Interferon regulatory factor 7 modulates virus clearance and immune responses to alphavirus encephalomyelitis.

IMPORTANCE: Viral encephalomyelitis outcome is dependent on host responses to neuronal infection. Interferon (IFN) is an important component of the innate response, and IFN regulatory factor (IRF) 7 is an inducible transcription factor for the synthesis of IFN-α. IRF7-deficient mice develop fatal paralysis after CNS infection with Sindbis virus, while wild-type mice recover. Irf7 -/- mice produce low levels of IFN-α but high levels of IFN-ß with induction of IFN-stimulated genes, so the reason for this difference is not understood. The current study shows that Irf7 -/- mice developed inflammation earlier but failed to clear virus from motor neuron-rich regions of the brainstem and spinal cord. Levels of IFN-γ and virus-specific antibody were comparable, indicating that IRF7 deficiency does not impair expression of these known viral clearance factors. Therefore, IRF7 is either necessary for the neuronal response to currently identified mediators of clearance or enables the production of additional antiviral factor(s) needed for clearance.

In Vitro and In Vivo Coinfection and Superinfection Dynamics of Mayaro and Zika Viruses in Mosquito and Vertebrate Backgrounds.

Globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Cocirculation of several arboviruses in the same geographic region provides an impetus to study the impacts of multiple concurrent infections within an individual vector mosquito. Here, we describe coinfection and superinfection with the Mayaro virus (Togaviridae, Alphavirus) and Zika virus (Flaviviridae, Flavivirus) in vertebrate and mosquito cells, as well as Aedes aegypti adult mosquitoes, to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination, and transmission. Aedes aegypti mosquitoes were able to be infected with and transmit both pathogens simultaneously. However, whereas Mayaro virus was largely unaffected by coinfection, it had a negative impact on infection and dissemination rates for Zika virus compared to single infection scenarios. Superinfection of Mayaro virus atop a previous Zika virus infection resulted in increased Mayaro virus infection rates. At the cellular level, we found that mosquito and vertebrate cells were also capable of being simultaneously infected with both pathogens. Similar to our findings in vivo, Mayaro virus negatively affected Zika virus replication in vertebrate cells, displaying complete blocking under certain conditions. Viral interference did not occur in mosquito cells. IMPORTANCE Epidemiological and clinical studies indicate that multiple arboviruses are cocirculating in human populations, leading to some individuals carrying more than one arbovirus at the same time. In turn, mosquitoes can become infected with multiple pathogens simultaneously (coinfection) or sequentially (superinfection). Coinfection and superinfection can have synergistic, neutral, or antagonistic effects on viral infection dynamics and ultimately have impacts on human health. Here we investigate the interaction between Zika virus and Mayaro virus, two emerging mosquito-borne pathogens currently circulating together in Latin America and the Caribbean. We find a major mosquito vector of these viruses-Aedes aegypti-can carry and transmit both arboviruses at the same time. Our findings emphasize the importance of considering co- and superinfection dynamics during vector-pathogen interaction studies, surveillance programs, and risk assessment efforts in epidemic areas.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.

Alphavirus Identification in Neotropical Bats.

Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Interleukin-17 contributes to Ross River virus-induced arthritis and myositis.

Arthritogenic alphaviruses are mosquito-borne viruses that are a major cause of infectious arthropathies worldwide, and recent outbreaks of chikungunya virus and Ross River virus (RRV) infections highlight the need for robust intervention strategies. Alphaviral arthritis can persist for months after the initial acute disease, and is mediated by cellular immune responses. A common strategy to limit inflammation and pathology is to dampen the overwhelming inflammatory responses by modulating proinflammatory cytokine pathways. Here, we investigate the contribution of interleukin-17 (IL-17), a cytokine involved in arthropathies such as rheumatoid arthritis, in the development RRV-induced arthritis and myositis. IL-17 was quantified in serum from RRV-infected patients, and mice were infected with RRV and joints and muscle tissues collected to analyse cellular infiltrates, tissue mRNA, cytokine expression, and joint and muscle histopathology. IL-17 expression was increased in musculoskeletal tissues and serum of RRV-infected mice and humans, respectively. IL-17-producing T cells and neutrophils contributed to the cellular infiltrate in the joint and muscle tissue during acute RRV disease in mice. Blockade of IL-17A/F using a monoclonal antibody (mAb) reduced disease severity in RRV-infected mice and led to decreased proinflammatory proteins, cellular infiltration in synovial tissues and cartilage damage, without affecting viral titers in inflamed tissues. IL-17A/F blockade triggered a shift in transcriptional profile of both leukocyte infiltrates and musculoskeletal stromal cells by downregulating proinflammatory genes. This study highlights a previously uncharacterized role for an effector cytokine in alphaviral pathology and points towards potential therapeutic benefit in targeting IL-17 to treat patients presenting with RRV-induced arthropathy.

