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1.
Proc Natl Acad Sci U S A ; 117(38): 23807-23814, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32873642

RESUMO

Avian-origin influenza viruses overcome the bottleneck of the interspecies barrier and infect humans through the evolution of variants toward more efficient replication in mammals. The dynamic adaptation of the genetic substitutions and the correlation with the virulence of avian-origin influenza virus in patients remain largely elusive. Here, based on the one-health approach, we retrieved the original virus-positive samples from patients with H7N9 and their surrounding poultry/environment. The specimens were directly deep sequenced, and the subsequent big data were integrated with the clinical manifestations. Unlike poultry/environment-derived samples with the consistent dominance of avian signature 627E of H7N9 polymerase basic protein 2 (PB2), patient specimens had diverse ratios of mammalian signature 627K, indicating the rapid dynamics of H7N9 adaptation in patients during the infection process. In contrast, both human- and poultry/environment-related viruses had constant dominance of avian signature PB2-701D. The intrahost dynamic adaptation was confirmed by the gradual replacement of 627E by 627K in H7N9 in the longitudinally collected specimens from one patient. These results suggest that host adaptation for better virus replication to new hosts, termed "genetic tuning," actually occurred in H7N9-infected patients in vivo. Notably, our findings also demonstrate the correlation between rapid host adaptation of H7N9 PB2-E627K and the fatal outcome and disease severity in humans. The feature of H7N9 genetic tuning in vivo and its correlation with the disease severity emphasize the importance of testing for the evolution of this avian-origin virus during the course of infection.


Assuntos
Adaptação Biológica/genética , Substituição de Aminoácidos/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Proteínas Virais/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , RNA Viral/genética , Análise de Sequência de RNA , Replicação Viral/genética
2.
HLA ; 96(3): 277-298, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32475052

RESUMO

We report detailed peptide-binding affinities between 438 HLA Class I and Class II proteins and complete proteomes of seven pandemic human viruses, including coronaviruses, influenza viruses and HIV-1. We contrast these affinities with HLA allele frequencies across hundreds of human populations worldwide. Statistical modelling shows that peptide-binding affinities classified into four distinct categories depend on the HLA locus but that the type of virus is only a weak predictor, except in the case of HIV-1. Among the strong HLA binders (IC50 ≤ 50), we uncovered 16 alleles (the top ones being A*02:02, B*15:03 and DRB1*01:02) binding more than 1% of peptides derived from all viruses, 9 (top ones including HLA-A*68:01, B*15:25, C*03:02 and DRB1*07:01) binding all viruses except HIV-1, and 15 (top ones A*02:01 and C*14:02) only binding coronaviruses. The frequencies of strongest and weakest HLA peptide binders differ significantly among populations from different geographic regions. In particular, Indigenous peoples of America show both higher frequencies of strongest and lower frequencies of weakest HLA binders. As many HLA proteins are found to be strong binders of peptides derived from distinct viral families, and are hence promiscuous (or generalist), we discuss this result in relation to possible signatures of natural selection on HLA promiscuous alleles due to past pathogenic infections. Our findings are highly relevant for both evolutionary genetics and the development of vaccine therapies. However they should not lead to forget that individual resistance and vulnerability to diseases go beyond the sole HLA allelic affinity and depend on multiple, complex and often unknown biological, environmental and other variables.


Assuntos
Infecções por Coronavirus/epidemiologia , Infecções por HIV/epidemiologia , Antígenos HLA/química , Influenza Humana/epidemiologia , Pandemias , Peptídeos/química , Pneumonia Viral/epidemiologia , Síndrome Respiratória Aguda Grave/epidemiologia , Proteínas Virais/química , África/epidemiologia , América/epidemiologia , Sequência de Aminoácidos , Ásia/epidemiologia , Austrália/epidemiologia , Betacoronavirus/genética , Betacoronavirus/imunologia , Biologia Computacional , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Europa (Continente)/epidemiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Antígenos HLA/classificação , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Peptídeos/genética , Peptídeos/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Vírus da SARS/genética , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Proteínas Virais/genética , Proteínas Virais/imunologia
3.
PLoS Pathog ; 16(6): e1008611, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511263

