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1.
PLoS Biol ; 17(10): e3000181, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31574080

RESUMO

Antagonistic interactions drive host-virus evolutionary arms races, which often manifest as recurrent amino acid changes (i.e., positive selection) at their protein-protein interaction interfaces. Here, we investigated whether combinatorial mutagenesis of positions under positive selection in a host antiviral protein could enhance its restrictive properties. We tested approximately 700 variants of human MxA, generated by combinatorial mutagenesis, for their ability to restrict Thogotovirus (THOV). We identified MxA super-restrictors with increased binding to the THOV nucleoprotein (NP) target protein and 10-fold higher anti-THOV restriction relative to wild-type human MxA, the most potent naturally occurring anti-THOV restrictor identified. Our findings reveal a means to elicit super-restrictor antiviral proteins by leveraging signatures of positive selection. Although some MxA super-restrictors of THOV were impaired in their restriction of H5N1 influenza A virus (IAV), other super-restrictor variants increased THOV restriction without impairment of IAV restriction. Thus, broadly acting antiviral proteins such as MxA mitigate breadth-versus-specificity trade-offs that could otherwise constrain their adaptive landscape.


Assuntos
Virus da Influenza A Subtipo H5N1/genética , Proteínas de Resistência a Myxovirus/genética , Nucleoproteínas/genética , Thogotovirus/genética , Proteínas Virais/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Evolução Molecular , Regulação da Expressão Gênica , Biblioteca Gênica , Células HEK293 , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Especificidade de Hospedeiro , Humanos , Virus da Influenza A Subtipo H5N1/metabolismo , Mutagênese , Proteínas de Resistência a Myxovirus/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Nucleoproteínas/metabolismo , Transdução de Sinais , Thogotovirus/metabolismo , Proteínas Virais/metabolismo
2.
Acta Biochim Pol ; 66(3): 329-336, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31531422

RESUMO

The potential emergence of deadly pandemic influenza viruses is unpredictable and most have emerged with no forewarning. The distinct epidemiological and pathological patterns of the Spanish (H1N1), pandemic-2009 (H1N1), and avian influenza (H5N1), known as bird flu, viruses may allow us to develop a 'template' for possible emergence of devastating pandemic strains. Here, we provide a detailed molecular dissection of the structural and nonstructural proteins of this triad of viruses. GenBank data for three representative strains were analyzed to determine the polymorphic amino acids, genetic distances, and isoelectric points, hydrophobicity plot, and protein modeling of various proteins. We propose that the most devastating pandemic strains may have full-length PB1-F2 protein with unique residues, highly cleavable HA, and a basic NS1. Any newly emerging strain should be compared with these three strains, so that resources can be directed appropriately.


Assuntos
Simulação por Computador , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Proteínas Virais/química , Animais , Aves , Transmissão de Doença Infecciosa , Genoma Viral , Humanos , Influenza Pandêmica, 1918-1919 , Vacinas contra Influenza , Influenza Aviária/epidemiologia , Influenza Aviária/prevenção & controle , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Pandemias , Conformação Proteica em alfa-Hélice , Proteínas Virais/genética
3.
Emerg Microbes Infect ; 8(1): 1370-1382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31526249

RESUMO

Egypt is a hotspot for avian influenza virus (AIV) due to the endemicity of H5N1 and H9N2 viruses. AIVs were isolated from 329 samples collected in 2016-2018; 48% were H9N2, 37.1% were H5N8, 7.6% were H5N1, and 7.3% were co-infections with 2 of the 3 subtypes. The 32 hemagglutinin (HA) sequences of the H5N1 viruses formed a well-defined lineage within clade 2.2.1.2. The 10 HA sequences of the H5N8 viruses belonged to a subclade within 2.3.4.4. The 11 HA of H9N2 isolates showed high sequence homology with other Egyptian G1-like H9N2 viruses. The prevalence of H5N8 viruses in ducks (2.4%) was higher than in chickens (0.94%). Genetic reassortment was detected in H9N2 viruses. Antigenic analysis showed that H9N2 viruses are homogenous, antigenic drift was detected among H5N1 viruses. AI H5N8 showed higher replication rate followed by H9N2 and H5N1, respectively. H5N8 was more common in Southern Egypt, H9N2 in the Nile Delta, and H5N1 in both areas. Ducks and chickens played a significant role in transmission of H5N1 viruses. The endemicity and co-circulation of H5N1, H5N8, and H9N2 AIV coupled with the lack of a clear control strategy continues to provide avenues for further virus evolution in Egypt.


