · 7.87 Impact Factor
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ABSTRACT: Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory-tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory-tract infection with hMPV, we demonstrate the accumulation of gamma interferon (IFN-gamma)-producing CD8+ T cells in the airways and lungs at day 7 post-infection (p.i.), associated with cytotoxic T lymphocytes (CTLs) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th1 cytokines IFN-gamma and interleukin (IL)-12 and antiviral cytokines (IFN-beta), as well as chemokines Mip-1alpha, Mip-1beta, Mig, IP-10 and CX3CL1. There was also a moderate increase in Th2-type cytokines IL-4 and IL-10 compared with uninfected mice. At 21 days p.i., a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response to the two related pathogens.
Journal of General Virology 05/2010; 91(Pt 5):1302-10. · 3.36 Impact Factor
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Journal of Medicinal Chemistry 10/2009; 52(24):7911-26. · 4.80 Impact Factor
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ABSTRACT: Alphaviruses such as chikungunya virus, Sindbis virus, o'nyong-nyong virus, Mayaro virus, and Ross River virus (RRV), are commonly associated with arthralgias and overt arthritides worldwide. Understanding the processes by which arthritogenic viruses cause disease is a prerequisite in the quest for better treatments. In this regard, we have recently established that monocyte/macrophages are mediators of alphavirus-induced arthritis in mice. We hypothesized that chemokines associated with monocyte/macrophage recruitment may play an important role in disease. The aim of the present investigations was to determine whether bindarit, an inhibitor of monocyte chemotactic protein (MCP) synthesis, could ameliorate alphavirus-induced rheumatic disease in mice.
Using our recently developed mouse model of RRV-induced arthritis, which has many characteristics of RRV disease (RRVD) in humans, the effects of bindarit treatment on RRVD in mice were determined via histologic analyses, immunohistochemistry, flow cytometry, real-time polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and electrophoretic mobility shift assay.
Bindarit-treated RRV-infected mice developed mild disease and had substantially reduced tissue destruction and inflammatory cell recruitment as compared with untreated RRV-infected mice. The virus load in the tissues was not affected by bindarit treatment. Bindarit exhibited its activity by down-regulating MCPs, which in turn led to inhibition of cell infiltration and lower production of NF-kappaB and tumor necrosis factor alpha, which are involved in mediating tissue damage.
Our data support the use of inhibitors of MCP production in the treatment of arthritogenic alphavirus syndromes and suggest that bindarit may be useful in treating RRVD and other alphavirus-induced arthritides in humans.
Arthritis & Rheumatism 08/2009; 60(8):2513-23. · 7.87 Impact Factor
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ABSTRACT: Human metapneumovirus (HMPV) has been identified as a worldwide agent of serious upper and lower respiratory tract infections in infants and young children. HMPV is second only to respiratory syncytial virus (RSV) as a leading cause of bronchiolitis, and, like RSV, consists of two major genotypes that cocirculate and vary among communities year to year. Children who have experienced acute HMPV infection may develop sequelae of wheezing and asthma; however, the features contributing to this pathology remain unknown. A possible mechanism for postbronchiolitis disease is that HMPV might persist in the lung providing a stimulus that could contribute to wheezing and asthma. Using immunohistochemistry to identify HMPV-infected cells in the lungs of mice, we show that HMPV mediates biphasic replication in respiratory epithelial cells then infection migrates to neuronal processes that innervate the lungs where the virus persists with no detectable infection in epithelial cells. After glucocorticoid treatment, the virus is reactivated from neural fibers and reinfects epithelial cells. The findings show that HMPV persists in neural fibers and suggest a mechanism for disease chronicity that has important implications for HMPV disease intervention strategies.
Journal of Virology 05/2009; 83(13):6837-48. · 5.40 Impact Factor
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ABSTRACT: The aetiology of asthma associated with viral infection is complex. The dynamics that contribute to disease pathogenesis are multifactorial and involve overlapping molecular and cellular mechanisms, particularly the immune response to respiratory virus infection or allergen sensitization. This review summarizes the evidence associated with factors that may contribute to the development or exacerbation of asthma including age, host factors, genetic polymorphisms, altered immune responses, and aspects of viral antigen expression. This review also provides an important perspective of key events linked to the development of asthmatic disease and related pulmonary inflammation from human and animal studies, and discusses their relationship as targets for disease intervention strategies.
Microbes and Infection 09/2008; 10(9):1014-23. · 3.10 Impact Factor
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ABSTRACT: Alphaviruses, such as chikungunya virus and Ross River virus (RRV), are associated with outbreaks of infectious rheumatic disease in humans worldwide. Using an established mouse model of disease that mimics RRV disease in humans, we showed that macrophage-derived factors are critical in the development of striated muscle and joint tissue damage. Histologic analyses of muscle and ankle joint tissues demonstrated a substantial reduction in inflammatory infiltrates in infected mice depleted of macrophages (i.e., "macrophage-depleted mice"). Levels of the proinflammatory factors tumor necrosis factor-alpha, interferon-gamma, and macrophage chemoattractant protein-1 were also dramatically reduced in tissue samples obtained from infected macrophage-depleted mice, compared with samples obtained from infected mice without macrophage depletion. These factors were also detected in the synovial fluid of patients with RRV-induced polyarthritis. Neutralization of these factors reduced the severity of disease in mice, whereas blocking nuclear factor kappaB by treatment with sulfasalazine ameliorated RRV inflammatory disease and tissue damage. To our knowledge, these findings are the first to demonstrate that macrophage-derived products play important roles in the development of arthritis and myositis triggered by alphavirus infection.
The Journal of Infectious Diseases 05/2008; 197(11):1585-93. · 6.41 Impact Factor
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ABSTRACT: Eosinophils are recruited to the lungs in response to respiratory syncytial virus (RSV) infection; however, their role in promoting antiviral host defense remains unclear. Here, we demonstrate that eosinophils express TLRs that recognize viral nucleic acids, are activated and degranulate after single-stranded RNA (ssRNA) stimulation of the TLR-7-MyD88 pathway, and provide host defense against RSV that is MyD88 dependent. In contrast to wild-type mice, virus clearance from lung tissue was more rapid in hypereosinophilic (interleukin-5 transgenic) mice. Transfer of wild-type but not MyD88-deficient eosinophils to the lungs of RSV-infected wild-type mice accelerated virus clearance and inhibited the development of airways hyperreactivity. Similar responses were observed when infected recipient mice were MyD88 deficient. Eosinophils isolated from infected hypereosinophilic MyD88-sufficient but not MyD88-deficient mice expressed greater amounts of IFN regulatory factor (IRF)-7 and eosinophil-associated ribonucleases EAR-1 and EAR-2. Hypereosinophilia in the airways of infected mice also correlated with increased expression of IRF-7, IFN-beta, and NOS-2, and inhibition of NO production with the NOS-2 inhibitor L-NMA partially reversed the accelerated virus clearance promoted by eosinophils. Collectively, our results demonstrate that eosinophils can protect against RSV in vivo, as they promote virus clearance and may thus limit virus-induced lung dysfunction.
Blood 10/2007; 110(5):1578-86. · 9.90 Impact Factor
