Potential of Ilhéus virus to emerge.

Ilhéus virus (ILHV)(Flaviviridae:Orthoflavivirus) is an arthropod-borne virus (arbovirus) endemic to Central and South America and the Caribbean. First isolated in 1944, most of our knowledge derives from surveillance and seroprevalence studies. These efforts have detected ILHV in a broad range of mosquito and vertebrate species, including humans, but laboratory investigations of pathogenesis and vector competence have been lacking. Here, we develop an immune intact murine model with several ages and routes of administration. Our model closely recapitulates human neuroinvasive disease with ILHV strain- and mouse age-specific virulence, as well as a uniformly lethal Ifnar-/- A129 immunocompromised model. Replication kinetics in several vertebrate and invertebrate cell lines demonstrate that ILHV is capable of replicating to high titers in a wide variety of potential host and vector species. Lastly, vector competence studies provide strong evidence for efficient infection of and potential transmission by Aedes species mosquitoes, despite ILHV's phylogenetically clustering with Culex vectored flaviviruses, suggesting ILHV is poised for emergence in the neotropics.

Pathophysiology of chikungunya virus infection associated with fatal outcomes.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes acute, subacute, and chronic human arthritogenic diseases and, in rare instances, can lead to neurological complications and death. Here, we combined epidemiological, virological, histopathological, cytokine, molecular dynamics, metabolomic, proteomic, and genomic analyses to investigate viral and host factors that contribute to chikungunya-associated (CHIK) death. Our results indicate that CHIK deaths are associated with multi-organ infection, central nervous system damage, and elevated serum levels of pro-inflammatory cytokines and chemokines compared with survivors. The histopathologic, metabolite, and proteomic signatures of CHIK deaths reveal hemodynamic disorders and dysregulated immune responses. The CHIKV East-Central-South-African lineage infecting our study population causes both fatal and survival cases. Additionally, CHIKV infection impairs the integrity of the blood-brain barrier, as evidenced by an increase in permeability and altered tight junction protein expression. Overall, our findings improve the understanding of CHIK pathophysiology and the causes of fatal infections.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

Yellow Fever Emergence: Role of Heterologous Flavivirus Immunity in Preventing Urban Transmission.

During major, recent yellow fever (YF) epidemics in Brazil, human cases were attributed only to spillover infections from sylvatic transmission with no evidence of human amplification. Furthermore, the historic absence of YF in Asia, despite abundant peridomestic Aedes aegypti and naive human populations, represents a longstanding enigma. We tested the hypothesis that immunity from dengue (DENV) and Zika (ZIKV) flaviviruses limits YF virus (YFV) viremia and transmission by Ae. aegypti . Prior DENV and ZIKV immunity consistently suppressed YFV viremia in experimentally infected macaques, leading to reductions in Ae. aegypti infection when mosquitoes were fed on infected animals. These results indicate that, in DENV- and ZIKV-endemic regions such as South America and Asia, flavivirus immunity suppresses YFV human amplification potential, reducing the risk of urban outbreaks. One-Sentence Summary: Immunity from dengue and Zika viruses suppresses yellow fever viremia, preventing infection of mosquitoes and reducing the risk of epidemics.

MVA-based vaccines are protective against lethal eastern equine encephalitis virus aerosol challenge in cynomolgus macaques.

MVA-based monovalent eastern equine encephalitis virus (MVA-BN-EEEV) and multivalent western, eastern, and Venezuelan equine encephalitis virus (MVA-BN-WEV) vaccines were evaluated in the cynomolgus macaque aerosol model of EEEV infection. Macaques vaccinated with two doses of 5 × 108 infectious units of the MVA-BN-EEEV or MVA-BN-WEV vaccine by the intramuscular route rapidly developed robust levels of neutralizing antibodies to EEEV that persisted at high levels until challenge at day 84 via small particle aerosol delivery with a target inhaled dose of 107 PFU of EEEV FL93-939. Robust protection was observed, with 7/8 animals receiving MVA-BN-EEEV and 100% (8/8) animals receiving MVA-BN-WEV surviving while only 2/8 mock vaccinated controls survived lethal challenge. Complete protection from viremia was afforded by both vaccines, with near complete protection from vRNA loads in tissues and any pathologic evidence of central nervous system damage. Overall, the results indicate both vaccines are effective in eliciting an immune response that is consistent with protection from aerosolized EEEV-induced disease.