RESUMO

Human infection with avian influenza A (H5N1) and (H7N9) viruses causes severe respiratory diseases. PB1-F2 protein is a critical virulence factor that suppresses early type I interferon response, but the mechanism of its action in relation to high pathogenicity is not well understood. Here we show that PB1-F2 protein of H7N9 virus is a particularly potent suppressor of antiviral signaling through formation of protein aggregates on mitochondria and inhibition of TRIM31-MAVS interaction, leading to prevention of K63-polyubiquitination and aggregation of MAVS. Unaggregated MAVS accumulated on fragmented mitochondria is prone to degradation by both proteasomal and lysosomal pathways. These properties are proprietary to PB1-F2 of H7N9 virus but not shared by its counterpart in WSN virus. A recombinant virus deficient of PB1-F2 of H7N9 induces more interferon ß in infected cells. Our findings reveal a subtype-specific mechanism for destabilization of MAVS and suppression of interferon response by PB1-F2 of H7N9 virus.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cães , Células HEK293 , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/patologia , Interferon beta/genética , Interferon beta/metabolismo , Células Madin Darby de Rim Canino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Agregação Patológica de Proteínas/genética , Células THP-1 , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética
4.
J Biomed Sci ; 27(1): 47, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32241276

RESUMO

BACKGROUND: Influenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms. METHODS: Four H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated. RESULTS: The HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses. CONCLUSIONS: Our study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.


Assuntos
Imunização , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/química , Influenza Humana/prevenção & controle , Vírus Reordenados/imunologia , Animais , Embrião de Galinha , Cães , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Vírus Reordenados/genética
5.
BMC Infect Dis ; 20(1): 154, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075579

RESUMO

BACKGROUND: The emergence of human infection with avian influenza A(H7N9) virus was reported in Wenshan City, southwestern China in 2017. The study describes the epidemiological and virological features of the outbreak and discusses the origin of the infection. METHODS: Poultry exposure and timelines of key events for each patient were collected. Samples derived from the patients, their close contacts, and environments were tested for influenza A(H7N9) virus by real-time reverse transcription polymerase chain reaction. Genetic sequencing and phylogenetic analysis were also conducted. RESULTS: Five patients were reported in the outbreak. An epidemiological investigation showed that all patients had been exposed at live poultry markets. The A(H7N9) isolates from these patients had low pathogenicity in avian species. Both epidemiological investigations of chicken sources and phylogenetic analysis of viral gene sequences indicated that the source of infection was from Guangxi Province, which lies 100 km to the east of Wenshan City. CONCLUSIONS: In the study, a sudden emergence of human cases of H7N9 was documented in urban area of Wenshan City. Chickens were an important carrier in the H7N9 virus spreading from Guangxi to Wenshan. Hygienic management of live poultry markets and virological screening of chickens transported across regions should be reinforced to limit the spread of H7N9 virus.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Filogenia , Adulto , Substituição de Aminoácidos , Animais , Pré-Escolar , China/epidemiologia , Surtos de Doenças , Feminino , Humanos , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Masculino , Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real
6.
Virus Genes ; 56(3): 396-400, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32034616

RESUMO

H7N9 low pathogenic avian influenza viruses (AIVs) emerged in China in 2013 and mutated into highly pathogenic strains in 2017, causing disease in infected birds and humans. Thus, the development of rapid, specific, and sensitive detection methods is urgently required. Herein, two specific monoclonal antibodies against H7N9 AIV were produced to develop a colloidal gold-based immunochromatographic test strip to detect H7N9 AIV. High specificity, repeatability, and sensitivity were achieved, with a detection limit of two hemagglutinin units or 102.55 50% tissue culture infective dose. This assay may represent a powerful tool to rapidly detect H7N9 influenza viruses in the future.


