N-Linked Glycans Shape Skin Immune Responses during Arthritis and Myositis after Intradermal Infection with Ross River Virus.

Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.

Decreased Virulence of Ross River Virus Harboring a Mutation in the First Cleavage Site of Nonstructural Polyprotein Is Caused by a Novel Mechanism Leading to Increased Production of Interferon-Inducing RNAs.

Infection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease.IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was highlighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen-associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease pathogenesis. This study also highlights the importance of the host innate immune response in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new approaches for rational attenuation of alphaviruses for construction of vaccine candidates.

Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction.

BACKGROUND: Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed. METHODS AND RESULTS: Using site-directed mutagenesis at the NS1-2A cleavage site of Murray Valley encephalitis virus (MVEV), we confirmed the dominant role of conserved octapeptide residues for efficient NS1-2A cleavage, while changes at variable and the P1' residues were mostly tolerated. However, digressions from the consensus cleavage motif derived from studies on DENV were also found. Thus, comparison of the impact on cleavage of mutations at the NS1-2A junction of MVEV and DENV showed virus-specific differences at both conserved and variable residues. CONCLUSION: We show, with subgenomic expression and infectious clone-derived mutants of MVEV that conserved residues in the flavivirus octapeptide motif can be replaced with a different amino acid without markedly reducing cleavage efficiency of NS1 and NS2A.

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.

MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies.

MHCII molecules are heterodimeric cell surface proteins composed of an α and ß chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα(-/-) (I-Aα(-/-)), MHC-IIAß(-/-) (I-ß(-/-)) and the double knockout (I-Aαxß(-/-)). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα(-/-), but not in I-ß(-/-) or I-Aαxß(-/-), mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα(-/-) mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα(-/-) bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα(-/-) mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα(-/-) compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα(-/-) mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.

Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis.

Dengue virus (DV) is the most widespread arbovirus, being endemic in over 100 countries, and is estimated to cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain can play a role in determining the virus virulence and subsequent clinical disease severity. Virus vector competence plays an integral role in virus transmission and is a critical factor in determining the severity and impact of DV outbreaks. Host genetic variations in immune-related genes, including the human leukocyte antigen, have also been shown to correlate with clinical disease and thus may play a role in regulating disease severity. The host's immune system, however, appears to be the primary factor in DV pathogenesis with the delicate interplay of innate and acquired immunity playing a crucial role. Although current research of DV pathogenesis has been limited by the lack of an appropriate animal model, the development of DV therapeutics has been a primary focus of research groups around the world. In the past decade advances in both the development of vaccines and anti-virals have increased in dramatically. This review summarises the current understanding of viral, vector and host factors which contribute to dengue virus pathogenesis and how this knowledge is critically important in the development of pharmaceutical interventions.

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.

Applications of animal models of infectious arthritis in drug discovery: a focus on alphaviral disease.

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.

Restricted Semliki Forest virus replication in perforin and Fas-ligand double-deficient mice.

Previously, we have shown that mice defective in granule exocytosis and/or Fas.L/Fas-mediated cytolytic pathways are significantly more resistant to alphavirus, Semliki Forest virus (SFV), infection compared with wild-type mice. Here, we evaluated SFV replication in different tissues of mice defective in both cytolytic pathways (perf(-/-)xgld) relative to that in wild-type counterparts and found that viral replication in perf(-/-)xgld mice is remarkably restricted. Although the mechanism responsible for this observation is yet to be established, the lower virus titres found in these mice indicate that the role of cytolytic effector molecules in antiviral immunity needs to be re-evaluated.

Cutting edge: lectin-like transcript-1 is a ligand for the inhibitory human NKR-P1A receptor.

Increasingly, roles are emerging for C-type lectin receptors in immune regulation. One receptor whose function has remained largely enigmatic is human NKR-P1A (CD161), present on NK cells and subsets of T cells. In this study, we demonstrate that the lectin-like transcript-1 (LLT1) is a physiologic ligand for NKR-P1A. LLT1-containing liposomes bind to NKR-P1A+ cells, and binding is inhibited by anti-NKR-P1A mAb. Additionally, LLT1 activates NFAT-GFP reporter cells expressing a CD3zeta-NKR-P1A chimeric receptor; reciprocally, reporter cells with a CD3zeta-LLT1 chimeric receptor are stimulated by NKR-P1A. Moreover, LLT1 on target cells can inhibit NK cytotoxicity via interactions with NKR-P1A.

A novel binding assay to assess specificity of monoclonal antibodies.

Dynabeads TALON are uniform superparamagnetic polystyrene beads of 1 microm diameter that bind 6-His-tagged recombinant proteins through Co++-affinity binding, and are normally used for protein purification. We have used these beads to bind 6-His-rNKp30 and 6-His-rNKp46 to use as a matrix for evaluating NKp30 and NKp46 mAb submitted to the 8th International Human Leukocyte Differentiation Antigen Workshop. We show that recombinant protein coated beads are an effective tool to evaluate the specificity and epitope reactivity of mAb.

Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan.

NKp30 (NCR3, CD337) is a natural cytotoxicity receptor, expressed on subsets of human peripheral blood NK cells, involved in NK cell killing of tumor cells and immature dendritic cells. The cellular ligand for NKp30 has remained elusive, although evidence that membrane-associated heparan sulfate (HS) proteoglycans are involved in the recognition of cellular targets by NKp30 was recently reported. The data presented in this report show conclusively that HS glycosaminoglycans (GAG) are not ligands for NKp30. We show that removing HS completely from the cell surface of human 293-EBNA cells with mammalian heparanase does not affect binding of rNKp30/human IgG1 Fc chimera complexes or binding of multimeric liposome-rNKp30 complexes. Removing HS from 293-EBNA cells, culture-generated DC, MM-170 malignant melanoma cells, or HeLa cells does not affect the NKp30-dependent killing of these cells by NK cells. We show further that the GAG-deficient hamster pgsA-745 cells that lack HS and the GAG-expressing parent CHO-K1 cells are both killed by NK cells, with killing of both cell lines inhibited to the same extent by anti-NKp30 mAb. From these results we conclude that HS GAG are not ligands for NKp30, leaving open the question as to the nature of the cellular ligand for this important NK cell activation receptor.