The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

Dynamics of chikungunya virus transmission in the first year after its introduction in Brazil: A cohort study in an urban community.

BACKGROUND: The first chikungunya virus (CHIKV) outbreaks during the modern scientific era were identified in the Americas in 2013, reaching high attack rates in Caribbean countries. However, few cohort studies have been performed to characterize the initial dynamics of CHIKV transmission in the New World. METHODOLOGY/PRINCIPAL FINDINGS: To describe the dynamics of CHIKV transmission shortly after its introduction in Brazil, we performed semi-annual serosurveys in a long-term community-based cohort of 652 participants aged ≥5 years in Salvador, Brazil, between Feb-Apr/2014 and Nov/2016-Feb/2017. CHIKV infections were detected using an IgG ELISA. Cumulative seroprevalence and seroincidence were estimated and spatial aggregation of cases was investigated. The first CHIKV infections were identified between Feb-Apr/2015 and Aug-Nov/2015 (incidence: 10.7%) and continued to be detected at low incidence in subsequent surveys (1.7% from Aug-Nov/2015 to Mar-May/2016 and 1.2% from Mar-May/2016 to Nov/206-Feb/2017). The cumulative seroprevalence in the last survey reached 13.3%. It was higher among those aged 30-44 and 45-59 years (16.1% and 15.6%, respectively), compared to younger (12.4% and 11.7% in <15 and 15-29 years, respectively) or older (10.3% in ≥60 years) age groups, but the differences were not statistically significant. The cumulative seroprevalence was similar between men (14.7%) and women (12.5%). Yet, among those aged 15-29 years, men were more often infected than women (18.1% vs. 7.4%, respectively, P = 0.01), while for those aged 30-44, a non-significant opposite trend was observed (9.3% vs. 19.0%, respectively, P = 0.12). Three spatial clusters of cases were detected in the study site and an increased likelihood of CHIKV infection was detected among participants who resided with someone with CHIKV IgG antibodies. CONCLUSIONS/SIGNIFICANCE: Unlike observations in other settings, the initial spread of CHIKV in this large urban center was limited and focal in certain areas, leaving a high proportion of the population susceptible to further outbreaks. Additional investigations are needed to elucidate the factors driving CHIKV spread dynamics, including understanding differences with respect to dengue and Zika viruses, in order to guide prevention and control strategies for coping with future outbreaks.

Complete genome sequence of Koutango virus strain DakAnD5443 isolated from Tatera kempi in 1968.

Koutango virus (KOUV), a close relative of West Nile virus, is highly neuroinvasive in animal models and has been associated with human disease. The complete genome of the KOUV prototype strain DakAnD5443 is reported here and may facilitate development of infectious clones for further characterization of this novel flavivirus.

Vaccination against SARS-CoV-2 Does Not Protect against the Development of Anosmia in a Hamster Model.

Anosmia, a total or partial loss of the ability to smell, is one of the most frequently documented sequelae of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Persistent anosmia is associated with a decrease in quality of life. Here, we assess the impact of virus lineage and vaccination status on anosmia development in the golden Syrian hamster model. To characterize anosmia driven by current variants, we assessed olfactory function in hamsters infected with SARS-CoV-2 lineages A, BA.2, BA.5, BQ.1, and BQ.1.1 using a buried food detection test. We found that significant anosmia occurs upon infection with all variants with a significant correlation between disease severity and degree of anosmia. Moreover, we found that vaccination with either the Pfizer (BNT16b2) or Moderna (mRNA-1273) mRNA vaccines does not protect against anosmia, despite protection against severe disease.

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

Immunogenicity and efficacy of vaccine boosters against SARS-CoV-2 Omicron subvariant BA.5 in male Syrian hamsters.