Assuntos
Coinfecção/veterinária , Monitoramento Epidemiológico/veterinária , Evolução Molecular , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H9N2/genética , Vírus Reordenados , Animais , Galinhas , Coinfecção/epidemiologia , Coinfecção/virologia , Patos , Egito/epidemiologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Aviária/transmissão , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Homologia de Sequência , Proteínas Virais/genética
4.
BMC Infect Dis ; 19(1): 676, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370782

RESUMO

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.


Assuntos
Colorimetria/métodos , Vírus da Influenza A/genética , Influenza Humana/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reações Cruzadas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Transcrição Reversa
5.
Vet Microbiol ; 235: 234-242, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383307

RESUMO

During 2012-2015, six H5N1 avian influenza viruses were isolated from domestic birds and the environment around Qinghai Lake. Phylogenetic analysis of HA genes revealed that A/chicken/Gansu/XG2/2012 (CK/GS/XG2/12) belonged to clade 2.3.2.1a, while A/environment/Qinghai/1/2013 (EN/QH/1/13), A/chicken/Qinghai/QH1/2015 (CK/QH/QH1/15), A/chicken/Qinghai/QH2/2015 (CK/QH/QH2/15), A/chicken/Qinghai/QH3/2015 (CK/QH/QH3/15), and A/goose/Qinghai/QH6/2015 (GS/QH/QH6/15) belonged to clade 2.3.2.1c. Further analysis of the internal genes of the isolates found that the PB2 gene of EN/QH/1/13 had 99.6% nucleotide identity with that of A/tiger/Jiangsu/1/2013 (H5N1), which clustered into an independent branch with PB2 from multiple subtypes. PB2, PB1, and M genes of CK/QH/QH3/15 were from H9N2, suggesting it was a reassortant of H5N1 and H9N2. Animal studies of three selected viruses revealed that CK/GS/XG2/12, EN/QH/1/13, and CK/QH/QH3/15 were highly lethal to chickens, with intravenous pathogenicity indexes (IVPIs) of 2.97, 2.81, and 3.00, respectively, and systemically replicated in chickens. In a mouse study, three selected H5N1 viruses were highly pathogenic to mice and readily replicated in the lungs, nasal turbinates, kidneys, spleens, and brains. Therefore, isolates in this study appear to be novel reassortants that were circulating at the interface of wild and domestic birds around Qinghai Lake and are lethal to chickens and mice. These data suggest that more extensive surveillance should be implemented, and matched vaccines should be chosen for the domestic birds in this area.


Assuntos
Animais Domésticos/virologia , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/epidemiologia , Lagos/virologia , Células A549 , Animais , Galinhas/virologia , China/epidemiologia , Cães , Patos/virologia , Evolução Molecular , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/mortalidade , Influenza Aviária/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Replicação Viral
6.
Virus Genes ; 55(6): 739-768, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31428925

RESUMO

Avian influenza viruses (AIVs) circulate globally, spilling over into domestic poultry and causing zoonotic infections in humans. Fortunately, AIVs are not yet capable of causing sustained human-to-human infection; however, AIVs are still a high risk as future pandemic strains, especially if they acquire further mutations that facilitate human infection and/or increase pathogenesis. Molecular characterization of sequencing data for known genetic markers associated with AIV adaptation, transmission, and antiviral resistance allows for fast, efficient assessment of AIV risk. Here we summarize and update the current knowledge on experimentally verified molecular markers involved in AIV pathogenicity, receptor binding, replicative capacity, and transmission in both poultry and mammals with a broad focus to include data available on other AIV subtypes outside of A/H5N1 and A/H7N9.


