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1.
Cell ; 157(2): 329-339, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24725402

RESUMO

Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.


Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Substituição de Aminoácidos , Animais , Furões , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Virus da Influenza A Subtipo H5N1/genética , Mutação , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Receptores Virais/metabolismo , Seleção Genética
2.
PLoS Pathog ; 19(2): e1011135, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36745654

RESUMO

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.


Assuntos
Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Aviária , Influenza Humana , Humanos , Animais , Camundongos , Virus da Influenza A Subtipo H5N1/genética , Neuraminidase/genética , Neuraminidase/metabolismo , Galinhas/metabolismo , Furões , Vírus da Influenza A/metabolismo , Mutação , Influenza Humana/genética
3.
PLoS Pathog ; 17(9): e1009566, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34555124

RESUMO

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Aviária/genética , Influenza Humana/genética , Zoonoses Virais/genética , Animais , Patos , Humanos , Pandemias
4.
Int J Mol Sci ; 24(3)2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36769336

RESUMO

Forty-five strains of AIVs were isolated from wild aquatic birds during their autumn migration through Moscow (Russia). The aim of this work is to study the dynamics of AIV genomes in their natural habitat. Viruses were isolated from fecal sample in embryonated chicken eggs; their complete genomes were sequenced, and a phylogenetic analysis was performed. The gene segments of the same lineage persisted over the years in the absence of persistence of complete viral genomes. The genes for internal proteins of the same lineage were often maintained by the viruses over few years; however, they were typically associated with the genes of novel HA and NA subtypes. Although frequent reassortment events were observed for any pair of internal genes, there was no reassortment between HA and NA segments. The differences in the persistence of phylogenetic lineages of surface and internal proteins and the different evolutionary strategy for these two types of genes of AIVs in primary hosts are discussed.


Assuntos
Vírus da Influenza A , Influenza Aviária , Animais , Filogenia , Moscou , Animais Selvagens , Aves
5.
J Virol ; 95(20): e0090621, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34319155

RESUMO

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Serina Endopeptidases/metabolismo , Animais , Brônquios/citologia , Linhagem Celular , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Pulmão/virologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeo Hidrolases/metabolismo , Proteólise , Mucosa Respiratória/metabolismo , Serina Endopeptidases/fisiologia , Replicação Viral
6.
Biochem J ; 478(2): 423-441, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33410905

RESUMO

The neuraminidases (NAs) of avian influenza viruses (IAVs) contain a second sialic acid-binding site (2SBS), historically known as the hemadsorption site, which is separated from the sialyl-hydrolase catalytic site and serves to facilitate NA catalytic activity towards multivalent sialyl-capped glycoconjugates. Transmission and adaptation of avian IAVs to humans decreases hemadsorption and catalytic activities of the NA. Here, we report the molecular recognition features of the NA 2SBS of two pandemic H1N1 IAVs, A/Brevig Mission /1/1918 (BM18) and A/California/04/2009 (CA09), differing by their 2SBS activity. Using explicit solvent MD simulation, molecular mechanics, and glycosidic conformation analysis we initially analyzed the interactions of BM18 2SBS with two sialyllacto-N-tetraose pentasaccharides, 3'SLN-LC and 6'SLN-LC, which are models for the glycan receptors of IAVs in birds and humans, respectively. These studies characterize the binding specificity of BM18 2SBS towards human-type and avian-type receptors and identifies the key amino acids that affects binding. We next compared the interactions of the 2SBSs of BM18 and CA09 with 6'SLN-LC, revealing the critical effect of amino acid 372 on binding. Our results expand the current knowledge of the molecular features of NA 2SBSs and its alteration during the adaptation of avian IAVs to humans.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Neuraminidase/química , Neuraminidase/metabolismo , Polissacarídeos/metabolismo , Sítios de Ligação , Simulação de Dinâmica Molecular , Polissacarídeos/química , Conformação Proteica , Ácidos Siálicos/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
7.
PLoS Pathog ; 15(6): e1007860, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181126

RESUMO

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.


