RESUMO
The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro, but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology.
Assuntos
Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Células Mieloides/virologia , Infecções por Orthomyxoviridae/genética , Replicação Viral/genética , Células A549 , Animais , Células Cultivadas , Cães , Feminino , Genes Virais/fisiologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Índice de Gravidade de DoençaRESUMO
Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.
Assuntos
Glicômica/métodos , Influenza Aviária/metabolismo , Influenza Humana/metabolismo , Pulmão/virologia , Orthomyxoviridae/metabolismo , Receptores Virais/metabolismo , Testes de Aglutinação , Animais , Aves , Galinhas , Eritrócitos/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/metabolismo , Vírus da Influenza A Subtipo H1N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Aviária/virologia , Influenza Humana/virologia , Lectinas/metabolismo , Pulmão/metabolismo , Orthomyxoviridae/isolamento & purificação , Orthomyxoviridae/patogenicidade , Polissacarídeos/metabolismo , Especificidade da Espécie , Suínos , VirulênciaRESUMO
Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.
Assuntos
Biomarcadores/química , Glicômica , Leite Humano/química , Polissacarídeos/química , Receptores Virais/análise , Animais , Sequência de Carboidratos , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Leite Humano/metabolismo , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismoRESUMO
An avian-like H3N2 influenza A virus (IAV) has recently caused sporadic canine influenza outbreaks in China and Korea, but the molecular mechanisms involved in the interspecies transmission of H3N2 IAV from avian to canine species are not well understood. Sequence analysis showed that residue 222 in haemagglutinin (HA) is predominantly tryptophan (W) in the closely related avian H3N2 IAV, but was leucine (L) in canine H3N2 IAV. In this study, reassortant viruses rH3N2-222L (canine-like) and rH3N2-222W (avian-like) with HA mutation L222W were generated using reverse genetics to evaluate the significance of the L222W mutation on receptor binding and host tropism of H3N2 IAV. Compared with rH3N2-222W, rH3N2-222L grew more rapidly in MDCK cells and had significantly higher infectivity in primary canine tracheal epithelial cells. Tissue-binding assays demonstrated that rH3N2-222L had a preference for canine tracheal tissues rather avian tracheal tissues, whereas rH3N2-222W favoured slightly avian rather canine tracheal tissues. Glycan microarray analysis suggested both rH3N2-222L and rH3N2-222W bound preferentially to α2,3-linked sialic acids. However, the rH3N2-222W had more than twofold less binding affinity than rH3N2-222L to a set of glycans with Neu5Aca2-3Galb1-4(Fuca-)-like or Neu5Aca2-3Galb1-3(Fuca-)-like structures. These data suggest the W to L mutation at position 222 of the HA could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinities of the HA to specific receptors with Neu5Aca2-3Galb1-4(Fuca-) or Neu5Aca2-3Galb1-3(Fuca-)-like structures that are present in dogs.
Assuntos
Doenças do Cão/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Mutação , Infecções por Orthomyxoviridae/veterinária , Animais , Sequência de Carboidratos , Linhagem Celular , China , Cães , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Leucina/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Triptofano/genéticaRESUMO
To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Mutação/genética , Infecções por Orthomyxoviridae/virologia , Receptores Virais/metabolismo , Replicação Viral , Animais , Sítios de Ligação , Bovinos , Células Cultivadas , Galinhas , Cães , Eritrócitos/metabolismo , Eritrócitos/virologia , Vetores Genéticos , Cobaias , Testes de Hemaglutinação , Cavalos , Rim/citologia , Rim/metabolismo , Rim/virologia , Camundongos , Análise em Microsséries , Modelos Moleculares , Mutagênese Sítio-Dirigida , Orthomyxoviridae/patogenicidade , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ovinos , TurquiaRESUMO
Viral vectors based on influenza virus, rabies virus (RV), and vaccinia virus (VV) were used to express large polypeptide segments derived from the Bacillus anthracis protective antigen (PA). For the infectious influenza virus vector and recombinant VV constructs, the receptor binding domain (RBD or domain 4) or the lethal and edema factor binding domain (LEF or domain 1') were engineered into functional chimeric hemagglutinin (HA) glycoproteins. In the case of the RV vector, the viral glycoprotein (G) was used as a carrier for RBD in an inactivated form of the vector. These constructs were examined by using multiple homologous and heterologous prime/boost immunization regimens in order to optimize the induction of alpha-PA antibody responses. Several immunization combinations were shown to induce high titers of antibody recognizing the anthrax RBD and LEF domains, as well as the full-length PA protein in mice. The heterologous prime/boost immunization regimens that involved an initial intranasal administration of a live influenza virus vector, followed by an intramuscular boost with either the killed RV vector or the VV vector, were particularly effective, inducing antigen-specific antibodies at levels severalfold higher than homologous or alternative heterologous protocols. Furthermore, sera from several groups of the immunized mice demonstrated neutralization activity in an in vitro anthrax toxin neutralization assay. In some cases, such toxin-neutralizing activity was notably high, indicating that the mechanisms by which immunity is primed by live influenza virus vectors may have beneficial properties.
