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1.
PLoS One ; 15(2): e0229326, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32078666

RESUMO

As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study.


Assuntos
Surtos de Doenças/veterinária , Patos/virologia , Vírus da Influenza A Subtipo H5N8/classificação , Vírus da Influenza A Subtipo H5N8/genética , Infecções por Orthomyxoviridae/veterinária , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodos , Animais , Genoma Viral , Infecções por Orthomyxoviridae/virologia , RNA Viral/análise , RNA Viral/genética , Análise de Sequência de DNA
2.
PLoS Pathog ; 16(1): e1008191, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951644

RESUMO

Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.


Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Animais , Camboja , Patos/virologia , Evolução Molecular , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Mutação , Filogenia , Proteínas Virais/genética
3.
Arch Virol ; 165(3): 643-659, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31925543

RESUMO

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain.


Assuntos
Aves/virologia , Patos/virologia , Genoma Viral/genética , Vírus da Influenza A Subtipo H3N8/genética , Influenza Aviária/virologia , África , Sequência de Aminoácidos , Migração Animal , Animais , Animais Selvagens/virologia , Sequência de Bases , Europa (Continente) , Variação Genética/genética , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Japão , América do Norte , Filogenia , Recombinação Genética/genética , Análise de Sequência de RNA
4.
Arch Virol ; 165(1): 87-96, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707455

RESUMO

In May 2017, high mortality of chickens and Muscovy ducks due to the H5N8 highly pathogenic avian influenza virus (HPAIV) was reported in the Democratic Republic of Congo (DR Congo). In this study, we assessed the molecular, antigenic, and pathogenic features in poultry of the H5N8 HPAIV from the 2017 Congolese outbreaks. Phylogenetic analysis of the eight viral gene segments revealed that all 12 DR Congo isolates clustered in clade 2.3.4.4B together with other H5N8 HPAIVs isolated in Africa and Eurasia, suggesting a possible common origin of these viruses. Antigenically, a slight difference was observed between the Congolese isolates and a representative virus from group C in the same clade. After intranasal inoculation with a representative DR Congo virus, high pathogenicity was observed in chickens and Muscovy ducks but not in Pekin ducks. Viral replication was higher in chickens than in Muscovy duck and Pekin duck organs; however, neurotropism was pronounced in Muscovy ducks. Our data confirmed the high pathogenicity of the DR Congo virus in chickens and Muscovy ducks, as observed in the field. National awareness and strengthening surveillance in the region are needed to better control HPAIVs.


Assuntos
Antígenos Virais/metabolismo , Vírus da Influenza A Subtipo H5N8/classificação , Vírus da Influenza A Subtipo H5N8/patogenicidade , Influenza Aviária/imunologia , Doenças das Aves Domésticas/virologia , África , Animais , Ásia , Galinhas , República Democrática do Congo , Patos/classificação , Patos/virologia , Europa (Continente) , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Influenza A Subtipo H5N8/genética , Vírus da Influenza A Subtipo H5N8/isolamento & purificação , Influenza Aviária/virologia , Filogenia , Filogeografia , Doenças das Aves Domésticas/imunologia , Especificidade da Espécie , Replicação Viral
5.
Arch Virol ; 165(3): 709-714, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31873767

RESUMO

Duck Tembusu virus (DTMUV) has caused significant economic losses in China since 2010. However, there is still a lack of effective methods to diagnose the disease caused by this virus, and especially to differentiate infection from vaccination. In this study, we established a novel indirect enzyme-linked immunosorbent assay (iELISA) and performed a retrospective serological survey for DTMUV in Anhui province, China. Our results show that the iELISA displayed high specificity sensitivity, and with no serological cross-reaction with other duck pathogens. These findings indicate that the newly developed iELISA could be a useful screening tool for large-scale monitoring of the epidemiology of DTMUV infection in ducks.


Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Infecções por Flavivirus/diagnóstico , Flavivirus/imunologia , Doenças das Aves Domésticas/diagnóstico , Proteínas não Estruturais Virais/imunologia , Animais , China , Patos/virologia , Flavivirus/isolamento & purificação , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/virologia , Doenças das Aves Domésticas/virologia , Estudos Retrospectivos , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética
6.
Emerg Microbes Infect ; 8(1): 377-380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31851878

RESUMO

Highly pathogenic avian influenza (HPAI) is a persistent threat to poultry, wild birds, humans, and other mammals. The continually evolving HPAI H5N6 virus has induced great losses in breeding industries in growing regions around the world. In this study, we confirmed an outbreak of the HPAI H5N6 virus in captive Pavo cristatus in Jiangxi Province, China. The causative agents H5N6 viruses were isolated and designated JS01, JS02, and K10. Animal experiments showed that all three isolates exhibited high pathogenicity to chickens, but they need adaption to effectively infect mice. A phylogenetic analysis showed that all three isolates were clustered in H5 clade 2.3.4.4c. No novel genetic reassortant was found in JS01, JS02, and K10 viruses. It was estimated that JS01, JS02, and K10 H5N6 viruses were direct descendants of the H5N6 virus circulating in South of China. The estimated divergence time from tMRCAs was anywhere between May 2014 to June 2016. Although the number of outbreaks of avian influenza decreased significantly in 2018, the threat from avian influenza to public health remains serious. Enhanced active surveillance is required to monitor the transmission and evolution of H5 influenza viruses.


Assuntos
Galinhas/virologia , Surtos de Doenças/veterinária , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Animais , China/epidemiologia , Patos/virologia , Feminino , Genoma Viral , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/epidemiologia , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia
7.
BMC Vet Res ; 15(1): 362, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31651323

RESUMO

BACKGROUND: Tembusu virus (TMUV) usually affects adult ducks, causing a severe drop of egg production. It has also been shown to be pathogenic in commercial Pekin ducklings below 7 weeks of age. Here, we report a TMUV-caused neurological disease in young egg-type ducklings and the pathogenicity of the egg-type duck-origin TMUV isolates in meat-type Pekin ducklings. RESULTS: The disease occurred in 25 to 40-day-old Jinding ducklings in China, and was characterized by paralysis. Gross lesions were lacking and microscopic lesions appeared chiefly in brain and spleen. Inoculation in embryonated duck eggs resulted in isolation of TMUV Y and GL. The clinical signs and microscopic lesions observed in the spontaneously infected egg-type ducks were repeated in Pekin ducklings by experimental infection. Notably, both Y and GL strains caused 100% mortality in the case of 2-day-old inoculation by intracerebral route. High mortalities (80 and 70%) also occurred following infection of the Y virus at 2 days of age by intramuscular route and at 9 days of age by intracerebral route. CONCLUSIONS: These findings demonstrate that the egg-type duck-origin TMUVs exhibit high pathogenicity in Pekin ducklings, and that the severity of the disease in ducklings is dependent on the infection route and the age of birds at the time of infection. The availability of the highly pathogenic TMUV strains provides a useful material with which to begin investigations into the molecular basis of TMUV pathogenicity in ducks.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/patogenicidade , Doenças das Aves Domésticas/virologia , Fatores Etários , Animais , Linhagem Celular , Cricetinae , Vias de Administração de Medicamentos/veterinária , Patos/virologia , Flavivirus/genética , Infecções por Flavivirus/patologia , Infecções por Flavivirus/virologia , Paralisia/veterinária , Paralisia/virologia , Doenças das Aves Domésticas/patologia
8.
Mater Sci Eng C Mater Biol Appl ; 105: 110052, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546360

RESUMO

Novel duck reovirus (NDRV) disease is a serious infectious disease for poultry, for which no effective therapy has been established. Therefore, development of novel antivirals against NDRV is urgently needed. In present study, we developed a complex wherein hypericin (HY), which shows broad-spectrum antiviral activity, was loaded onto graphene oxide (GO), which has a high drug-loading capacity and low cytotoxicity. The antiviral activity of the complex (GO/HY) was studied in DF-1 cells and in ducklings infected with the NDRV TH11 strain. GO/HY showed a dose-dependent inhibition of NDRV replication, which may be attributed to direct virus inactivation or inhibition of virus attachment. Western blotting and indirect immunofluorescence assay (IFA) showed markedly suppressed protein expression in GO/HY-treated NDRV-infected DF-1 cells. Moreover, GO/HY prolonged the survival time of the ducklings by reducing pathological lesions caused by the infection and inhibiting viral replication in the liver and lungs. These results suggest that GO/HY has antiviral activity against NDRV both in vitro and in vivo.