Detection and genome characterization of Middelburg virus strains isolated from CSF and whole blood samples of humans with neurological manifestations in South Africa.

BACKGROUND: The Old world Alphavirus, Middelburg virus (MIDV), is not well known and although a few cases associated with animal illness have previously been described from Southern Africa, there has been no investigation into the association of the virus with human illness. The current study aimed to investigate possible association of MIDV infection with febrile or neurological manifestations in hospitalized or symptomatic patients fromGauteng, South Africa. METHODS: This study is a descriptive retrospective and prospective laboratory based study. Archived cerebrospinal fluid (CSF) samples submitted to the National Health Laboratory Service (NHLS), Tshwane Academic division for viral investigation from public sector hospitals in Gauteng as well as EDTA (ethylenediaminetetraacetic acid) whole blood samples from ad hoc cases of veterinary students, presenting with neurological and febrile illness, were selected and screened for the presence of alphaviruses using real-time reverse transcription(rtRT) PCR.Virus isolations from rtRT-PCR positive samples were conducted in Vero cell culture and used to obtain full genome sequences. Basic descriptive statistical analysis was conducted using EpiInfo. RESULTS: MIDV was detected by rtRT-PCR in 3/187 retrospective CSF specimens obtained from the NHLS from hospitalised patients in the Tshwane region of Gauteng and 1/2 EDTA samples submitted in the same year (2017) from ad hoc query arbovirus cases from veterinary students from the Faculty of Veterinary Science University of Pretoria.Full genome sequences were obtained for virus isolates from two cases; one from an EDTA whole blood sample (ad hoc case) and another from a CSF sample (NHLS sample).Two of the four Middelburg virus positive cases,for which clinical information was available, had other comorbidities or infections at the time of infection. CONCLUSION: Detection of MIDV in CSF of patients with neurological manifestations suggests that the virus should be investigated as a human pathogen with the potential of causing or contributing to neurological signs in children and adults.

Mutations at the Alphavirus E1'-E2 Interdimer Interface Have Host-Specific Phenotypes.

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane, and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at the following two interfaces: (i) the intradimer interactions between E2 and E1 of the same heterodimer and (ii) the interdimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1'). We hypothesized that the interdimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (chikungunya piggyback [CPB]) where we replaced six interdimeric residues in the E2 protein of Sindbis virus (wild-type [WT] SINV) with those from the E2 protein from chikungunya virus and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to the capsid protein and contained defects in particle morphology compared with virus derived from mosquito cells. Using cryo-electron microscopy (cryo-EM), we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the interdimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCE Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This finding is important because it identifies a region in the spike that could be a target for antiviral drug design.

Attenuation of Getah Virus by a Single Amino Acid Substitution at Residue 253 of the E2 Protein that Might Be Part of a New Heparan Sulfate Binding Site on Alphaviruses.

The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo. Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the three-dimensional structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.

A systematic review and meta-analysis of the potential non-human animal reservoirs and arthropod vectors of the Mayaro virus.

