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Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production.
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Vírus Chikungunya , RNA Polimerase Dependente de RNA , Vírion , Replicação Viral , Vírus Chikungunya/genética , Vírus Chikungunya/metabolismo , Replicação Viral/fisiologia , Animais , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , Vírion/metabolismo , Febre de Chikungunya/virologia , Febre de Chikungunya/metabolismo , Humanos , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Linhagem Celular , Domínios ProteicosRESUMO
Arthropod-borne viruses (arboviruses) constitute a significant ongoing public health threat, as the mechanisms of pathogenesis remain incompletely understood. Cardiovascular symptomatology is emerging as an important manifestation of arboviral infection. We have recently studied the cardiac tropism and mechanisms implicated in cardiac damage in mice for the alphavirus chikungunya virus (CHIKV), and we therefore sought to evaluate the cardiac tropism of other emerging alphaviruses and arboviruses. Using human primary cardiac cells, we found that arboviruses from diverse viral families were able to replicate within these cells. Interestingly, we noted that while the closely related alphavirus Mayaro virus (MAYV) could replicate to high titers in primary human cardiac microvascular endothelial cells, pulmonary, and brain endothelial cells, the Indian Ocean Lineage of CHIKV (CHIKV-IOL) was completely restricted in all endothelial cells tested. Upon further investigation, we discovered that this restriction occurs at both entry and egress stages. Additionally, we observed that compared to CHIKV, MAYV may antagonize or evade the innate immune response more efficiently in human cardiac endothelial cells to increase infection. Overall, this study explores the tropism of arboviruses in human primary cardiac cells and characterizes the strain-specific restriction of CHIKV-IOL in human endothelial cells. Further work is needed to understand how the differential restriction of alphaviruses in human endothelial cells impacts pathogenesis in a living model, as well as the specific host factors responsible.
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Chikungunya virus (CHIKV) is a mosquito-transmitted, RNA virus that causes an often-severe musculoskeletal illness characterized by fever, joint pain, and a range of debilitating symptoms. The virus has re-emerged as a global health threat in recent decades, spreading from its origin in Africa across Asia and the Americas, leading to widespread outbreaks impacting millions of people. Despite more than 50 years of research into the pathogenesis of CHIKV, there is still no curative treatment available. Current management of CHIKV infections primarily involves providing supportive care to alleviate symptoms and improve the patient's quality of life. Given the ongoing threat of CHIKV, there is an urgent need to better understand its pathogenesis. This understanding is crucial for deciphering the mechanisms underlying the disease and for developing effective strategies for both prevention and management. This review aims to provide a comprehensive overview of CHIKV and its pathogenesis, shedding light on the complex interactions of viral genetics, host factors, immune responses, and vector-related factors. By exploring these intricate connections, the review seeks to contribute to the knowledge base surrounding CHIKV, offering insights that may ultimately lead to more effective prevention and management strategies for this re-emerging global health threat.