The SARS-CoV-2 Omicron subvariant BA.5 rapidly spread worldwide and replaced BA.1/BA.2 in many countries, becoming globally dominant. BA.5 has unique amino acid substitutions in the spike protein that both mediate immune escape from neutralizing antibodies produced by immunizations and increase ACE2 receptor binding affinity. In a comprehensive, long-term (up to 9 months post primary vaccination), experimental vaccination study using male Syrian hamsters, we evaluate neutralizing antibody responses and efficacy against BA.5 challenge after primary vaccination with Ad26.COV2.S (Janssen) or BNT162b2 (Pfizer/BioNTech) followed by a homologous or heterologous booster with mRNA-1273 (Moderna) or NVX-CoV2373 (Novavax). Notably, one high or low dose of Ad26.COV2.S provides more durable immunity than two primary doses of BNT162b2, and the NVX-CoV2373 booster provides the strongest augmentation of immunity, reduction in BA.5 viral replication, and disease. Our data demonstrate the immunogenicity and efficacy of different prime/boost vaccine regimens against BA.5 infection in an immune-competent model and provide new insights regarding COVID-19 vaccine strategies.

Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection.

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Morphologic and Genetic Characterization of Ilheus Virus, a Potential Emergent Flavivirus in the Americas.

Ilheus virus (ILHV) is a mosquito-borne flavivirus circulating throughout Central and South America and the Caribbean. It has been detected in several mosquito genera including Aedes and Culex, and birds are thought to be its primary amplifying and reservoir host. Here, we describe the genomic and morphologic characterization of ten ILHV strains. Our analyses revealed a high conservation of both the 5'- and 3'-untranslated regions but considerable divergence within the open reading frame. We also showed that ILHV displays a typical flavivirus structural and genomic organization. Our work lays the foundation for subsequent ILHV studies to better understand its transmission cycles, pathogenicity, and emergence potential.

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'- O methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'- O MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo , using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive to type I interferon (IFN-I) in vitro . Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'- O methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. Importance: Similar to other coronaviruses, disruption of SARS-CoV-2 NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo , our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1, but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'- O methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Vaccination Decreases the Infectious Viral Load of Delta Variant SARS-CoV-2 in Asymptomatic Patients.

The Delta variant of SARS-CoV-2 has caused many breakthrough infections in fully vaccinated individuals. While vaccine status did not generally impact the number of viral RNA genome copies in nasopharyngeal swabs of breakthrough patients, as measured by Ct values, it has been previously found to decrease the infectious viral load in symptomatic patients. We quantified the viral RNA, infectious virus, and anti-spike IgA in nasopharyngeal swabs collected from individuals asymptomatically infected with the Delta variant of SARS-CoV-2. Vaccination decreased the infectious viral load, but not the amount of viral RNA. Furthermore, vaccinees with asymptomatic infections had significantly higher levels of anti-spike IgA in their nasal secretions compared to unvaccinated individuals with asymptomatic infections. Thus, vaccination may decrease the transmission risk of Delta, and perhaps other variants, despite not affecting the amount of viral RNA measured in nasopharyngeal swabs.

Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models.

Emergence of SARS-CoV-2 variants of concern (VOCs), including the highly transmissible Omicron and Delta strains, has posed constant challenges to the current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified messenger RNA (mRNA) vaccine that expresses the more conserved viral nucleoprotein (mRNA-N) and show that mRNA-N vaccination alone can induce modest control of SARS-CoV-2. Critically, combining mRNA-N with the clinically proven S-expressing mRNA vaccine (mRNA-S+N) induced robust protection against both Delta and Omicron variants. In the hamster models of SARS-CoV-2 VOC challenge, we demonstrated that, compared to mRNA-S alone, combination mRNA-S+N vaccination not only induced more robust control of the Delta and Omicron variants in the lungs but also provided enhanced protection in the upper respiratory tract. In vivo CD8+ T cell depletion suggested a potential role for CD8+ T cells in protection conferred by mRNA-S+N vaccination. Antigen-specific immune analyses indicated that N-specific immunity, as well as augmented S-specific immunity, was associated with enhanced protection elicited by the combination mRNA vaccination. Our findings suggest that combined mRNA-S+N vaccination is an effective approach for promoting broad protection against SARS-CoV-2 variants.