Improving our understanding of Mayaro virus (MAYV) ecology is critical to guide surveillance and risk assessment. We conducted a PRISMA-adherent systematic review of the published and grey literature to identify potential arthropod vectors and non-human animal reservoirs of MAYV. We searched PubMed/MEDLINE, Embase, Web of Science, SciELO and grey-literature sources including PAHO databases and dissertation repositories. Studies were included if they assessed MAYV virological/immunological measured occurrence in field-caught, domestic, or sentinel animals or in field-caught arthropods. We conducted an animal seroprevalence meta-analysis using a random effects model. We compiled granular georeferenced maps of non-human MAYV occurrence and graded the quality of the studies using a customized framework. Overall, 57 studies were eligible out of 1523 screened, published between the years 1961 and 2020. Seventeen studies reported MAYV positivity in wild mammals, birds, or reptiles and five studies reported MAYV positivity in domestic animals. MAYV positivity was reported in 12 orders of wild-caught vertebrates, most frequently in the orders Charadriiformes and Primate. Sixteen studies detected MAYV in wild-caught mosquito genera including Haemagogus, Aedes, Culex, Psorophora, Coquillettidia, and Sabethes. Vertebrate animals or arthropods with MAYV were detected in Brazil, Panama, Peru, French Guiana, Colombia, Trinidad, Venezuela, Argentina, and Paraguay. Among non-human vertebrates, the Primate order had the highest pooled seroprevalence at 13.1% (95% CI: 4.3-25.1%). From the three most studied primate genera we found the highest seroprevalence was in Alouatta (32.2%, 95% CI: 0.0-79.2%), followed by Callithrix (17.8%, 95% CI: 8.6-28.5%), and Cebus/Sapajus (3.7%, 95% CI: 0.0-11.1%). We further found that MAYV occurs in a wide range of vectors beyond Haemagogus spp. The quality of evidence behind these findings was variable and prompts calls for standardization of reporting of arbovirus occurrence. These findings support further risk emergence prediction, guide field surveillance efforts, and prompt further in-vivo studies to better define the ecological drivers of MAYV maintenance and potential for emergence.

Orally acquired cyclic dinucleotides drive dSTING-dependent antiviral immunity in enterocytes.

Enteric pathogens overcome barrier immunity within the intestinal environment that includes the endogenous flora. The microbiota produces diverse ligands, and the full spectrum of microbial products that are sensed by the epithelium and prime protective immunity is unknown. Using Drosophila, we find that the gut presents a high barrier to infection, which is partially due to signals from the microbiota, as loss of the microbiota enhances oral viral infection. We report cyclic dinucleotide (CDN) feeding is sufficient to protect microbiota-deficient flies from enhanced oral infection, suggesting that bacterial-derived CDNs induce immunity. Mechanistically, we find CDN protection is dSTING- and dTBK1-dependent, leading to NF-kB-dependent gene expression. Furthermore, we identify the apical nucleoside transporter, CNT2, as required for oral CDN protection. Altogether, our studies define a role for bacterial products in priming immune defenses in the gut.

Serological Responses in Patients Infected with Mayaro Virus and Evaluation of Cross-Protective Responses against Chikungunya Virus.

Mayaro virus (MAYV) is an alphavirus endemic to both Latin America and the Caribbean. Recent reports have questioned the ability of MAYV and its close relative, Chikungunya virus (CHIKV), to generate cross-reactive, neutralizing antibodies to one another. Since CHIKV was introduced to South America in 2013, discerning whether individuals have cross-reactive antibodies or whether they have had exposures to both viruses previously has been difficult. Using samples obtained from people infected with MAYV prior to the introduction of CHIKV in the Americas, we performed neutralizing assays and observed no discernable neutralization of CHIKV by sera from patients previously infected with MAYV. These data suggest that a positive CHIKV neutralization test cannot be attributed to prior exposure to MAYV and that previous exposure to MAYV may not be protective against a subsequent CHIKV infection.

Evaluation of Antiviral Activity of Cyclic Ketones against Mayaro Virus.

Mayaro virus (MAYV) is a neglected arthropod-borne virus found in the Americas. MAYV infection results in Mayaro fever, a non-lethal debilitating disease characterized by a strong inflammatory response affecting the joints and muscles. MAYV was once considered endemic to forested areas in Brazil but has managed to adapt and spread to urban regions using new vectors, such as Aedes aegypti, and has the potential to cause serious epidemics in the future. Currently, there are no vaccines or specific treatments against MAYV. In this study, the antiviral activity of a series of synthetic cyclic ketones were evaluated for the first time against MAYV. Twenty-four compounds were screened in a cell viability assay, and eight were selected for further evaluation. Effective concentration (EC50) and selectivity index (SI) were calculated and compound 9-(5-(4-chlorophenyl]furan-2-yl)-3,6-dimethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2))-dione (9) (EC50 = 21.5 µmol·L-1, SI = 15.8) was selected for mechanism of action assays. The substance was able to reduce viral activity by approximately 70% in both pre-treatment and post-treatment assays.