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Febre de Chikungunya , Vírus Chikungunya , Humanos , Vírus Chikungunya/patogenicidade , Vírus Chikungunya/genética , Febre de Chikungunya/virologia , Febre de Chikungunya/epidemiologia , Animais , Virulência , Mosquitos Vetores/virologia , Interações Hospedeiro-PatógenoRESUMO
Severe COVID-19 has been associated with coinfections with bacterial and fungal pathogens. Notably, patients with COVID-19 who develop Staphylococcus aureus bacteremia exhibit higher rates of mortality than those infected with either pathogen alone. To understand this clinical scenario, we collected and examined S. aureus blood and respiratory isolates from a hospital in New York City during the early phase of the pandemic from both SARS-CoV-2+ and SARS-CoV-2- patients. Whole genome sequencing of these S. aureus isolates revealed broad phylogenetic diversity in both patient groups, suggesting that SARS-CoV-2 coinfection was not associated with a particular S. aureus lineage. Phenotypic characterization of the contemporary collection of S. aureus isolates from SARS-CoV-2+ and SARS-CoV-2- patients revealed no notable differences in several virulence traits examined. However, we noted a trend toward overrepresentation of S. aureus bloodstream strains with low cytotoxicity in the SARS-CoV-2+ group. We observed that patients coinfected with SARS-CoV-2 and S. aureus were more likely to die during the acute phase of infection when the coinfecting S. aureus strain exhibited high or low cytotoxicity. To further investigate the relationship between SARS-CoV-2 and S. aureus infections, we developed a murine coinfection model. These studies revealed that infection with SARS-CoV-2 renders mice susceptible to subsequent superinfection with low cytotoxicity S. aureus. Thus, SARS-CoV-2 infection sensitizes the host to coinfections, including S. aureus isolates with low intrinsic virulence. IMPORTANCE: The COVID-19 pandemic has had an enormous impact on healthcare across the globe. Patients who were severely infected with SARS-CoV-2, the virus causing COVID-19, sometimes became infected with other pathogens, which is termed coinfection. If the coinfecting pathogen is the bacterium Staphylococcus aureus, there is an increased risk of patient death. We collected S. aureus strains that coinfected patients with SARS-CoV-2 to study the disease outcome caused by the interaction of these two important pathogens. We found that both in patients and in mice, coinfection with an S. aureus strain lacking toxicity resulted in more severe disease during the early phase of infection, compared with infection with either pathogen alone. Thus, SARS-CoV-2 infection can directly increase the severity of S. aureus infection.
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COVID-19 , Coinfecção , SARS-CoV-2 , Infecções Estafilocócicas , Staphylococcus aureus , COVID-19/complicações , COVID-19/microbiologia , Coinfecção/microbiologia , Coinfecção/virologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Infecções Estafilocócicas/microbiologia , Humanos , Animais , Camundongos , SARS-CoV-2/genética , Filogenia , Feminino , Cidade de Nova Iorque/epidemiologia , Masculino , Virulência , Pessoa de Meia-Idade , Sequenciamento Completo do Genoma , Bacteriemia/microbiologia , Modelos Animais de Doenças , IdosoRESUMO
In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein ß-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo. The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.IMPORTANCEArboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects.
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Febre de Chikungunya , Vírus Chikungunya , Proteínas do Envelope Viral , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Animais , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/química , Camundongos , Febre de Chikungunya/virologia , Humanos , Internalização do Vírus , Conformação Proteica , Receptores Virais/metabolismo , Receptores Virais/genética , Mutação , Linhagem Celular , Ligação Proteica , Simulação de Dinâmica MolecularRESUMO
The genomic determinants that contribute to orthobunyavirus infection and pathogenesis are not well-defined. In this study, we harnessed the process of reassortment to understand which viral factors drive change in the replication and pathogenesis of La Crosse virus (LACV). We systematically reassorted the genomic segments of two genetically similar Lineage I LACV isolates into six unique reassortants. Despite the parental isolates having high levels of RNA and protein consensus, the reassortants demonstrate how minimal changes in RNA and protein structure can have significant changes in viral growth and reproduction in vitro in mammalian and insect models. We observed that swapping the S segment between isolates led to differences in replication and assembly resulting in one non-rescuable reassortant and one viable reassortant that exhibited an increase in viral growth dynamics. Switching the M segment led to changes in viral plaque phenotype and growth kinetics. L segment reassortants similarly differed in changes in viral growth dynamics. We further explored the M segment reassortants in a neonate mouse model and observed a role for the M segment in neuroinflammation and virulence. Through reassortment of the La Crosse virus genomic segments, we are able to further understand how genomic determinants of infection and pathogenesis operate in orthobunyaviruses. Future investigations will focus on identifying the specific molecular elements that govern the observed phenotypes in vitro and in vivo . Importance: La Crosse virus is the leading cause of pediatric arboviral encephalitis in the United States, yet it is largely unknown how each of the three genomic segments contribute to pathogenesis and disease. Our study utilizes genomic reassortment between two similar Lineage I LACV isolates to understand genomic determinants for differences in infection and pathogenesis phenotypes in vitro and in vivo. By identifying roles for each segment in observed outcomes, we are able to plan further studies for molecular characterization of these phenotypes. Additionally, it is imperative to continue to characterize orthobunyavirus function since climate change will expand the range and prevalence of arthropod-borne diseases such as LACV in the United States.