Assuntos
Coloide de Ouro , Imunoensaio/métodos , Subtipo H7N9 do Vírus da Influenza A , Influenza Humana/diagnóstico , Influenza Humana/virologia , Fitas Reagentes , Animais , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Camundongos , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
7.
PLoS Pathog ; 16(2): e1008330, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32084248

RESUMO

Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs.


Assuntos
Vírus da Influenza A/genética , Influenza Humana/genética , Suínos/virologia , Animais , Galinhas , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Influenza Aviária/genética , Influenza Humana/virologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Infecções por Orthomyxoviridae , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Suínos/genética , Suínos/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
8.
Microb Pathog ; 140: 103940, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31863839

RESUMO

H9N2 viruses can cause great economic losses to the domestic poultry industry when co-infected with other influenza viruses or pathogens. . To better understand the molecular characteristics of H9N2 avian influenza viruses (AIVs) and analyze the genetic evolutionary relationship, we isolated three H9N2 subtypes AIVs from nasopharyngeal swab specimens from the three cases reported in Anhui province since 2015, and systematically reviewed the genome-wide data of 21 poultry--isolated H9N2 viruses during 1998-2017. The six internal genes of three human-isolated viruses and recent poultry-isolated viruses (since 2014) in Anhui province presented high gene homologies with HPAI H7N9, even including H10N8 and H5N6. The three human-isolated H9N2 AIVs and poultry-isolated viruses (since 2008) in Anhui province were highly similar, and classified into genotype S. Seven N-linked potential glycosylation sites in the HA protein were detected in the three human-isolated viruses, which also appeared in poultry-isolated H9N2 AIVs. None of the human-isolated H9N2 AIVs had the I368V mutation in PB1 protein, but all the poultry-isolated H9N2 viruses in 2017 carried this mutation. Multidisciplinary, cross-regional and cross-sectoral approaches are warranted to address complex public health challenges and achieve the goal of 'one health'.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/virologia , Aves Domésticas/virologia , Animais , Galinhas , China/epidemiologia , Genoma Viral , Humanos , Incidência , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/transmissão , Influenza Humana/transmissão , Influenza Humana/virologia , Filogenia , Doenças das Aves Domésticas/transmissão , Doenças das Aves Domésticas/virologia , Prevalência
9.
Transbound Emerg Dis ; 67(2): 758-768, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31631569

RESUMO

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1-like M and PB2 genes for the earlier F/98-like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1-like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98-like counterpart, while the G1-like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1-like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses.


Assuntos
Galinhas/virologia , Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Vírus Reordenados , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Animais , Embrião de Galinha , Cães , Feminino , Genótipo , Células HEK293 , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Vírus da Influenza A Subtipo H9N2/fisiologia , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Virulência
10.
J Virol ; 94(2)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666373

RESUMO

The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD50) of >7.5 log10 50% egg infectious dose (EID50), whereas A/chicken/Hunan/S1220/2017 (CK/S1220) is virulent in mice, with an MLD50 of 3.2 log10 EID50 In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP, carrying the nucleoprotein (NP) of CK/SD098, was avirulent in mice, with an MLD50 of >107.5 EID50 The NPs of these two viruses differ by two amino acids, at positions 286 and 437. We further demonstrated that the amino acid mutations A286V and T437M of NP independently slowed the process of NP import to and export from the nucleus and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccines and antiviral drugs against influenza viruses.IMPORTANCE The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality rate of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggested that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice and the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza viruses is a polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Substituição de Aminoácidos , Animais , Galinhas , Cães , Células HEK293 , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Proteínas do Nucleocapsídeo , Infecções por Orthomyxoviridae/genética , Proteínas de Ligação a RNA/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/metabolismo , Proteínas do Core Viral/genética
11.
Cell Rep ; 29(8): 2217-2228.e5, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31747596