Assuntos
Vacinas contra Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/imunologia , Antitoxinas/imunologia , Toxinas Bacterianas/imunologia , Vetores Genéticos , Orthomyxoviridae/genética , Animais , Vacinas contra Antraz/genética , Feminino , Imunização Secundária/métodos , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Raiva/genética , Vacinação/métodos , Vaccinia virus/genéticaRESUMO
During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Virais de Fusão/metabolismo , Animais , Linhagem Celular , DNA Complementar/genética , Cães , Fluoresceínas , Células HeLa , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Mutação/genética , Dobramento de ProteínaRESUMO
The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.
Assuntos
Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Mutação de Sentido Incorreto , Proteínas Virais/genética , Proteínas Virais/metabolismo , Substituição de Aminoácidos/genética , Animais , Anticorpos Antivirais/sangue , Sítios de Ligação , Peso Corporal , Linhagem Celular , Embrião de Galinha , Cães , Testes de Inibição da Hemaglutinação , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Estrutura Terciária de Proteína , Virulência , Replicação Viral/fisiologiaRESUMO
Type I interferon (IFNα/ß) pathways are fine-tuned to elicit antiviral protection while minimizing immunopathology; however, the initiating stimuli, target tissues, and underlying mechanisms are unclear. Using models of physiological and dysregulated IFNα/ß receptor (IFNAR1) surface expression, we show here that IFNAR1-dependent signals set the steady-state IFN signature in both hematopoietic and stromal cells. Increased IFNAR1 levels promote a lung environment refractory to early influenza virus replication by elevating the baseline interferon signature. Commensal microbiota drive the IFN signature specifically in lung stroma, as shown by antibiotic treatment and fecal transplantation. Bone marrow chimera experiments identify lung stromal cells as crucially important for early antiviral immunity and stroma-immune cell interaction for late antiviral resistance. We propose that the microbiota-driven interferon signature in lung epithelia impedes early virus replication and that IFNAR1 surface levels fine-tune this signature. Our findings highlight the interplay between bacterial and viral exposure, with important implications for antibiotic use.
Assuntos
Antibacterianos/farmacologia , Vírus da Influenza A , Influenza Humana/imunologia , Influenza Humana/microbiologia , Pulmão/imunologia , Microbiota/imunologia , Receptor de Interferon alfa e beta/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Quimera/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Transplante de Microbiota Fecal , Regulação Viral da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/imunologia , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Interferon Tipo I/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA-Seq , Receptor de Interferon alfa e beta/genética , Células Estromais/imunologia , Células Estromais/metabolismo , Células Estromais/microbiologia , Células Estromais/virologiaRESUMO
We investigated geographic disparity in numbers of hantavirus pulmonary syndrome (HPS) cases in the United States. The 12-year incidences of HPS (cases/100,000 for total and rural residents) by state did not parallel the number of cases per state. The state with the greatest overall incidence was New Mexico, with Montana ranking second. When rural incidence based on rural human population sizes were compared, New Mexico also had the highest incidence, but Utah, Nevada, Montana, Arizona, and Colorado, in that order, also had high incidences. From these evaluations, it is clear that, in order to allow a precise risk assessment of acquiring HPS, we must first understand the host-virus cycle and we must have data regarding more exact conditions of human behaviors and exposure to the etiologic agent.
Assuntos
Reservatórios de Doenças/veterinária , Síndrome Pulmonar por Hantavirus/epidemiologia , Síndrome Pulmonar por Hantavirus/transmissão , Zoonoses , Animais , Humanos , Incidência , Densidade Demográfica , Medição de Risco , Roedores , População Rural , Estados Unidos/epidemiologiaRESUMO
The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/virologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Embrião de Galinha , Citocinas/imunologia , Citocinas/metabolismo , Eritrócitos/virologia , Feminino , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Suínos , Virulência/genética , Replicação Viral , Redução de PesoRESUMO
Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage [Neu5Ac(α2,3)Gal]. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential.