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Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus (CHIKV) has established the importance of the nsP4 residue cysteine 483 in maintaining viral genetic fidelity. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the Coxsackievirus B3 3 polymerase. We rescued each variant in both mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to polymerase host-specific replication and genetic stability. Surprisingly, in mosquito cells, these variants in the ring finger and palm domain were replication competent and produced viral structural proteins, but they were unable to produce infectious progeny, indicating a yet uncharacterized role for the polymerase in viral assembly. Finally, we have identified additional residues in the nsP4 palm domain that influence the genetic diversity of the viral progeny, potentially via an alteration in NTP binding and/or discrimination by the polymerase. Taken together, these studies highlight that distinct nsP4 subdomains regulate multiple processes of the alphavirus life cycle, placing nsP4 in a central role during the switch from RNA synthesis to packaging and assembly.
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Patients with coronavirus disease 2019 (COVID-19) present increased risk for ischemic cardiovascular complications up to 1 year after infection. Although the systemic inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques remains unknown. Here we report that SARS-CoV-2 viral RNA is detectable and replicates in coronary lesions taken at autopsy from severe COVID-19 cases. SARS-CoV-2 targeted plaque macrophages and exhibited a stronger tropism for arterial lesions than adjacent perivascular fat, correlating with macrophage infiltration levels. SARS-CoV-2 entry was increased in cholesterol-loaded primary macrophages and dependent, in part, on neuropilin-1. SARS-CoV-2 induced a robust inflammatory response in cultured macrophages and human atherosclerotic vascular explants with secretion of cytokines known to trigger cardiovascular events. Our data establish that SARS-CoV-2 infects coronary vessels, inducing plaque inflammation that could trigger acute cardiovascular complications and increase the long-term cardiovascular risk.
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Alphaviruses infect both mammals and insects, yet the distinct mechanisms that alphaviruses use to infect different hosts are not well defined. In this study, we characterize CHIKV E1 variants in the fusion loop (E1-M88L) and hinge region (E1-N20Y) in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. Through cell culture assays, we found that CHIKV E1-N20Y enhanced infectivity in mosquito cells while the CHIKV E1-M88L variant enhanced virus binding and infectivity in both BHK-21 and C6/36 cells, and led to changes in the virus cholesterol-dependence in BHK-21 cells. Given these in vitro results and that residue E1-M88L is in a defined Mxra8 interacting domain, we hypothesized that this residue may be important for receptor usage. However, while the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, it was attenuated in vitro in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type dependent manner to alter entry. Finally, using molecular dynamics to understand how potential changes in the E1 glycoprotein may impact the CHIKV glycoprotein E1-E2 complex, we found that E1-M88L and other E1 domain II variants lead to changes in both E1 and E2 dynamics. Taken together, these studies show that key residues in the CHIKV E1 fusion loop and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry. Importance: Arthropod-borne viruses (arboviruses) are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus class II glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, to address these gaps in our knowledge, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo . Here, we show that changes in the CHIKV E1 fusion loop and hinge contribute to host-specific entry and E1-E2 dynamics, furthering our knowledge of how alphaviruses infect mammals and insects.
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IMPORTANCE: Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.