Assuntos
Marcadores Genéticos/genética , Influenza Aviária/genética , Influenza Humana/genética , Zoonoses/genética , Animais , Aves/genética , Aves/virologia , Farmacorresistência Viral/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Influenza Humana/virologia , Pandemias , Aves Domésticas/genética , Aves Domésticas/virologia , Zoonoses/virologia
7.
Vet Microbiol ; 235: 21-24, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282375

RESUMO

Occurrence of avian influenza (AI) with Neuraminidase (NA) mutations which confer reduced neuraminidase inhibitor (NAI) susceptibility has remained a cause of concern. The susceptibility to NAIs of 67 highly pathogenic avian influenza H5N1 viruses isolated during 2006-2012 in India was tested in phenotypic fluorescence-based NA inhibition assay, sequence analysis and in ovo. One isolate showed a novel NA I117T amino acid substitution (N2 numbering) and eight isolates showed previously known NAI-resistance marker mutations (I117V, E119D, N294S, total 9/67). The overall incidence of resistant variants was 13.4%. The novel I117T substitution reduced oseltamivir susceptibility by 18.6-fold and zanamivir susceptibility by 11.8-fold, compared to the wild type AI H5N1virus, thus showed cross-resistance to both oseltamivir and zanamivir in NA inhibition assays. However, the other two isolates with I117V substitution were sensitive to both the NAIs. In addition, the comparison of growth of the I117T and I117V variants in presence of NAI's in the in ovo assays exhibited difference in growth levels. The present study reports the natural occurrence of a novel I117T mutation in AI H5N1 virus conferring cross-resistance to oseltamivir and zanamivir highlighting the urgent need of antiviral surveillance of AI viruses.


Assuntos
Antivirais/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Neuraminidase/genética , Oseltamivir/farmacologia , Proteínas Virais/genética , Zanamivir/farmacologia , Substituição de Aminoácidos , Animais , Galinhas , Farmacorresistência Viral , Índia , Virus da Influenza A Subtipo H5N1/genética , Concentração Inibidora 50 , Mutação de Sentido Incorreto , Zigoto
8.
Transbound Emerg Dis ; 66(6): 2507-2516, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31357255

RESUMO

Vietnamese poultry are host to co-circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and two H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV-infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.


Assuntos
Comércio , Influenza Aviária/epidemiologia , Produtos Avícolas , Aves Domésticas , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Reação em Cadeia da Polimerase , Prevalência , RNA Viral/análise , Fatores de Risco , Vietnã/epidemiologia
9.
Emerg Microbes Infect ; 8(1): 823-826, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31164049

RESUMO

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus is associated with wild fowl migration in East Asian-Australasian (EA) and Central Asian (CA) flyways. However, the spread of H5N1 virus between the two flyways is still unclear. Here, the movements of wild waterfowl were obtained from satellite tracking data covering seven bar-headed geese and three great black-headed gulls breeding in the Qinghai Lake area (along the EA flyway), and 20 whooper swans wintering in the Sanmenxia Reservoir area (at the CA flyway). From the 2688 samples that were screened from wild birds at Qinghai Lake after an outbreak of H5N1 in July 2015, four genomes of H5N1 virus were obtained from bar-headed geese. The results of phylogenetic analysis indicated that these H5N1 viruses belonged to clade 2.3.2.1c and their gene fragments were highly homologous with A/whooper swan/Henan/SMX1/2015 (H5N1) virus (ranging from 99.76% to 100.00%) isolated from a dead whooper swan from the Sanmenxia Reservoir area along the EA flyway in January 2015. Furthermore, the coincidental timing of the H5N1 outbreak with spring migration, together with phylogenetic evidence, provided new evidence of the east-to-west spread of HPAI H5N1 between the EA and CA migratory flyways of China.