RESUMO

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,600 human infections, posing a threat to public health. An emerging concern is whether H7N9 AIVs will cause pandemics among humans. Molecular analysis of hemagglutinin (HA), which is a critical determinant of interspecies transmission, shows that the current H7N9 AIVs are still dual-receptor tropic, indicating limited human-to-human transmission potency. Mutagenesis and structural studies reveal that a G186V substitution is sufficient for H7N9 AIVs to acquire human receptor-binding capacity, and a Q226L substitution would favor binding to both avian and human receptors only when paired with A138/V186/P221 hydrophobic residues. These data suggest a different evolutionary route of H7N9 viruses compared to other AIV-subtype HAs.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Animais , Aves , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas/genética , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Viruses ; 11(11)2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717393

RESUMO

The prevalence and variation of the H9N2 avian influenza virus (AIV) pose a threat to public health. A total of eight viruses isolated from farmed poultry in South China during 2017-2018 were selected as representative strains for further systematic study. Phylogenetic analyses indicated that these prevalent viruses belong to the Y280-like lineage and that the internal genes are highly similar to those of recently circulating human H7N9 viruses. The receptor-binding assay showed that most of the H9N2 isolates preferentially bound to the human-like receptor, increasing the risk of them crossing the species barrier and causing human infection. Our in vitro, multi-step growth curve results indicate these viruses can effectively replicate in mammalian cells. Infection in mice showed that three viruses effectively replicated in the lung of mice. Infection in swine revealed that the viruses readily replicated in the upper respiratory tract of pig and effectively induced viral shedding. Our findings suggested that the H9N2 AIVs circulating in poultry recently acquired an enhanced ability to transmit from avian to mammalians, including humans. Based on our findings, we propose that it is essential to strengthen the efforts to surveil and test the pathogenicity of H9N2 AIVs.


Assuntos
Vírus da Influenza A Subtipo H9N2 , Influenza Aviária/transmissão , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/transmissão , Animais , Aves , China/epidemiologia , Genes Virais , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Camundongos , Infecções por Orthomyxoviridae/veterinária , Filogenia , Aves Domésticas/virologia , Receptores Virais/genética , Suínos , Replicação Viral
13.
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
14.
J Virol ; 94(1)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597767

RESUMO

The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-ß) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.


Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Proteínas não Estruturais Virais/genética , Animais , Aves , Células Dendríticas/imunologia , Células Dendríticas/virologia , Cães , Células Epiteliais/virologia , Regulação da Expressão Gênica , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H7N2/classificação , Vírus da Influenza A Subtipo H7N2/genética , Vírus da Influenza A Subtipo H7N2/imunologia , Subtipo H7N9 do Vírus da Influenza A/classificação , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células Madin Darby de Rim Canino , Filogenia , Cultura Primária de Células , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas não Estruturais Virais/classificação , Proteínas não Estruturais Virais/imunologia
15.
J Virol ; 94(1)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597771

RESUMO

Efficient human-to-human transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission are still not fully understood. In this study, we compared the respiratory droplet transmissibilities of four H7N9 viruses that are genetic closely related and found that these viruses have dissimilar transmissibilities in guinea pigs: A/Anhui/1/2013 (AH/1) transmitted efficiently, whereas the other three viruses did not transmit. The three nontransmissible viruses have one to eight amino acid differences compared with the AH/1 virus. To investigate which of these amino acids is important for transmission, we used reverse genetics to generate a series of reassortants and mutants in the AH/1 background and tested their transmissibility in guinea pigs. We found that the neuraminidase (NA) of the nontransmissible virus A/chicken/Shanghai/S1053/2013 had low enzymatic activity that impaired the transmission of AH/1 virus, and three amino acid mutations-V292I and K627E in PB2 and D156E in M1-independently abolished the transmission of the AH/1 virus. We further found that an NA reassortant and three single-amino-acid mutants replicated less efficiently than the AH/1 virus in A549 cells and that the amino acid at position 156 of M1 affected the morphology of H7N9 viruses. Our study identifies key amino acids in PB2 and M1 that play important roles in H7N9 influenza virus transmission and provides new insights into the transmissibility of influenza virus.IMPORTANCE Efficient transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission remain poorly understood. H7N9 influenza viruses, which emerged in 2013 in China, have caused over 1,560 human infection cases, showing clear pandemic potential. Previous studies have shown that the H7N9 viruses differ in their transmissibility in animal models. In this study, we found two amino acids in PB2 (292V and 627K) and one in M1 (156D) that are extremely important for H7N9 virus transmission. Of note, PB2 292V and M1 156D appear in most H7N9 viruses, and the PB2 627K mutation could easily occur when the H7N9 virus replicates in humans. Our study thus identifies new amino acids that are important for influenza virus transmission and suggests that just a few key amino acid changes can render the H7N9 virus transmissible in mammals.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Neuraminidase/genética , Infecções por Orthomyxoviridae/transmissão , Vírus Reordenados/genética , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Células A549 , Substituição de Aminoácidos , Animais , Expressão Gênica , Cobaias , Humanos , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Mutação , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/metabolismo , Vírus Reordenados/patogenicidade , Genética Reversa , Relação Estrutura-Atividade , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
16.
Emerg Microbes Infect ; 8(1): 1465-1478, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608791