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Proteases Semelhantes à Papaína de Coronavírus , Interações entre Hospedeiro e Microrganismos , Glicoproteínas de Membrana , Proteínas de Membrana , Proteínas de Membrana Transportadoras , SARS-CoV-2 , Replicação Viral , Humanos , COVID-19/virologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/metabolismoRESUMO
Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat, with limited antiviral treatments or vaccines available. La Crosse virus (LACV) from the Bunyavirales order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the Togaviridae family, we hypothesized that LACV would share similar entry mechanisms with CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol-modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol dependent, while replication was less affected by cholesterol manipulation. In addition, we generated single-point mutants in the LACV Gc ij loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc ij loop impaired virus infectivity and attenuated LACV replication in vitro and in vivo. Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolves in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, providing evidence for the Gc glycoprotein as a contributor to LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to replication and pathogenesis. IMPORTANCE Vector-borne viruses are significant health threats that lead to devastating disease worldwide. The emergence of arboviruses, in addition to the lack of effective antivirals or vaccines, highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contains strong structural similarities at the tip of domain II. Here, we show that the bunyavirus La Crosse virus uses a cholesterol-dependent entry pathway similar to the alphavirus chikungunya virus, and residues in the ij loop are important for virus infectivity in vitro and replication in mice. These studies show that genetically diverse viruses may use similar pathways through conserved structure domains, suggesting that these viruses may be targets for broad-spectrum antivirals in multiple arboviral families.
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Arbovírus , Encefalite da Califórnia , Vírus La Crosse , Animais , Camundongos , Antivirais/farmacologia , Glicoproteínas/genética , Vírus La Crosse/genética , Mosquitos Vetores , Estados Unidos , Replicação ViralRESUMO
Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.
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Febre de Chikungunya , Vírus Chikungunya , Doenças Transmissíveis , Cardiopatias , Vasculite , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Inflamação , Infecção Persistente , Replicação ViralRESUMO
COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome, and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA) is detectable and replicates in coronary atherosclerotic lesions taken at autopsy from patients with severe COVID-19. SARS-CoV-2 localizes to plaque macrophages and shows a stronger tropism for arterial lesions compared to corresponding perivascular fat, correlating with the degree of macrophage infiltration. In vitro infection of human primary macrophages highlights that SARS-CoV-2 entry is increased in cholesterol-loaded macrophages (foam cells) and is dependent, in part, on neuropilin-1 (NRP-1). Furthermore, although viral replication is abortive, SARS-CoV-2 induces a robust inflammatory response that includes interleukins IL-6 and IL-1ß, key cytokines known to trigger ischemic cardiovascular events. SARS-CoV-2 infection of human atherosclerotic vascular explants recapitulates the immune response seen in cultured macrophages, including pro-atherogenic cytokine secretion. Collectively, our data establish that SARS-CoV-2 infects macrophages in coronary atherosclerotic lesions, resulting in plaque inflammation that may promote acute CV complications and long-term risk for CV events.
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Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat with little to no antiviral treatments. La Crosse virus (LACV) from the Bunyavirales order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the Togaviridae family, we hypothesized that LACV would share similar entry mechanisms to CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol-dependent while replication was less affected by cholesterol manipulation. In addition, we generated single point mutants in the LACV ij loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc ij loop impaired virus infectivity and attenuate LACV in vitro and in vivo . Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolution in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, supporting the Gc glycoprotein as a target for LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to infectivity and pathogenesis. Importance: Vector-borne arboviruses are significant health threats leading to devastating disease worldwide. This emergence and the fact that there are little to no vaccines or antivirals targeting these viruses highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contain strong structural similarities in the tip of domain II. Here we show that the bunyavirus La Crosse virus uses similar mechanisms to entry as the alphavirus chikungunya virus and residues in the ij loop are important for virus infectivity. These studies show that genetically diverse viruses use similar mechanisms through concerned structure domains, suggesting these may be a target for broad-spectrum antivirals to multiple arbovirus families.
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Although microbial populations in the gut microbiome are associated with COVID-19 severity, a causal impact on patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. We first demonstrate SARS-CoV-2 infection induces gut microbiome dysbiosis in mice, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, including blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.