Assuntos
Anseriformes/fisiologia , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/epidemiologia , Migração Animal , Animais , Animais Selvagens/fisiologia , Animais Selvagens/virologia , Anseriformes/virologia , Ásia/epidemiologia , Austrália/epidemiologia , China/epidemiologia , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/transmissão , Influenza Aviária/virologia , Filogenia , Estações do Ano
10.
Talanta ; 201: 419-425, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122444

RESUMO

Detection and identification of DNA by PCR has opened tremendous possibilities and allows detection of minute quantities of DNA highly specifically. However, PCR remains confined to laboratory settings because of the need of thermocyclers and other analytical equipment. This led to development of isothermal amplification techniques, among which Pad Lock Probe (PLP)-based Rolling Circle Amplification (RCA) has several advantages, but typically also requires a laboratory apparatus of some sort to measure DNA amplification. To circumvent this limitation, while still taking advantage of PLP-based RCA, we developed a colorimetric assay that relies on pH change. Using this assay, we can detect DNA in the low picomolar range and obtain results observable with the naked eye in only 20 min without any requirement for a thermocycler or other complex device, making it a particularly portable assay.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , DNA Viral/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Bacteriófago M13/genética , Calibragem , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química , Virus da Influenza A Subtipo H5N1/genética , Limite de Detecção , Hibridização de Ácido Nucleico , Fenolsulfonaftaleína/química , Polimorfismo de Nucleotídeo Único , Ratos
11.
Artigo em Inglês | MEDLINE | ID: mdl-30961818

RESUMO

This study aimed to investigate the prevalence of influenza A viruses in birds and humans residing in the same localities of Sharkia Province, Egypt and the risk factors' assessment in poultry farms. A total of 100 birds comprised of 50 chickens, 25 ducks and 25 wild egrets were sampled. Swab samples were collected from 65 people (50 poultry farm workers and 15 hospitalized patients). All samples were screened for the presence of influenza A viruses using isolation and molecular assays. Avian influenza viruses were only detected in chicken samples (18%) and molecularly confirmed as subtype H5. The infection rate was higher in broilers (40%) than layers (8.6%). Influenza A (H1) pdm09 virus was detected in a single human case (1.54%). All the isolated AI H5 viruses were clustered into clade (2.2.1.2) and shared a high similarity rate at nucleotides and amino acid levels. In addition, they had a multi-basic amino acid motif (ـــPQGEKRRKKR/GLFـــ) at the H5 gene cleavage site that exhibited point mutations. Chicken breed, movement of workers from one flock to another, lack of utensils' disinfection and the introduction of new birds to the farm were significant risk factors associated with highly pathogenic AI H5 virus infection in poultry farms (p ≤ 0.05). Other factors showed no significant association. The HPAI H5 viruses are still endemic in Egypt with continuous mutation. Co-circulation of these viruses in birds and pdm09 viruses in humans raises alarm for the emergence of reassortant viruses that are capable of potentiating pandemics.


Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Sequência de Aminoácidos/genética , Animais , Galinhas/virologia , Patos/virologia , Egito/epidemiologia , Fazendas , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Masculino , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , Prevalência , Fatores de Risco
12.
Emerg Microbes Infect ; 8(1): 650-661, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31014196

RESUMO

Since November 2008, we have conducted active avian influenza surveillance in Bangladesh. Clades 2.2.2, 2.3.4.2, and 2.3.2.1a of highly pathogenic avian influenza H5N1 viruses have all been identified in Bangladeshi live poultry markets (LPMs), although, since the end of 2014, H5N1 viruses have been exclusively from clade 2.3.2.1a. In June 2015, a new reassortant H5N1 virus (H5N1-R1) from clade 2.3.2.1a was identified, containing haemagglutinin, neuraminidase, and matrix genes of H5N1 viruses circulating in Bangladesh since 2011, plus five other genes of Eurasian-lineage low pathogenic avian influenza A (LPAI) viruses. Here we report the status of circulating avian influenza A viruses in Bangladeshi LPMs from March 2016 to January 2018. Until April 2017, H5N1 viruses exclusively belonged to H5N1-R1 clade 2.3.2.1a. However, in May 2017, we identified another reassortant H5N1 (H5N1-R2), also of clade 2.3.2.1a, wherein the PA gene segment of H5N1-R1 was replaced by that of another Eurasian-lineage LPAI virus related to A/duck/Bangladesh/30828/2016 (H3N8), detected in Bangladeshi LPM in September 2016. Currently, both reassortant H5N1-R1 and H5N1-R2 co-circulate in Bangladeshi LPMs. Furthermore, some LPAI viruses isolated from LPMs during 2016-2017 were closely related to those from ducks in free-range farms and wild birds in Tanguar haor, a wetland region of Bangladesh where ducks have frequent contact with migratory birds. These data support a hypothesis where Tanguar haor-like ecosystems provide a mechanism for movement of LPAI viruses to LPMs where reassortment with poultry viruses occurs adding to the diversity of viruses at this human-animal interface.


