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Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.
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The globular head domain of influenza virus surface protein hemagglutinin (HA1) is the major target of neutralizing antibodies elicited by vaccines. As little as one amino acid substitution in the HA1 can result in an antigenic drift of influenza viruses, indicating the dominance of some epitopes in the binding of HA to polyclonal serum antibodies. Therefore, identifying dominant binding epitopes of HA is critical for selecting seasonal influenza vaccine viruses. In this study, we have developed a biolayer interferometry (BLI)-based assay to determine dominant binding epitopes of the HA1 in antibody response to influenza vaccines using a panel of recombinant HA1 proteins of A(H1N1)pdm09 virus with each carrying a single amino acid substitution. Sera from individuals vaccinated with the 2010-2011 influenza trivalent vaccines were analyzed for their binding to the HA1 panel and hemagglutination inhibition (HI) activity against influenza viruses with cognate mutations. Results revealed an over 50% reduction in the BLI binding of several mutated HA1 compared to the wild type and a strong correlation between dominant residues identified by the BLI and HI assays. Our study demonstrates a method to systemically analyze antibody immunodominance in the humoral response to influenza vaccines.
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PURPOSE: Multiple sclerosis (MS) is an immune-mediated, neuroinflammatory disease of the central nervous system and in industrialised countries is the most common cause of progressive neurological disability in working age persons. While treatable, there is substantial interindividual heterogeneity in disease activity and response to treatment. Currently, the ability to predict at diagnosis who will have a benign, intermediate or aggressive disease course is very limited. There is, therefore, a need for integrated predictive tools to inform individualised treatment decision making. PARTICIPANTS: Established with the aim of addressing this need for individualised predictive tools, FutureMS is a nationally representative, prospective observational cohort study of 440 adults with a new diagnosis of relapsing-remitting MS living in Scotland at the time of diagnosis between May 2016 and March 2019. FINDINGS TO DATE: The study aims to explore the pathobiology and determinants of disease heterogeneity in MS and combines detailed clinical phenotyping with imaging, genetic and biomarker metrics of disease activity and progression. Recruitment, baseline assessment and follow-up at year 1 is complete. Here, we describe the cohort design and present a profile of the participants at baseline and 1 year of follow-up. FUTURE PLANS: A third follow-up wave for the cohort has recently begun at 5 years after first visit and a further wave of follow-up is funded for year 10. Longer-term follow-up is anticipated thereafter.
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Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Adulto , Biomarcadores , Estudios de Cohortes , Progresión de la Enfermedad , Humanos , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Estudios ProspectivosRESUMEN
Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.
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COVID-19 , Animales , COVID-19/genética , Células CACO-2 , Chlorocebus aethiops , Humanos , Poliadenilación , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , SARS-CoV-2 , Análisis de Secuencia de ARN , Células VeroRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.
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COVID-19/genética , SARS-CoV-2/genética , Transcripción Genética/genética , Animales , Células CACO-2 , Línea Celular , Chlorocebus aethiops , Epigénesis Genética , Genoma Viral/genética , Humanos , Evasión Inmune , Sistemas de Lectura Abierta , ARN Viral/genética , Transcriptoma , Células Vero , Proteínas Virales/genéticaRESUMEN
To better understand the antibody landscape changes following influenza virus natural infection and vaccination, we developed a high-throughput multiplex influenza antibody detection assay (MIADA) containing 42 recombinant hemagglutinins (rHAs) (ectodomain and/or globular head domain) from pre-2009 A(H1N1), A(H1N1)pdm09, A(H2N2), A(H3N2), A(H5N1), A(H7N7), A(H7N9), A(H7N2), A(H9N2), A(H13N9), and influenza B viruses. Panels of ferret antisera, 227 paired human sera from vaccinees (children and adults) in 5 influenza seasons (2010 to 2018), and 17 paired human sera collected from real-time reverse transcription-PCR (rRT-PCR)-confirmed influenza A(H1N1)pdm09, influenza A(H3N2), or influenza B virus-infected adults were analyzed by the MIADA. Ferret antisera demonstrated clear strain-specific antibody responses to exposed subtype HA. Adults (19 to 49 years old) had broader antibody landscapes than young children (<3 years old) and older children (9 