RESUMO

The ANP32A is responsible for mammalian-restricted influenza virus polymerase activity. However, the mechanism of ANP32A modulation of polymerase activity remains poorly understood. Here, we report that chicken ANP32A (chANP32A) -X1 and -X2 stimulated mammalian-restricted PB2 627E polymerase activity in a dose-dependent manner. Distinct effects of ANP32A constructs suggested that the 180VK181 residues within chANP32A-X1 are necessary but not sufficient to stimulate PB2 627E polymerase activity. The PB2 N567D, T598V, A613V or F636L mutations promoted PB2 627E polymerase activity and chANP32A-X1 showed additive effects, providing further support that species-specific regulation of ANP32A might be only relevant with the PB2 E627K mutation. Rescue of cycloheximide-mediated inhibition showed that ANP32A is species-specific for modulation of vRNA but not mRNA and cRNA, demonstrating chANP32A-X1 compensated for defective cRNPs produced by PB2 627E virus in mammalian cells. The promoter mutations of cRNA enhanced the restriction of PB2 627E polymerase in mammalian cells, which could be restored by chANP32A-X1, indicating that ANP32A is likely to regulate the interaction of viral polymerase with RNA promoter. Coimmunoprecipitation showed that ANP32A did not affect the primary cRNPs assembly. We propose a model that chANP32A-X1 regulates PB2 627E polymerase for suitable interaction with cRNA promoter for vRNA replication.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Vírus da Influenza A Subtipo H9N2/enzimologia , Influenza Aviária/metabolismo , Influenza Humana/metabolismo , Doenças das Aves Domésticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Mutação , Proteínas Nucleares , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Ligação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Especificidade da Espécie , Proteínas Virais/genética , Replicação Viral
17.
Influenza Other Respir Viruses ; 13(6): 610-617, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31487118

RESUMO

BACKGROUND: Avian influenza A (H7N9) remains circulating in China. For countries at risk of introduction of H7N9, such as Vietnam, early detection of H7N9 virus is essential for the early containment of the virus. Insulated isothermal reverse transcriptase PCR (iiRT-PCR) is a portable PCR system that can be deployed under field conditions to identify pathogens at the sampling site. Applying PCR at the sampling site will greatly reduce the time to obtain a diagnostic result which allows the veterinary authority to take immediate action to contain disease spreading. OBJECTIVE: To determine analytical and diagnostic sensitivity and specificity of the portable iiRT-PCR for H7N9 virus detection. METHODS: A panel of 59 virus isolates, including H7N9, avian influenza viruses of subtype H1 to H13, swine and human influenza viruses, Newcastle disease virus, and infectious bursal disease virus, were tested by H7 and N9 iiRT-PCR reagents, using probes and primers specific to H7 or N9, in comparison with laboratory-based real-time RT-PCR assays to determine analytical sensitivity and specificity. Fifty oropharyngeal samples from experimentally infected chicken and ducks with H7N9 and 50 non-infected control swabs were tested by the H7 iiRT-PCR to determine diagnostic sensitivity and specificity. RESULTS: The H7 and N9 iiRT-PCR reagents yielded comparable levels of analytical sensitivity and specificity with real-time RT-PCR for the detection of H7N9 virus. Diagnostic sensitivity and specificity of H7 iiRT-PCR were 98% and 100%, respectively. CONCLUSION: The observed high sensitivity and specificity of iiRT-PCR for H7N9 detection show its potential for early detection of H7N9 in risk-based surveillance.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Técnicas de Diagnóstico Molecular/veterinária , Reação em Cadeia da Polimerase/veterinária , Animais , Galinhas , Patos , Subtipo H7N9 do Vírus da Influenza A/classificação , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Orofaringe/virologia , Testes Imediatos , Sensibilidade e Especificidade , Vietnã
19.
Int J Infect Dis ; 88: 80-87, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499209