Assuntos
Adaptação Fisiológica/imunologia , Animais Selvagens/virologia , Furões/virologia , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/fisiologia , Influenza Aviária/transmissão , Replicação Viral/fisiologia , Aminoácidos/metabolismo , Animais , Antígenos Virais/imunologia , Aves/virologia , Agregação Eritrocítica , Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A/patogenicidade , Influenza Aviária/imunologia , Influenza Aviária/patologia , Influenza Aviária/virologia , Influenza Humana/virologia , Camundongos , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Sistema Respiratório/patologia , Sistema Respiratório/virologia , Especificidade da Espécie , Virulência , Eliminação de Partículas ViraisRESUMO
We have utilized glycan microarray technology to determine the receptor binding properties of early isolates from the recent 2009 H1N1 human pandemic (pdmH1N1), and compared them to North American swine influenza isolates from the same year, as well as past seasonal H1N1 human isolates. We showed that the pdmH1N1 strains, as well as the swine influenza isolates examined, bound almost exclusively to glycans with α2,6-linked sialic acid with little binding detected for α2,3-linked species. This is highlighted by pair-wise comparisons between compounds with identical glycan backbones, differing only in the chemistry of their terminal linkages. The overall similarities in receptor binding profiles displayed by pdmH1N1 strains and swine isolates indicate that little or no adaptation appeared to be necessary in the binding component of HA for transmission from pig to human, and subsequent human to human spread.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/veterinária , Receptores Virais/metabolismo , Doenças dos Suínos/virologia , Ligação Viral , Aglutinação , Animais , Sítios de Ligação , Eritrócitos , Hemaglutininas/química , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Influenza Humana/epidemiologia , Análise em Microsséries/métodos , Modelos Moleculares , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Pandemias , Filogenia , Ligação Proteica , Conformação Proteica , Estações do Ano , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Avian influenza viruses (AIV) are an important emerging threat to public health. It is thought that sialic acid (sia) receptors are barriers in cross-species transmission where the binding preferences of AIV and human influenza viruses are sias α2,3 versus α2,6, respectively. In this study, we show that a normal fully differentiated, primary human bronchial epithelial cell model is readily infected by low pathogenic H5N1, H5N2 and H5N3 AIV, which primarily bind to sia α2,3 moieties, and replicate in these cells independent of specific sias on the cell surface. NHBE cells treated with neuraminidase prior to infection are infected by AIV despite removal of sia α2,3 moieties. Following AIV infection, higher levels of IP-10 and RANTES are secreted compared to human influenza virus infection, indicating differential chemokine expression patterns, a feature that may contribute to differences in disease pathogenesis between avian and human influenza virus infections in humans.
Assuntos
Aves/virologia , Brônquios/patologia , Células Epiteliais/virologia , Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Influenza Humana/virologia , Ácido N-Acetilneuramínico/metabolismo , Adolescente , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Quimiocinas/metabolismo , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cílios/patologia , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Aviária/patologia , Influenza Humana/patologia , Masculino , Neuraminidase/farmacologia , Receptores de Superfície Celular/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Eliminação de Partículas Virais/efeitos dos fármacos , Eliminação de Partículas Virais/fisiologiaRESUMO
The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8(+) T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8(+) T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8(+) T cells, which recognize multiple strains of influenza viruses. These CD8(+) T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Vetores Genéticos , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Vacinas contra Antraz/imunologia , Formação de Anticorpos , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Reações Cruzadas , Feminino , Testes de Inibição da Hemaglutinação , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Virais/imunologiaRESUMO
A panel of eight single amino acid deletion mutants was generated within the first 24 residues of the fusion peptide domain of the of the hemagglutinin (HA) of A/Aichi/2/68 influenza A virus (H3N2 subtype). The mutant HAs were analyzed for folding, cell surface transport, cleavage activation, capacity to undergo acid-induced conformational changes, and membrane fusion activity. We found that the mutant DeltaF24, at the C-terminal end of the fusion peptide, was expressed in a non-native conformation, whereas all other deletion mutants were transported to the cell surface and could be cleaved into HA1 and HA2 to activate membrane fusion potential. Furthermore, upon acidification these cleaved HAs were able to undergo the characteristic structural rearrangements that are required for fusion. Despite this, all mutants were inhibited for fusion activity based on two separate assays. The results indicate that the mutant fusion peptide domains associate with target membranes in a non-functional fashion, and suggest that structural features along the length of the fusion peptide are likely to be relevant for optimal membrane fusion activity.