to 17 years old) both at baseline and post-vaccination. Influenza vaccination and infection induced the strongest antibody responses specific to HA(s) of exposed strain/subtype viruses and closely related strains; they also induced cross-reactive antibodies to an unexposed influenza virus subtype(s), including novel viruses. Subsequent serum adsorption confirmed that the cross-reactive antibodies against novel subtype HAs were mainly induced by exposures to A(H1N1)/A(H3N2) influenza A viruses. In contrast, adults infected by influenza B viruses mounted antibody responses mostly specific to two influenza B virus lineage HAs. Median fluorescence intensities (MFIs) and seroconversion in MIADA had good correlations with the titers and seroconversion measured by hemagglutination inhibition and microneutralization assays. Our study demonstrated that antibody landscape analysis by the MIADA can be used for influenza vaccine evaluations and characterization of influenza virus infections.IMPORTANCE Repeated influenza vaccination and natural infections generate complex immune profiles in humans that require antibody landscape analysis to assess immunity and evaluate vaccines. However, antibody landscape analyses are difficult to perform using traditional assays. Here, we developed a high-throughput, serum-sparing, multiplex influenza antibody detection assay (MIADA) and analyzed the antibody landscapes following influenza vaccination and infection. We showed that adults had broader antibody landscapes than children. Influenza vaccination and infection not only induced the strongest antibody responses to the hemagglutinins of the viruses of exposure, but also induced cross-reactive antibodies to novel influenza viruses that can be removed by serum adsorption. There is a good correlation between the median fluorescence intensity (MFI) measured by MIADA and hemagglutination inhibition/microneutralization titers. Antibody landscape analysis by the MIADA can be used in influenza vaccine evaluations, including the development of universal influenza vaccines and the characterization of influenza virus infections.
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Anticuerpos Antivirales/sangre , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/inmunología , Vacunación , Adolescente , Adulto , Animales , Niño , Preescolar , Reacciones Cruzadas , Hurones , Ensayos Analíticos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Adulto JovenRESUMEN
Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.
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Evolución Molecular , Subtipo H9N2 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/inmunología , Gripe Aviar/virología , Gripe Humana/virología , Fenotipo , Replicación Viral/genética , Animales , Asia , China , Modelos Animales de Enfermedad , Femenino , Variación Genética , Humanos , Gripe Aviar/inmunología , Gripe Aviar/transmisión , Gripe Humana/inmunología , Gripe Humana/transmisión , Masculino , Mamíferos , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Aves de Corral/virología , Enfermedades de las Aves de Corral/virología , VietnamRESUMEN
Of the eighteen hemagglutinin (HA) subtypes (H1-H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed.
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BACKGROUND: The development of serologic assays that can rapidly assess human exposure to novel influenza viruses remains a public health need. Previously, we developed an 11-plex magnetic fluorescence microsphere immunoassay (MAGPIX) by using globular head domain recombinant hemagglutinins (rHAs) with serum adsorption using two ectodomain rHAs. METHODS: We compared sera collected from two cohorts with novel influenza exposures: animal shelter staff during an A(H7N2) outbreak in New York City in 2016-2017 (n = 119 single sera) and poultry workers from a live bird market in Bangladesh in 2012-2014 (n = 29 pairs). Sera were analyzed by microneutralization (MN) assay and a 20-plex MAGPIX assay with rHAs from 19 influenza strains (11 subtypes) combined with serum adsorption using 8 rHAs from A(H1N1) and A(H3N2) viruses. Antibody responses were analyzed to determine the novel influenza virus exposure. RESULTS: Among persons with novel influenza virus exposures, the median fluorescence intensity (MFI) against the novel rHA from exposed influenza virus had the highest correlation with MN titers to the same viruses and could be confirmed by removal of cross-reactivity from seasonal H1/H3 rHAs following serum adsorption. Interestingly, in persons with exposures to novel influenza viruses, age and MFIs against exposed novel HA were negatively correlated, whereas in persons without exposure to novel influenza viruses, age and MFI against novel HAs were positively correlated. CONCLUSIONS: This 20-plex high-throughput assay with serum adsorption will be a useful tool to detect novel influenza virus infections during influenza outbreak investigations and surveillance, especially when well-paired serum samples are not available.