RESUMO

OBJECTIVES: Eight additional provinces in western China reported human infections for the first time during the fifth wave of human H7N9 infections. The aim of this study was to analyze the epidemiological and virological characteristics of this outbreak. METHODS: The epidemiological data of H7N9 cases from the newly affected western Chinese provinces were collected and analyzed. Full-length genome sequences of H7N9 virus were downloaded from the GenBank and GISAID databases, and phylogenetic, genotyping, and genetic analyses were conducted. RESULTS: The peak of human infections in the newly affected western Chinese provinces was delayed by 4 months compared to the eastern Chinese provinces, and both low pathogenic (LP) and highly pathogenic (HP) H7N9-infected cases were found. The LP- and HP-H7N9 virus belonged to 10 different genotypes (including four new genotypes), of which G11 and G3 were the dominant genotypes, respectively. Almost all of these viruses originated from eastern and southern China and were most probably imported from neighboring provinces. Genetic characteristics of the circulating viruses were similar to those of the viruses from previously affected provinces during Wave Five. CONCLUSIONS: A delayed peak of human infections was observed in the newly affected western Chinese provinces, and reassortment has been ongoing since the introduction of H7N9 viruses. This study highlights the importance of continued surveillance of the circulation and evolution of H7N9 virus in western China.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , China/epidemiologia , Surtos de Doenças , Genoma Viral , Genótipo , Humanos , Subtipo H7N9 do Vírus da Influenza A/classificação , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/epidemiologia , Filogenia
20.
Math Biosci Eng ; 16(5): 3393-3410, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31499620

RESUMO

In 2017, the low pathogenic avian influenza H7N9 virus in China had mutated into high pathogenicity to domestic poultry, and led to a large number of poultry death and human cases. To evaluate the effect of virus mutation on the transmission of avian influenza H7N9 virus, this paper takes Guangdong province for the research area, takes domestic poultry, virus in the domestic poultry survival environment and human beings for the research objects, and establishes a non-autonomous dynamical model. By fitting model with the newly confirmed human cases in Guangdong province, the model we established is confirmed and applied to explain the dynamics of historical human cases. By carrying on parameter estimation, it is deduced that at least 5279376 human beings in Guangdong province had been infected with avian influenza H7N9 virus from March 2013 to September 2017, but most of them were not confirmed, since they had no obvious symptoms or had been cured as common influenza. And comparing with the low pathogenic avian influenza H7N9 virus (H7N9 LPAIV), the transmission rate of the highly pathogenic avian influenza H7N9 virus (H7N9 HPAIV) to human is almost unchanged, but to domestic poultry is about 3.87 times higher. Also, we calculate the basic reproduction number ℜ0 = 1.3042, which indicates that the virus will persist in Guangdong province with time. Besides, we also perform some sensitivity analysis of the newly confirmed human cases and ℜ0 in terms of model parameters and conclude that reducing the birth population of domestic poultry, speeding up the circulation of domestic poultry in the market and raising the rate of disease-related death of domestic poultry are benefit to control the transmission of the avian influenza H7N9 virus.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/transmissão , Influenza Humana/virologia , Mutação , Algoritmos , Migração Animal , Animais , Número Básico de Reprodução , China/epidemiologia , Surtos de Doenças , Epidemias , Humanos , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Modelos Teóricos , Aves Domésticas , Temperatura
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