Favipiravir Inhibits Mayaro Virus Infection in Mice.

Mayaro virus (MAYV) is an emergent alphavirus that causes MAYV fever. It is often associated with debilitating symptoms, particularly arthralgia and myalgia. MAYV infection is becoming a considerable health issue that, unfortunately, lacks a specific antiviral treatment. Favipiravir, a broad-spectrum antiviral drug, has recently been shown to exert anti-MAYV activity in vitro. In the present study, the potential of Favipiravir to inhibit MAYV replication in an in vivo model was evaluated. Immunocompetent mice were orally administrated 300 mg/kg/dose of Favipiravir at pre-, concurrent-, or post-MAYV infection. The results showed a significant reduction in infectious viral particles and viral RNA transcripts in the tissues and blood of the pre- and concurrently treated infected mice. A significant reduction in the presence of both viral RNA transcript and infectious viral particles in the tissue and blood of pre- and concurrently treated infected mice was observed. By contrast, Favipiravir treatment post-MAYV infection did not result in a reduction in viral replication. Interestingly, Favipiravir strongly decreased the blood levels of the liver disease markers aspartate- and alanine aminotransferase in the pre- and concurrently treated MAYV-infected mice. Taken together, these results suggest that Favipiravir is a potent antiviral drug when administered in a timely manner.

Development of a reverse transcription recombinase-aided amplification assay for detection of Getah virus.

GETV, an arbo-borne zoonotic virus of the genus Alphavirus, which causes diarrhea and reproduction disorders in swine, lead to serious economic losses to the swine industry in China. At present, the existing methods for GETV detection are time-consuming and low sensitivity, so, a rapid, accurate and sensitive GETV detection method is urgently needed. In this study, a fluorescent reverse transcription recombinase-assisted amplification method (RT-RAA) was successfully established for the rapid detection of GETV. The sensitivity of this method to GETV was 8 copies/reaction and 20 TCID50/reaction. No cross-reaction with other viruses. A total of 118 samples were prepared for GETV detection using fluorescent RT-RAA and SYBR Green I RT-qPCR, the coincidence rate of the two methods was 100%. The results suggest that the RT-RAA method is rapid, sensitive and specific for GETV detection and can be applied in the clinical.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

Altered Spatial and Temporal Gait Parameters in Mice Infected with Ross River Virus.

Infection with mosquito-borne arthritogenic alphaviruses, such as Ross River virus (RRV) and Barmah Forest virus (BFV), can lead to long-lasting rheumatic disease. Existing mouse models that recapitulate the disease signs and immunopathogenesis of acute RRV and BFV infection have consistently shown relevance to human disease. However, these mouse models, which chiefly model hindlimb dysfunction, may be prone to subjective interpretation when scoring disease. Assessment is therefore time-consuming and requires experienced users. The DigiGait system provides video-based measurements of movement, behavior, and gait dynamics in mice and small animals. Previous studies have shown DigiGait to be a reliable system to objectively quantify changes in gait in other models of pain and inflammation. Here, for the first time, we determine measurable differences in the gait of mice with infectious arthritis using the DigiGait system. Statistically significant differences in paw area and paw angle were detected during peak disease in RRV-infected mice. Significant differences in temporal gait parameters were also identified during the period of peak disease in RRV-infected mice. These trends were less obvious or absent in BFV-infected mice, which typically present with milder disease signs than RRV-infected mice. The DigiGait system therefore provides an objective model of variations in gait dynamics in mice acutely infected with RRV. DigiGait is likely to have further utility for murine models that develop severe forms of infectious arthritis resulting in hindlimb dysfunction like RRV. IMPORTANCE Mouse models that accurately replicate the immunopathogenesis and clinical disease of alphavirus infection are vital to the preclinical development of therapeutic strategies that target alphavirus infection and disease. Current models rely on subjective scoring made through experienced observation of infected mice. Here, we demonstrate how the DigiGait system, and interventions on mice to use this system, can make an efficient objective assessment of acute disease progression and changes in gait in alphavirus-infected mice. Our study highlights the importance of measuring gait parameters in the assessment of models of infectious arthritis.