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Anticuerpos Antivirales/sangre , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Pruebas Serológicas/métodos , Adsorción , Animales , Bangladesh , Estudios de Cohortes , Glicoproteínas Hemaglutininas del Virus de la Influenza/sangre , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Subtipo H7N2 del Virus de la Influenza A/inmunología , Subtipo H7N2 del Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Ciudad de Nueva York , Suero/virologíaRESUMEN
A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.
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Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Neuraminidasa/química , Neuraminidasa/inmunología , Animales , Anticuerpos Monoclonales , Antígenos Virales/genética , Antígenos Virales/inmunología , Dominio Catalítico , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Genes Virales/genética , Glicosilación , Hong Kong , Humanos , Inmunogenicidad Vacunal , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/prevención & control , Ratones , Modelos Moleculares , Mutación , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/inmunología , Conformación Proteica , Análisis de Secuencia de Proteína , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
BACKGROUND: Human immunodeficiency virus (HIV)-infected persons are at a higher risk of severe influenza. Although we have shown that a standard-dose intradermal influenza vaccine versus a standard-dose intramuscular influenza vaccine does not result in differences in hemagglutination-inhibition titers in this population, a comprehensive examination of cell-mediated immune responses remains lacking. METHODS: Serological, antigen-specific B-cell, and interleukin 2-, interferon γ-, and tumor necrosis factor α-secreting T-cell responses were assessed in 79 HIV-infected men and 79 HIV-uninfected men. RESULTS: The route of vaccination did not affect the immunoglobulin A and immunoglobulin G (IgG) plasmablast or memory B-cell response, although these were severely impaired in the group with a CD4+ T-cell count of <200 cells/µL. The frequencies of IgG memory B cells measured on day 28 after vaccination were highest in the HIV-uninfected group, followed by the group with a CD4+ T-cell count of ≥200 cells/µL and the group with a CD4+ T-cell count of <200 cells/µL. The route of vaccination did not affect the CD4+ or CD8+ T-cell responses measured at various times after vaccination. CONCLUSIONS: The route of vaccination had no effect on antibody responses, antibody avidity, T-cell responses, or B-cell responses in HIV-infected or HIV-uninfected subjects. With the serological and cellular immune responses to influenza vaccination being impaired in HIV-infected individuals with a CD4+ T-cell count of <200 cells/µL, passive immunization strategies need to be explored to protect this population. CLINICAL TRIALS REGISTRATION: NCT01538940.
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Infecciones por VIH/inmunología , Inmunidad Celular/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/normas , Gripe Humana/prevención & control , Adulto , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Linfocitos B/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Infecciones por VIH/complicaciones , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunoglobulina A , Inmunoglobulina G , Subtipo H1N1 del Virus de la Influenza A/inmunología , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Masculino , Persona de Mediana Edad , Tailandia , Factor de Necrosis Tumoral alfa/metabolismo , VacunaciónRESUMEN
The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that, compared to the 2013 virus HAs, the fifth-wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9) HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth-wave H7 HAs analyzed in this study.IMPORTANCE The A(H7N9) viruses circulating in China are of great public health concern. Here, we report a molecular and structural study of the major surface proteins from several recent A(H7N9) influenza viruses. Our results improve the understanding of these evolving viruses and provide important information on their receptor preference that is central to ongoing pandemic risk assessment.
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Epidemias/estadística & datos numéricos , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H7N9 del Virus de la Influenza A/metabolismo , Gripe Humana/epidemiología , China/epidemiología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H7N9 del Virus de la Influenza A/química , Subtipo H7N9 del Virus de la Influenza A/clasificación , Subtipo H7N9 del Virus de la Influenza A/genética , Modelos Moleculares , Mutación , Filogenia , Polisacáridos/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.