Assuntos
Evolução Molecular , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Aves Domésticas , Vírus Reordenados/classificação , Vírus Reordenados/genética , Animais , Bangladesh/epidemiologia , Variação Genética , Genótipo , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/epidemiologia , Epidemiologia Molecular , Vírus Reordenados/isolamento & purificação
13.
Emerg Microbes Infect ; 8(1): 262-271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866780

RESUMO

The continuing pandemic threat posed by avian influenza A/H5N1 viruses calls for improved insights into their evolution during human infection. We performed whole genome deep sequencing of respiratory specimens from 44 H5N1-infected individuals from Indonesia and found substantial within-host viral diversity. At nearly 30% of genome positions multiple amino acids were observed within or across samples, including positions implicated in aerosol transmission between ferrets. Amino acid variants detected our cohort were often found more frequently in available H5N1 sequences of human than avian isolates. We additionally identified previously unreported amino acid variants and multiple variants that increased in proportion over time in available sequential samples. Given the importance of the polymerase complex for host adaptation, we tested 121 amino acid variants found in the PB2, PB1 and PA subunits for their effects on polymerase activity in human cells. We identified multiple single amino acid variants in all three polymerase subunits that substantially increase polymerase activity including some with effects comparable to that of the widely recognized adaption and virulence marker PB2-E627 K. These results indicate highly dynamic evolutionary processes during human H5N1 virus infection and the potential existence of previously undocumented adaptive pathways.


Assuntos
Substituição de Aminoácidos , Virus da Influenza A Subtipo H5N1/classificação , Influenza Humana/virologia , Sequenciamento Completo do Genoma/métodos , Evolução Molecular , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Indonésia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/genética
14.
PLoS Pathog ; 15(3): e1007601, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30883607

RESUMO

Influenza viruses (IVs) tend to rapidly develop resistance to virus-directed vaccines and common antivirals targeting pathogen determinants, but novel host-directed approaches might preclude resistance development. To identify the most promising cellular targets for a host-directed approach against influenza, we performed a comparative small interfering RNA (siRNA) loss-of-function screen of IV replication in A549 cells. Analysis of four different IV strains including a highly pathogenic avian H5N1 strain, an influenza B virus (IBV) and two human influenza A viruses (IAVs) revealed 133 genes required by all four IV strains. According to gene enrichment analyses, these strain-independent host genes were particularly enriched for nucleocytoplasmic trafficking. In addition, 360 strain-specific genes were identified with distinct patterns of usage for IAVs versus IBV and human versus avian IVs. The strain-independent host genes served to define 43 experimental and otherwise clinically approved drugs, targeting reportedly fourteen of the encoded host factors. Amongst the approved drugs, the urea-based kinase inhibitors (UBKIs) regorafenib and sorafenib exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were active also against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV1) and vesicular stomatitis virus (VSV) and showed antiviral efficacy in human primary respiratory cells. An in vitro resistance development analysis for regorafenib failed to detect IV resistance development against this drug. Taken together, the otherwise clinically approved UBKIs regorafenib and sorafenib possess high and broad-spectrum antiviral activity along with substantial robustness against resistance development and thus constitute attractive host-directed drug candidates against a range of viral infections including influenza.


Assuntos
Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Replicação Viral/fisiologia , Células A549 , Transporte Ativo do Núcleo Celular/fisiologia , Antivirais , Interações Hospedeiro-Patógeno , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vírus da Influenza B/genética , Vírus da Influenza B/imunologia , Influenza Humana , Orthomyxoviridae/patogenicidade , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/metabolismo , Piridinas/farmacologia , Interferência de RNA/imunologia , Vírus de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Sorafenibe/farmacologia , Ureia/metabolismo
15.
Viruses ; 11(3)2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909490

RESUMO

Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2⁻3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection.