The C-Terminal Domain of Salmonid Alphavirus Nonstructural Protein 2 (nsP2) Is Essential and Sufficient To Block RIG-I Pathway Induction and Interferon-Mediated Antiviral Response.

Salmonid alphavirus (SAV) is an atypical alphavirus that has a considerable impact on salmon and trout farms. Unlike other alphaviruses, such as the chikungunya virus, SAV is transmitted without an arthropod vector, and it does not cause cell shutoff during infection. The mechanisms by which SAV escapes the host immune system remain unknown. By studying the role of SAV proteins on the RIG-I signaling cascade, the first line of defense of the immune system during infection, we demonstrated that nonstructural protein 2 (nsP2) effectively blocks the induction of type I interferon (IFN). This inhibition, independent of the protease activity carried by nsP2, occurs downstream of IRF3, which is the transcription factor allowing the activation of the IFN promoter and its expression. The inhibitory effect of nsP2 on the RIG-I pathway depends on the localization of nsP2 in the host cell nucleus, which is linked to two nuclear localization sequences (NLS) located in its C-terminal part. The C-terminal domain of nsP2 by itself is sufficient and necessary to block IFN induction. Mutation of the NLS of nsP2 is deleterious to the virus. Finally, nsP2 does not interact with IRF3, indicating that its action is possible through a targeted interaction within discrete areas of chromatin, as suggested by its punctate distribution observed in the nucleus. These results therefore demonstrate a major role for nsP2 in the control by SAV of the host cell's innate immune response. IMPORTANCE The global consumption of fish continues to rise, and the future demand cannot be met by capture fisheries alone due to limited stocks of wild fish. Aquaculture is currently the world's fastest-growing food production sector, with an annual growth rate of 6 to 8%. Recurrent outbreaks of SAV result in significant economic losses with serious environmental consequences for wild stocks. While the clinical and pathological signs of SAV infection are fairly well known, the molecular mechanisms involved are poorly described. In the present study, we focus on the nonstructural protein nsP2 and characterize a specific domain containing nuclear localization sequences that are critical for the inhibition of the host innate immune response mediated by the RIG-I pathway.

Capsid-E2 Interactions Rescue Core Assembly in Viruses That Cannot Form Cytoplasmic Nucleocapsid Cores.

Alphavirus capsid proteins (CPs) have two domains: the N-terminal domain (NTD), which interacts with the viral RNA, and the C-terminal domain (CTD), which forms CP-CP interactions and interacts with the cytoplasmic domain of the E2 spike protein (cdE2). In this study, we examine how mutations in the CP NTD affect CP CTD interactions with cdE2. We changed the length and/or charge of the NTD of Ross River virus CP and found that changing the charge of the NTD has a greater impact on core and virion assembly than changing the length of the NTD. The NTD CP insertion mutants are unable to form cytoplasmic cores during infection, but they do form cores or core-like structures in virions. Our results are consistent with cdE2 having a role in core maturation during virion assembly and rescuing core formation when cytoplasmic cores are not assembled. We go on to find that the isolated cores from some mutant virions are now assembly competent in that they can be disassembled and reassembled back into cores. These results show how the two domains of CP may have distinct yet coordinated roles. IMPORTANCE Structural viral proteins have multiple roles during entry and assembly. The capsid protein (CP) of alphaviruses has one domain that interacts with the viral genome and another domain that interacts with the E2 spike protein. In this work, we determined that the length and/or charge of the CP affects cytoplasmic core formation. However, defects in cytoplasmic core formation can be overcome by E2-CP interactions, thus assembling a core or core-like complex in the virion. In the absence of both cytoplasmic cores and CP-E2 interactions, CP is not even packaged in the released virions, but some infectious particles are still released, presumably as RNA packaged in a glycoprotein-containing membrane shell. This suggests that the virus has multiple mechanisms in place to ensure the viral genome is surrounded by a capsid core during its life cycle.

Alphaviruses: Host pathogenesis, immune response, and vaccine & treatment updates.

Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.