RESUMEN
The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
Asunto(s)
COVID-19/virología , Contención de Riesgos Biológicos/métodos , SARS-CoV-2 , Células A549 , Animales , Chlorocebus aethiops , Cricetinae , Prueba de Complementación Genética/métodos , Genoma Viral , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , ARN Viral , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Células Vero , Virulencia , Replicación ViralRESUMEN
Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.
Asunto(s)
Virus de la Encefalitis Equina del Oeste , Especificidad del Huésped , Protocadherinas , Receptores Virales , Animales , Femenino , Humanos , Masculino , Ratones , Aves/metabolismo , Aves/virología , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Virus de la Encefalitis Equina del Oeste/clasificación , Virus de la Encefalitis Equina del Oeste/metabolismo , Virus de la Encefalitis Equina del Oeste/patogenicidad , Encefalomielitis Equina/epidemiología , Encefalomielitis Equina/virología , Proteínas Relacionadas con Receptor de LDL/metabolismo , Neuronas/metabolismo , Neuronas/virología , Fenotipo , Protocadherinas/metabolismo , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Zoonosis Virales/epidemiología , Zoonosis Virales/virologíaRESUMEN
The B.1.1.7 variant (also known as Alpha) of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the UK in the summer of 2020. The prevalence of this variant increased rapidly owing to an increase in infection and/or transmission efficiency1. The Alpha variant contains 19 nonsynonymous mutations across its viral genome, including 8 substitutions or deletions in the spike protein that interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that of the 8 individual spike protein substitutions, only N501Y resulted in consistent fitness gains for replication in the upper airway in a hamster model as well as in primary human airway epithelial cells. The N501Y substitution recapitulated the enhanced viral transmission phenotype of the eight mutations in the Alpha spike protein, suggesting that it is a major determinant of the increased transmission of the Alpha variant. Mechanistically, the N501Y substitution increased the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil, South Africa and elsewhere2,3, our results indicate that N501Y substitution is an adaptive spike mutation of major concern.
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Sustitución de Aminoácidos , COVID-19/transmisión , COVID-19/virología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Unión Competitiva , Bronquios/citología , Células Cultivadas , Cricetinae , Humanos , Masculino , Mesocricetus , Modelos Moleculares , Mutación , Unión Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Replicación ViralRESUMEN
Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.
Asunto(s)
Mosquitos Vectores , Virus de los Bosques Semliki , Animales , Proteínas Relacionadas con Receptor de LDL , Ligandos , Ratones , Receptores de LDL , Virus de los Bosques Semliki/metabolismo , Virus Sindbis/fisiologíaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
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COVID-19/transmisión , COVID-19/virología , Aptitud Genética , Mutación , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Cricetinae , Modelos Animales de Enfermedad , Humanos , Pulmón/virología , Masculino , Mesocricetus/virología , Modelos Biológicos , Mucosa Nasal/virología , Pruebas de Neutralización , Estabilidad Proteica , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Técnicas de Cultivo de Tejidos , Tráquea/virología , Carga Viral , Virión/química , Virión/patogenicidad , Virión/fisiología , Replicación Viral/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
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COVID-19/virología , Furina/metabolismo , Mutación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/patología , COVID-19/fisiopatología , Línea Celular , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/fisiopatología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Replicación Viral/genéticaRESUMEN
The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).
Asunto(s)
Adyuvantes Inmunológicos , Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/prevención & control , SARS-CoV-2/inmunología , Vacunas de Subunidad/inmunología , Compuestos de Alumbre , Animales , Anticuerpos Antivirales/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , COVID-19/virología , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Modelos Animales de Enfermedad , Inmunidad Celular , Inmunidad Humoral , Macaca mulatta/inmunología , Masculino , Oligodesoxirribonucleótidos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , EscualenoRESUMEN
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.
Asunto(s)
COVID-19 , Síndrome del Cromosoma X Frágil , Humanos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Péptidos/metabolismo , Proteínas de Unión al ARN/genética , SARS-CoV-2RESUMEN
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.
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Vacunas contra la COVID-19 , COVID-19 , Cricetinae , Animales , Humanos , Ratones , ARN Mensajero/genética , Vacunas contra la COVID-19/genética , Vacunas de ARNm , SARS-CoV-2/genética , COVID-19/prevención & control , Regiones no Traducidas 3' , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.
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COVID-19 , Furina , Proteolisis , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Secuencias de Aminoácidos/genética , Animales , COVID-19/virología , Chlorocebus aethiops , Furina/química , Humanos , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Eliminación de Secuencia , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Replicación Viral/genéticaRESUMEN
Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL map to genome regions of known immunological significance (major histocompatibility and immunoglobulin heavy chain locus), Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and identify Methyl-CpG binding domain protein 1 under this locus as a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells.