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Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/veterinaria , Animales , Células Cultivadas , Enfermedades de los Perros/virología , Perros/virología , Células Epiteliales/virología , Hurones/virología , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/fisiología , Ratones , Neuraminidasa/genética , Neuraminidasa/metabolismo , Filogenia , Conformación Proteica , Estados Unidos/epidemiología , Replicación ViralRESUMEN
The emergence of A(H7N9) virus strains with resistance to neuraminidase (NA) inhibitors highlights a critical need to discover new countermeasures for treatment of A(H7N9) virus-infected patients. We previously described an anti-NA mAb (3c10-3) that has prophylactic and therapeutic efficacy in mice lethally challenged with A(H7N9) virus when delivered intraperitoneally (i.p.). Here we show that intrananasal (i.n.) administration of 3c10-3 protects 100% of mice from mortality when treated 24h post-challenge and further characterize the protective efficacy of 3c10-3 using a nonlethal A(H7N9) challenge model. Administration of 3c10-3 i.p. 24h prior to challenge resulted in a significant decrease in viral lung titers and deep sequencing analysis indicated that treatment did not consistently select for viral variants in NA. Furthermore, prophylactic administration of 3c10-3 did not inhibit the development of protective immunity to subsequent homologous virus re-challenge. Taken together, 3c10-3 highlights the potential use of anti-NA mAb to mitigate influenza virus infection.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Factores Inmunológicos/administración & dosificación , Subtipo H7N9 del Virus de la Influenza A/inmunología , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/terapia , Administración Intranasal , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Modelos Animales de Enfermedad , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Pulmón/virología , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
UNLABELLED: During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (HPAI) A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015, the virus was detected in wild birds in Canada and the United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA-approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses and to continually assess future changes that may increase their pandemic potential. IMPORTANCE: The H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N2 del Virus de la Influenza A/química , Subtipo H5N8 del Virus de la Influenza A/química , Neuraminidasa/química , Neuraminidasa/metabolismo , Animales , Animales Salvajes/virología , Asia/epidemiología , Canadá/epidemiología , Brotes de Enfermedades , Europa (Continente)/epidemiología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H5N1 del Virus de la Influenza A/enzimología , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N2 del Virus de la Influenza A/enzimología , Subtipo H5N2 del Virus de la Influenza A/genética , Subtipo H5N8 del Virus de la Influenza A/enzimología , Subtipo H5N8 del Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Gripe Aviar/virología , Gripe Humana/virología , Neuraminidasa/genética , América del Norte/epidemiología , Filogenia , Aves de Corral , Virus ReordenadosRESUMEN
The sub-division of the posterior-most territory of the neural plate results in the formation of two distinct neural structures, the hindbrain and the spinal cord. Although many of the molecular signals regulating the development of these individual structures have been elucidated, the mechanisms involved in delineating the boundary between the hindbrain and spinal cord remain elusive. Two molecules, retinoic acid (RA) and the Cdx4 transcription factor have been previously implicated as important regulators of hindbrain and spinal cord development, respectively. Here, we provide evidence that suggests multiple regulatory interactions occur between RA signaling and the Cdx4 transcription factor to establish the anterior-posterior (AP) position of the transition between the hindbrain and spinal cord. Using chemical inhibitors to alter RA concentrations and morpholinos to knock-down Cdx4 function in zebrafish, we show that Cdx4 acts to prevent RA degradation in the presumptive spinal cord domain by suppressing expression of the RA degradation enzyme, Cyp26a1. In the hindbrain, RA signaling modulates its own concentration by activating the expression of cyp26a1 and inhibiting the expansion of cdx4. Therefore, interactions between Cyp26a1 and Cdx4 modulate RA levels along the AP axis to segregate the posterior neural plate into the hindbrain and spinal cord territories.