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Bacteriemia , COVID-19 , Coinfecção , Microbioma Gastrointestinal , Camundongos , Animais , Disbiose/microbiologia , Antibacterianos , SARS-CoV-2 , BactériasRESUMO
The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate in a mouse model that SARS-CoV-2 infection can induce gut microbiome dysbiosis, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Comparison with stool samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.
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Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 (ACE2) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.
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COVID-19 , Enzima de Conversão de Angiotensina 2/genética , Humanos , Organoides , SARS-CoV-2RESUMO
Chikungunya virus (CHIKV) is a reemerging arthropod-borne alphavirus and a serious threat to human health. Therefore, efforts toward elucidating how this virus causes disease and the molecular mechanisms underlying steps of the viral replication cycle are crucial. Using an in vivo transmission system that allows intrahost evolution, we identified an emerging CHIKV variant carrying a mutation in the E1 glycoprotein (V156A) in the serum of mice and saliva of mosquitoes. E1 V156A has since emerged in humans during an outbreak in Brazil, cooccurring with a second mutation, E1 K211T, suggesting an important role for these residues in CHIKV biology. Given the emergence of these variants, we hypothesized that they function to promote CHIKV infectivity and subsequent disease. Here, we show that E1 V156A and E1 K211T modulate virus attachment and fusion and impact binding to heparin, a homolog of heparan sulfate, a key entry factor on host cells. These variants also exhibit differential neutralization by antiglycoprotein monoclonal antibodies, suggesting structural impacts on the particle that may be responsible for altered interactions at the host membrane. Finally, E1 V156A and E1 K211T exhibit increased titers in an adult arthritic mouse model and induce increased foot-swelling at the site of injection. Taken together, this work has revealed new roles for E1 where discrete regions of the glycoprotein are able to modulate cell attachment and swelling within the host. IMPORTANCE Alphaviruses represent a growing threat to human health worldwide. The reemerging alphavirus chikungunya virus (CHIKV) has rapidly spread to new geographic regions in the last several decades, causing overwhelming outbreaks of disease, yet there are no approved vaccines or therapeutics. The CHIKV glycoproteins are key determinants of CHIKV adaptation and virulence. In this study, we identify and characterize the emerging E1 glycoprotein variants, V156A and K211T, that have since emerged in nature. We demonstrate that E1 V156A and K211T function in virus attachment to cells, a role that until now has only been attributed to specific residues of the CHIKV E2 glycoprotein. We also demonstrate E1 V156A and K211T increase foot-swelling of the ipsilateral foot in mice infected with these variants. Observing that these variants and other pathogenic variants occur at the E1-E1 interspike interface, we highlight this structurally important region as critical for multiple steps during CHIKV infection. Together, these studies further define the function of E1 in CHIKV infection and can inform the development of therapeutic or preventative strategies.
Assuntos
Vírus Chikungunya/fisiologia , Vírus Chikungunya/patogenicidade , Proteínas do Envelope Viral/metabolismo , Ligação Viral , Aedes/virologia , Animais , Anticorpos Monoclonais/imunologia , Febre de Chikungunya/patologia , Febre de Chikungunya/transmissão , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Vírus Chikungunya/imunologia , Modelos Animais de Doenças , Heparina/metabolismo , Humanos , Inflamação , Camundongos , Mutação , Testes de Neutralização , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Internalização do Vírus , Replicação ViralRESUMO
Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.
Assuntos
Alphavirus/fisiologia , Flavivirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Vírion/metabolismo , Replicação Viral , Células A549 , Alphavirus/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Animais , Culicidae/virologia , Flavivirus/efeitos dos fármacos , Humanos , Interferon Tipo I/deficiência , Camundongos , Camundongos Mutantes , Mutação , Domínios Proteicos , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vírion/genética , Montagem de Vírus/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/genética , Zika virus/efeitos dos fármacos , Zika virus/fisiologia , Infecção por Zika virus/virologiaRESUMO
Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.