Assuntos
Interações Hospedeiro-Patógeno/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/transmissão , Mutação de Sentido Incorreto , Proteínas Virais/genética , Adaptação Fisiológica , Substituição de Aminoácidos , Animais , Aves , Egito , Humanos , Indonésia , Virus da Influenza A Subtipo H5N1/enzimologia , Influenza Aviária/virologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Replicação Viral
16.
Mol Biol Evol ; 36(7): 1580-1595, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30854550

RESUMO

Subspecies nomenclature systems of pathogens are increasingly based on sequence data. The use of phylogenetics to identify and differentiate between clusters of genetically similar pathogens is particularly prevalent in virology from the nomenclature of human papillomaviruses to highly pathogenic avian influenza (HPAI) H5Nx viruses. These nomenclature systems rely on absolute genetic distance thresholds to define the maximum genetic divergence tolerated between viruses designated as closely related. However, the phylogenetic clustering methods used in these nomenclature systems are limited by the arbitrariness of setting intra and intercluster diversity thresholds. The lack of a consensus ground truth to define well-delineated, meaningful phylogenetic subpopulations amplifies the difficulties in identifying an informative distance threshold. Consequently, phylogenetic clustering often becomes an exploratory, ad hoc exercise. Phylogenetic Clustering by Linear Integer Programming (PhyCLIP) was developed to provide a statistically principled phylogenetic clustering framework that negates the need for an arbitrarily defined distance threshold. Using the pairwise patristic distance distributions of an input phylogeny, PhyCLIP parameterizes the intra and intercluster divergence limits as statistical bounds in an integer linear programming model which is subsequently optimized to cluster as many sequences as possible. When applied to the hemagglutinin phylogeny of HPAI H5Nx viruses, PhyCLIP was not only able to recapitulate the current WHO/OIE/FAO H5 nomenclature system but also further delineated informative higher resolution clusters that capture geographically distinct subpopulations of viruses. PhyCLIP is pathogen-agnostic and can be generalized to a wide variety of research questions concerning the identification of biologically informative clusters in pathogen phylogenies. PhyCLIP is freely available at http://github.com/alvinxhan/PhyCLIP, last accessed March 15, 2019.


Assuntos
Técnicas Genéticas , Filogenia , Programação Linear , Software , Virus da Influenza A Subtipo H5N1/genética
17.
Viruses ; 11(2)2019 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-30813415

RESUMO

Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H7N9 viruses pose a severe threat to public health through zoonotic infection, causing severe respiratory disease in humans. While HPAI H5N1 human infections have typically been reported in Asian countries, avian H7N9 human infections have been reported mainly in China. However, Canada reported a case of fatal human infection by the HPAI H5N1 virus in 2014, and two cases of human illness associated with avian H7N9 virus infection in 2015. While the genomes of the causative viruses A/Alberta/01/2014 (H5N1) (AB14 (H5N1)) and A/British Columbia/1/2015 (H7N9) (BC15 (H7N9)) are reported, the isolates had not been evaluated for their pathogenicity in animal models. In this study, we characterized the pathogenicity of AB14 (H5N1) and BC15 (H7N9) and found that both strain isolates are highly lethal in mice. AB14 (H5N1) caused systemic viral infection and erratic proinflammatory cytokine gene expression in different organs. In contrast, BC15 (H7N9) replicated efficiently only in the respiratory tract, and was a potent inducer for proinflammatory cytokine genes in the lungs. Our study provides experimental evidence to complement the specific human case reports and animal models for evaluating vaccine and antiviral candidates against potential influenza pandemics.


Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Doença Relacionada a Viagens , Animais , Aves/virologia , Canadá/epidemiologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Reação em Cadeia da Polimerase , Replicação Viral
18.
Viruses ; 11(3)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917500

RESUMO

Highly pathogenic avian influenza (HPAI) and Newcastle disease are economically important avian diseases worldwide. Effective vaccination is critical to control these diseases in poultry. Live attenuated Newcastle disease virus (NDV) vectored vaccines have been developed for bivalent vaccination against HPAI viruses and NDV. These vaccines have been generated by inserting the hemagglutinin (HA) gene of avian influenza virus into NDV genomes. In laboratory settings, several experimental NDV-vectored vaccines have protected specific pathogen-free chickens from mortality, clinical signs, and virus shedding against H5 and H7 HPAI viruses and NDV challenges. NDV-vectored H5 vaccines have been licensed for poultry vaccination in China and Mexico. Recently, an antigenically chimeric NDV vector has been generated to overcome pre-existing immunity to NDV in poultry and to provide early protection of poultry in the field. Prime immunization of one-day-old poults with a chimeric NDV vector followed by boosting with a conventional NDV vector has shown to protect broiler chickens against H5 HPAI viruses and a highly virulent NDV. This novel vaccination approach can provide efficient control of HPAI viruses in the field and facilitate poultry vaccination.


Assuntos
Vetores Genéticos , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/genética , Influenza Aviária/prevenção & controle , Vírus da Doença de Newcastle/genética , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/imunologia , Influenza Aviária/virologia , Influenza Humana/virologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Eliminação de Partículas Virais
19.
Influenza Other Respir Viruses ; 13(4): 407-414, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30714323

RESUMO

AIM: Egypt is the habitat for a large number of bird species and serves as a vital stopover for millions of migratory birds during their annual migration between the Palearctic and Afrotropical ecozones. Surveillance for avian influenza viruses (AIVs) is critical to assessing risks for potential spreading of these viruses among domestic poultry. Surveillance for AIV among hunted and captured wild birds in Egypt was conducted in order to understand the characteristics of circulating viruses. METHODS: Sampling of wild bird species occurred in two locations along the Mediterranean Coast of Egypt in the period from 2014 to 2016. A total of 1316 samples (cloacal and oropharyngeal swabs) were collected from 20 different species of hunted or captured resident and migratory birds sold at live bird markets. Viruses were propagated then sequenced. Phylogenetic analysis and receptor binding affinities were studied. RESULTS: Eighteen AIVs (1.37%) were isolated from migratory Anseriformes at live bird markets. Further characterization of the viral isolates identified five hemagglutinin (H3, H5, H7, H9, and H10) and five neuraminidase (N1, N2, N3, N6, and N9) subtypes, which were related to isolates reported in the Eurasian region. Two of the 18 isolates were highly pathogenic H5N1 viruses related to clade 2.2.1, while three isolates were G1-like H9N2 viruses. CONCLUSIONS: Our data show significant diversity of AIVs in Anserifromes sold at live bird markets in Egypt. This allows for genetic exchanges between imported and enzootic viruses and put the exposed humans at a higher risk of infection.


Assuntos
Monitoramento Epidemiológico/veterinária , Influenza Aviária/epidemiologia , Aves Domésticas/virologia , Animais , Animais Selvagens/virologia , Cloaca/virologia , Egito/epidemiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Filogenia , Sequenciamento Completo do Genoma
20.
Arch Virol ; 164(4): 1027-1036, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30740636

RESUMO

This paper describes a preclinical study analyzing the immunogenicity and protective efficacy of Kazfluvac®, an adjuvant-based inactivated pandemic influenza A/H5N1 virus vaccine. In this study, laboratory animals (ferrets and mice) were vaccinated by the intramuscular or intraperitoneal route at an interval of 14 days with two doses of the vaccine containing different concentrations of influenza virus hemagglutinin (HA) protein. HA protein without adjuvant (aluminum hydroxide and Merthiolate) was used as a control. As a negative control, we utilized PBS. We assessed the protective efficacy of the candidate vaccine by analyzing the response to challenge with the influenza virus strain A/chicken/Astana/6/05 (H5N1). Our experimental results revealed substantially reduced clinical disease and an increased antibody response, as determined by hemagglutination-inhibition (HAI) test and microneutralization assay (MNA). This study showed that the candidate vaccine is safe and elicits an antigen-dose-dependent serum antibody response. In summary, we determined the optimum antigen dose in a Kazfluvac® adjuvant formulation required for induction of heightened immunogenicity and protective efficacy to mitigate H5N1 disease in experimental animals, suggesting its readiness for clinical studies in humans.


Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/imunologia , Feminino , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Pandemias , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia
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