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Proteínas de Homeodominio/fisiología , Rombencéfalo/embriología , Médula Espinal/embriología , Tretinoina/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Sistema Enzimático del Citocromo P-450/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Ácido Retinoico 4-Hidroxilasa , Transducción de Señal , Factores de Transcripción , Transcripción Genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
UNLABELLED: During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. IMPORTANCE: Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H10N8 del Virus de la Influenza A/genética , Modelos Moleculares , Proteínas Recombinantes/genética , Animales , Aves , Clonación Molecular , Cristalización , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H10N8 del Virus de la Influenza A/metabolismo , Análisis por Micromatrices , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad de la EspecieRESUMEN
A(H3N2) influenza viruses have circulated in humans since 1968, and antigenic drift of the hemagglutinin (HA) protein continues to be a driving force that allows the virus to escape the human immune response. Since the major antigenic sites of the HA overlap into the receptor binding site (RBS) of the molecule, the virus constantly struggles to effectively adapt to host immune responses, without compromising its functionality. Here, we have structurally assessed the evolution of the A(H3N2) virus HA RBS, using an established recombinant expression system. Glycan binding specificities of nineteen A(H3N2) influenza virus HAs, each a component of the seasonal influenza vaccine between 1968 and 2012, were analyzed. Results suggest that while its receptor-binding site has evolved from one that can bind a broad range of human receptor analogs to one with a more restricted binding profile for longer glycans, the virus continues to circulate and transmit efficiently among humans.
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Evolución Molecular , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H3N2 del Virus de la Influenza A/fisiología , Receptores Virales/metabolismo , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión ProteicaRESUMEN
UNLABELLED: In late 2011, an A(H3N8) influenza virus infection resulted in the deaths of 162 New England harbor seals. Virus sequence analysis and virus receptor binding studies highlighted potential markers responsible for mammalian adaptation and a mixed receptor binding preference (S. J. Anthony, J. A. St Leger, K. Pugliares, H. S. Ip, J. M. Chan, Z. W. Carpenter, I. Navarrete-Macias, M. Sanchez-Leon, J. T. Saliki, J. Pedersen, W. Karesh, P. Daszak, R. Rabadan, T. Rowles, W. I. Lipkin, MBio 3:e00166-00112, 2012, http://dx.doi.org/10.1128/mBio.00166-12). Here, we present a detailed structural and biochemical analysis of the surface antigens of the virus. Results obtained with recombinant proteins for both the hemagglutinin and neuraminidase indicate a true avian receptor binding preference. Although the detection of this virus in new species highlights an increased potential for cross-species transmission, our results indicate that the A(H3N8) virus currently poses a low risk to humans. IMPORTANCE: Cross-species transmission of zoonotic influenza viruses increases public health concerns. Here, we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. The results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses as these zoonotic influenza viruses continue to circulate and adapt to new hosts.
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Antígenos Virales/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H3N8 del Virus de la Influenza A/fisiología , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/veterinaria , Phoca/virología , Proteínas Virales/metabolismo , Acoplamiento Viral , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H3N8 del Virus de la Influenza A/química , Subtipo H3N8 del Virus de la Influenza A/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Neuraminidasa/química , New England , Infecciones por Orthomyxoviridae/virología , Polisacáridos/análisis , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Proteínas Virales/químicaRESUMEN
The relative performance of ELISA using globular head domain (GH) and ectodomain hemagglutinins (HAs) as antigens to detect influenza A virus IgG antibody responses was assessed. Assay sensitivity and subtype cross-reactivity were evaluated using sera collected from recipients of monovalent H5N1 vaccine and A(H1N1)pdm09 virus-infected persons. Assay specificity was determined using collections of sera from either individuals unexposed to either H5N1 or A(H1N1)pdm09 viruses or exposed to H5N1 or A(H1N1)pdm09 viruses through vaccination or infection, respectively. ELISA using GH HA showed a similar degree of sensitivity, significantly higher specificity, and significantly lower subtype cross-reactivity compared to ELISA using ectodomain HA.