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Ratones de Colaboración Cruzada , Sitios de Carácter Cuantitativo , Ratones , Humanos , Animales , Sitios de Carácter Cuantitativo/genética , Ratones de Colaboración Cruzada/genética , Activación de Linfocitos , Inmunoglobulina G/genética , Homeostasis/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genéticaRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales , Péptidos y Proteínas de Señalización Intracelular , SARS-CoV-2 , Proteínas no Estructurales Virales , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , COVID-19/virología , Interferón Tipo I/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Proteínas no Estructurales Virales/metabolismo , Animales , CricetinaeRESUMEN
While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/genética , Glucógeno Sintasa Quinasa 3 , Humanos , Mutación , Nucleocápside , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2-infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.
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Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , SARS-CoV-2/inmunología , Animales , COVID-19/patología , Vacunas contra la COVID-19/uso terapéutico , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Genética Inversa , Pase Seriado , Replicación ViralRESUMEN
The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over five million people worldwide as of December 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics.
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COVID-19 , Modelos Animales de Enfermedad , Macaca nemestrina , Enfermedades de los Monos/virología , Animales , COVID-19/inmunología , COVID-19/patología , COVID-19/fisiopatología , COVID-19/virología , Humanos , Inmunidad Humoral , Pulmón/inmunología , Pulmón/virología , Masculino , Enfermedades de los Monos/inmunología , Enfermedades de los Monos/patología , Enfermedades de los Monos/fisiopatología , Linfocitos T/inmunologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a wide range of disease severity, ranging from asymptomatic infection to a life-threating illness, particularly in the elderly population and individuals with comorbid conditions. Among individuals with serious coronavirus 2019 (COVID-19) disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19-induced ARDS and evaluate therapeutic strategies. We report two cases of ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that had pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic, and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. Notable increases in circulating cytokines were observed in three of four infected, aged AGMs but not in infected RMs. All the AGMs had increased levels of plasma IL-6 compared with baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2. Together, our results indicate that both RMs and AGMs are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations, including ARDS.
Asunto(s)
COVID-19/etiología , Pulmón/virología , SARS-CoV-2/patogenicidad , Envejecimiento , Animales , Chlorocebus aethiops/virología , Infecciones por Coronavirus/tratamiento farmacológico , Citocinas/metabolismo , Humanos , Pulmón/patología , Macaca mulatta/virología , Carga Viral/métodosRESUMEN
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.
Asunto(s)
Regiones no Traducidas 3'/genética , Virus Chikungunya/genética , ARN Viral/química , ARN Viral/genética , Animales , Línea Celular , Fiebre Chikungunya/virología , Chlorocebus aethiops , Culicidae , Efecto Citopatogénico Viral , Genoma Viral , Mutación , Conformación de Ácido Nucleico , Análisis de Secuencia , Células Vero , Replicación Viral/genéticaRESUMEN
BACKGROUND: Virus infections result in a range of clinical outcomes for the host, from asymptomatic to severe or even lethal disease. Despite global efforts to prevent and treat virus infections to limit morbidity and mortality, the continued emergence and re-emergence of new outbreaks as well as common infections such as influenza persist as a health threat. Challenges to the prevention of severe disease after virus infection include both a paucity of protective vaccines as well as the early identification of individuals with the highest risk that may require supportive treatment. METHODS: We completed a screen of mice from the Collaborative Cross (CC) that we infected with influenza, severe acute respiratory syndrome-coronavirus, and West Nile virus. RESULTS: The CC mice exhibited a range of disease manifestations upon infections, and we used this natural variation to identify strains with mortality after infection and strains exhibiting no mortality. We then used comprehensive preinfection immunophenotyping to identify global baseline immune correlates of protection from mortality to virus infection. CONCLUSIONS: These data suggest that immune phenotypes might be leveraged to identify humans at highest risk of adverse clinical outcomes upon infection, who may most benefit from intensive clinical interventions, in addition to providing insight for rational vaccine design.
Asunto(s)
Mortalidad , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/mortalidad , Animales , Ratones de Colaboración Cruzada , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Virus de la Influenza A/inmunología , Gripe Humana , Masculino , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/mortalidad , ARN , Infecciones por Virus ARN/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Síndrome Respiratorio Agudo Grave/mortalidad , Linfocitos T/inmunología , Linfocitos T/metabolismo , Vacunas Virales/inmunología , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/mortalidad , Virus del Nilo Occidental/inmunologíaRESUMEN
We aerosolized severe acute respiratory syndrome coronavirus 2 and determined that its dynamic aerosol efficiency surpassed those of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome. Although we performed experiment only once across several laboratories, our findings suggest retained infectivity and virion integrity for up to 16 hours in respirable-sized aerosols.