Assuntos
Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Neuraminidase , Receptores Virais , Proteínas Virais , Vírion , Animais , Sítios de Ligação , Chlorocebus aethiops , Cães , Humanos , Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Células Madin Darby de Rim Canino , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
8.
J Virol ; 94(1)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597765

RESUMO

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K582I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells.IMPORTANCE Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here, we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having a relatively low pH optimum of fusion cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that the infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells, and the extracellular pH. Given significant variation of levels of HA stability among animal, human, and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity.


Assuntos
Endossomos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Vírus Reordenados/genética , Substituição de Aminoácidos , Animais , Cães , Endossomos/virologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Regulação da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração de Íons de Hidrogênio , Virus da Influenza A Subtipo H5N1/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Vírus Reordenados/metabolismo , Vírus Reordenados/patogenicidade , Relação Estrutura-Atividade , Tropismo Viral/genética , Replicação Viral
9.
J Allergy Clin Immunol ; 142(3): 804-814, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29132960

RESUMO

BACKGROUND: The hygiene hypothesis is the leading concept to explain the current asthma epidemic, which is built on the observation that a lack of bacterial contact early in life induces allergic TH2 immune responses. OBJECTIVE: Because little is known about the contribution of respiratory tract viruses in this context, we evaluated the effect of prior influenza infection on the development of allergic asthma. METHODS: Mice were infected with influenza and, once recovered, subjected to an ovalbumin- or house dust mite-induced experimental asthma protocol. Influenza-polarized effector memory T (Tem) cells were transferred adoptively to allergen-sensitized animals before allergen challenge. A comprehensive in silico analysis assessed homologies between virus- and allergen-derived proteins. Influenza-polarized Tem cells were stimulated ex vivo with candidate peptides. Mice were immunized with a pool of virus-derived T-cell epitopes. RESULTS: In 2 murine models we found a long-lasting preventive effect against experimental asthma features. Protection could be attributed about equally to CD4+ and CD8+ Tem cells from influenza-infected mice. An in silico bioinformatic analysis identified 4 influenza- and 3 allergen-derived MHC class I and MHC class II candidate T-cell epitopes with potential antigen-specific cross-reactivity between influenza and allergens. Lymphocytes from influenza-infected mice produced IFN-γ and IL-2 but not IL-5 on stimulation with the aforementioned peptides. Immunization with a mixture of the influenza peptides conferred asthma protection, and peptide-immunized mice transferred protection through CD4+ and CD8+ Tem cells. CONCLUSION: For the first time, our results illustrate heterologous immunity of virus-infected animals toward allergens. This finding extends the original hygiene hypothesis.


Assuntos
Alérgenos/imunologia , Asma/imunologia , Vírus da Influenza A Subtipo H1N1/metabolismo , Infecções por Orthomyxoviridae/imunologia , Peptídeos/imunologia , Animais , Epitopos de Linfócito T/imunologia , Feminino , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Pyroglyphidae/imunologia , Linfócitos T/imunologia
10.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27847361

RESUMO

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. IMPORTANCE: The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity.


Assuntos
Adaptação Biológica/genética , Ebolavirus/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Mutação , África Ocidental/epidemiologia , Substituição de Aminoácidos , Linhagem Celular , Replicação do DNA , Surtos de Doenças , Ebolavirus/classificação , Genoma Viral , Humanos , Modelos Moleculares , Filogenia , Conformação Proteica , Transcrição Gênica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
11.
J Virol ; 91(11)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28356532

RESUMO

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-ß-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins.IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno , Virus da Influenza A Subtipo H5N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Interferons/imunologia , Fusão de Membrana , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células A549 , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Linhagem Celular , Cães , Patos , Células Epiteliais/efeitos dos fármacos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Concentração de Íons de Hidrogênio , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/química , Subtipo H7N9 do Vírus da Influenza A/genética , Interferon beta/imunologia , Células Madin Darby de Rim Canino , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Internalização do Vírus , Replicação Viral
13.
PLoS Pathog ; 12(6): e1005544, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27322618

RESUMO

Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(ß4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of ß-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10 levels may represent a putative therapy to overcome regeneration failure after IV-induced lung injury.