Assuntos
Portadores de Fármacos , Patos , Grafite , Orthoreovirus Aviário/metabolismo , Perileno/análogos & derivados , Doenças das Aves Domésticas , Infecções por Reoviridae , Animais , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Patos/metabolismo , Patos/virologia , Grafite/química , Grafite/farmacologia , Perileno/química , Perileno/farmacologia , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/tratamento farmacológico , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/patologia , Infecções por Reoviridae/veterinária
9.
Arch Virol ; 164(11): 2837-2841, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31494776

RESUMO

Since January 2019, abnormal molting has been observed frequently in approximately 40-day-old Pekin ducks in China. To investigate the possible involvement of a virus, we tested the prevalence of duck circovirus (DuCV), goose hemorrhagic polyomavirus (GHPyV), and goose parvovirus (GPV) in 11 molt cases in two provinces. GPV was detected in all cases, particularly in all samples collected from the feather area. The complete genome sequences of three GPV strains were determined and found to have 52 nucleotide changes relative to GPVs associated with short beak and dwarfism syndrome of Pekin ducks. These data will enhance our understanding of GPV diversity and outcomes of GPV infection in Pekin ducks.


Assuntos
Patos/virologia , Gansos/virologia , Muda/fisiologia , Parvovirinae/isolamento & purificação , Doenças das Aves Domésticas/virologia , Animais , China/epidemiologia , Circovirus/genética , Circovirus/isolamento & purificação , Genoma Viral/genética , Parvovirinae/genética , Polyomavirus/genética , Polyomavirus/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia
10.
Virol J ; 16(1): 112, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488178

RESUMO

BACKGROUND: Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. METHODS: In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. RESULTS: The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 µg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. CONCLUSIONS: An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.


Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/genética , Avastrovirus/isolamento & purificação , Patos/virologia , Genética Reversa/métodos , Cultura de Vírus/métodos , Animais , Infecções por Astroviridae/virologia , Avastrovirus/crescimento & desenvolvimento , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Genoma Viral , Plasmídeos , RNA Viral/genética , Transfecção , Tropismo Viral , Vírion/genética
11.
BMC Infect Dis ; 19(1): 762, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477028

RESUMO

BACKGROUND: Avian influenza A (H5N6) virus poses a great threat to the human health since it is capable to cross the species barrier and infect humans. Although human infections are believed to largely originate from poultry contaminations, the transmissibility is unclear and only limited information was available on poultry environment contaminations, especially in Fujian Province. METHODS: A total of 4901 environmental samples were collected and tested for Avian Influenza Virus (AIV) from six cities in Fujian Province through the Fujian Influenza Surveillance System from 2013 to 2017. Two patient-related samples were taken from Fujian's first confirmed H5N6 human case and his backyard chicken feces in 2017. Chi-square test or Fisher's exact probability test was used to compare the AIV and the viral subtype positive rates among samples from different Surveillance cities, surveillance sites, sample types, and seasons. Phylogenetic tree analysis and molecular analysis were conducted to track the viral transmission route of the human infection and to map out the evolutions of H5N6 in Fujian. RESULTS: The overall positive rate of the H5 subtype AIVs was 4.24% (208/4903). There were distinctive differences (p < 0.05) in the positive rates in samples from different cities, sample sites, sample types and seasons. The viruses from the patient and his backyard chicken feces shared high homologies (99.9-100%) in all the eight gene segments. Phylogenetic trees also showed that these two H5N6 viruses were closely related to each other, and were classified into the same genetic clade 2.3.4.4 with another six H5N6 isolates from the environmental samples. The patient's H5N6 virus carried genes from H6N6, H5N8 and H5N6 viruses originated from different areas. The R294K or N294S substitution was not detected in the neuraminidase (NA). The S31 N substitution in the matrix2 (M2) gene was detected but only in one strain from the environmental samples. CONCLUSIONS: The H5 subtype of AIVs has started circulating in the poultry environments in Fujian Province. The patient's viral strain originated from the chicken feces in his backyard. Genetic reassortment in H5N6 viruses in Fujian Province was indicated. The H5N6 viruses currently circulating in Fujian Province were still commonly sensitive to Oseltamivir and Zanamivir, but the resistance against Amantadine has emerged.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Aves Domésticas/virologia , Animais , Embrião de Galinha , Galinhas/virologia , China/epidemiologia , Patos/virologia , Meio Ambiente , Microbiologia Ambiental , Genes Virais , Abrigo para Animais/normas , Humanos , Vírus da Influenza A/genética , Influenza Aviária/diagnóstico , Influenza Aviária/epidemiologia , Tipagem Molecular , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Fatores de Risco
12.
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
13.
Transbound Emerg Dis ; 66(6): 2605-2610, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31402584