Assuntos
Células Epiteliais/virologia , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/virologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células-Tronco/virologia , Animais , Separação Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/metabolismo
14.
J Virol ; 90(3): 1569-77, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26608319

RESUMO

UNLABELLED: The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T492S, N722D, R752K, and S1132F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCE: Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased its conformational stability and increased pH optimum of membrane fusion activity. This finding represents the first formal evidence of alteration of the HA fusion activity/stability during interspecies transmission of influenza viruses under natural settings.


Assuntos
Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Internalização do Vírus/efeitos dos fármacos , Adaptação Biológica , Animais , Aves , Fusão Celular , Linhagem Celular , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Aviária/virologia , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Suínos , Doenças dos Suínos/virologia
15.
J Gen Virol ; 97(7): 1511-1519, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27075405

RESUMO

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species. We reveal that NiV growth substantially differs in primary cells between pigs and humans, with a more rapid spread of infection in human airway epithelia. Increased replication, correlated with higher endogenous expression levels of the main NiV entry receptor ephrin-B2, not only significantly differed between airway cells of the two species but also varied between cells from different human donors. To our knowledge, our study provides the first experimental evidence of species-specific and individual differences in NiV receptor expression and replication kinetics in primary airway epithelial cells. It remains to be determined whether and how these differences contribute to the viral host range and pathogenicity.


Assuntos
Efrina-B2/metabolismo , Células Epiteliais/virologia , Infecções por Henipavirus/transmissão , Vírus Nipah/fisiologia , Receptores Virais/metabolismo , Mucosa Respiratória/virologia , Replicação Viral/fisiologia , Animais , Células Cultivadas , Infecções por Henipavirus/virologia , Especificidade de Hospedeiro , Humanos , Vírus Nipah/patogenicidade , Mucosa Respiratória/citologia , Especificidade da Espécie , Suínos , Doenças dos Suínos/virologia , Internalização do Vírus
16.
J Virol ; 89(23): 12211-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26378170

RESUMO

Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differs from the putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for adaptation of avian HA to mammals. We found that introduction of avian-virus-like amino acids at five other HA positions (positions 62, 81, 92, 144, and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.


Assuntos
Substituição de Aminoácidos/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Influenza Humana/genética , Modelos Moleculares , Pandemias/história , Análise por Conglomerados , Biologia Computacional , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , História do Século XX , Humanos , Modelos Genéticos , Filogenia
17.
J Virol ; 89(8): 4170-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25631088

RESUMO

UNLABELLED: The H2N2/1957 and H3N2/1968 pandemic influenza viruses emerged via the exchange of genomic RNA segments between human and avian viruses. The avian hemagglutinin (HA) allowed the hybrid viruses to escape preexisting immunity in the human population. Both pandemic viruses further received the PB1 gene segment from the avian parent (Y. Kawaoka, S. Krauss, and R. G. Webster, J Virol 63:4603-4608, 1989), but the biological significance of this observation was not understood. To assess whether the avian-origin PB1 segment provided pandemic viruses with some selective advantage, either on its own or via cooperation with the homologous HA segment, we modeled by reverse genetics the reassortment event that led to the emergence of the H3N2/1968 pandemic virus. Using seasonal H2N2 virus A/California/1/66 (Cal) as a surrogate precursor human virus and pandemic virus A/Hong Kong/1/68 (H3N2) (HK) as a source of avian-derived PB1 and HA gene segments, we generated four reassortant recombinant viruses and compared pairs of viruses which differed solely by the origin of PB1. Replacement of the PB1 segment of Cal by PB1 of HK facilitated viral polymerase activity, replication efficiency in human cells, and contact transmission in guinea pigs. A combination of PB1 and HA segments of HK did not enhance replicative fitness of the reassortant virus compared with the single-gene PB1 reassortant. Our data suggest that the avian PB1 segment of the 1968 pandemic virus served to enhance viral growth and transmissibility, likely by enhancing activity of the viral polymerase complex. IMPORTANCE: Despite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the emergence of the H3N2/1968 virus from its putative human and avian precursors. We show that the avian PB1 segment increased activity of the viral polymerase and facilitated viral replication. Our results suggest that in addition to the acquisition of antigenically novel HA (i.e., antigenic shift), enhanced viral polymerase activity is required for the emergence of pandemic influenza viruses from their seasonal human precursors.


Assuntos
Surtos de Doenças/história , Vírus da Influenza A Subtipo H3N2 , Influenza Aviária/epidemiologia , Influenza Aviária/transmissão , Influenza Humana/epidemiologia , Proteínas Virais/genética , Zoonoses/epidemiologia , Zoonoses/transmissão , Animais , Sequência de Bases , Aves , Cães , Cobaias , Células HEK293 , História do Século XX , Humanos , Influenza Aviária/virologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Genética Reversa , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Proteínas Virais/fisiologia , Zoonoses/virologia
18.
J Virol ; 89(10): 5395-405, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25741006

RESUMO

UNLABELLED: Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galß1-4GlcNAcß (3'SLN) and Neu5Acα2-3Galß1-3GlcNAcß (SiaLe(c)) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3'SLN and Neu5Acα2-3Galß1-4(Fucα1-3)GlcNAcß (SiaLe(x)), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. IMPORTANCE: Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical "poultry-like" receptor, 3'SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Sequência de Aminoácidos , Animais , Animais Selvagens/virologia , Anseriformes/virologia , Sequência de Carboidratos , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Neuraminidase/metabolismo , Oligossacarídeos/metabolismo , Filogenia , Aves Domésticas/virologia , Ligação Proteica , Receptores Virais/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Virulência/fisiologia
19.
J Virol ; 88(16): 8735-42, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24899203

RESUMO

UNLABELLED: Mutation D701N in the PB2 protein is known to play a prominent role in the adaptation of avian influenza A viruses to mammalian hosts. In contrast, little is known about the nearby mutations S714I and S714R, which have been observed in some avian influenza viruses highly pathogenic for mammals. We have generated recombinant H5N1 viruses with PB2 displaying the avian signature 701D or the mammalian signature 701N and serine, isoleucine, and arginine at position 714 and compared them for polymerase activity and virus growth in avian and mammalian cells, as well as for pathogenicity in mice. Mutation D701N led to an increase in polymerase activity and replication efficiency in mammalian cells and in mouse pathogenicity, and this increase was significantly enhanced when mutation D701N was combined with mutation S714R. Stimulation by mutation S714I was less distinct. These observations indicate that PB2 mutation S714R, in combination with the mammalian signature at position 701, has the potential to promote the adaptation of an H5N1 virus to a mammalian host. IMPORTANCE: Influenza A/H5N1 viruses are avian pathogens that have pandemic potential, since they are spread over large parts of Asia, Africa, and Europe and are occasionally transmitted to humans. It is therefore of high scientific interest to understand the mechanisms that determine the host specificity and pathogenicity of these viruses. It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-α binding and entry of PB2 into the nucleus, where virus replication and transcription take place.


Assuntos
Adaptação Fisiológica/genética , Virus da Influenza A Subtipo H5N1/genética , Mamíferos/virologia , Mutação/genética , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Animais , Cães , Células HEK293 , Humanos , Influenza Humana/genética , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mamíferos/genética , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia
20.
PLoS Pathog ; 9(2): e1003188, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23468627

RESUMO

Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-ß significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-ß release in AM in a protein kinase R- (PKR-) and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-ß and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-ß-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Influenza Humana/metabolismo , Interferon beta/metabolismo , Macrófagos Alveolares/metabolismo , Pneumonia Viral/metabolismo , Mucosa Respiratória/metabolismo , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Adulto , Animais , Apoptose , Modelos Animais de Doenças , Humanos , Influenza Humana/imunologia , Influenza Humana/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mosaicismo , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
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