RESUMO

Wild and domestic aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). All subtypes of AIVs, including 16 hemagglutinin (HA) and nine neuraminidase (NA), have been isolated from the waterfowls. The H5 viruses in wild birds display distinct biological differences from their highly pathogenic H5 counterparts. Here, we isolated seven H5N3 AIVs including three from wild birds and four from domestic ducks in China from 2015 to 2018. The isolation sites of all the seven viruses were located in the region of the East Asian-Australasian Migratory Flyway. Phylogenetic analysis indicated that the surface genes of these viruses originated from the wild bird H5 HA subtype and the N3 Eurasian lineage. The internal genes of the seven H5N3 isolates are derived from the five gene donors isolated from the wild birds or ducks in Eastern-Asia region. They were also divided into five genotypes according to their surface genes and internal gene combinations. Interestingly, two of the seven H5N3 viruses contributed their partial internal gene segments (PB1, M and NS) to the newly emerged H7N4 reassortants, which have caused first human H7N4 infection in China in 2018. Moreover, we found that the H5N3 virus used in this study react with the anti-serum of the H5 subtype vaccine isolate (Re-11 and Re-12) and reacted well with the Re-12 anti-serum. Our findings suggest that worldwide intensive surveillance and the H5 vaccination (Re-11 and Re-12) in domestic ducks are needed to monitor the emergence of novel H5N3 reassortants in wild birds and domestic ducks and to prevent H5N3 viruses transmission from the apparently healthy wild birds and domestic ducks to chickens.


Assuntos
Animais Selvagens/virologia , Patos/virologia , Vírus da Influenza A/genética , Influenza Aviária/virologia , Vírus Reordenados/genética , Animais , China/epidemiologia , Genótipo , Influenza Aviária/epidemiologia , Filogenia
14.
Vet Microbiol ; 235: 151-163, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282373

RESUMO

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.


Assuntos
Proteínas Cdc20/metabolismo , Chaperonina com TCP-1/metabolismo , Orthoreovirus/genética , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/veterinária , Proteínas não Estruturais Virais/metabolismo , Animais , Apoptose , Caspase 8/genética , Caspase 8/metabolismo , Proteínas Cdc20/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Chaperonina com TCP-1/genética , Patos/virologia , Fibroblastos/virologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Interferente Pequeno , Células Vero , Proteínas não Estruturais Virais/genética
15.
Transbound Emerg Dis ; 66(6): 2209-2217, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31309743

RESUMO

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is also a public concern due to sporadic human infections being reported in China. In the current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were most closely related (99.6%-99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4%-99.9% homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to 2018. This indicated that AI470 was introduced into Vietnam from China. In addition, molecular characteristics related to mammalian adaptation among the recent human H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are cause for concern since H5N6 HPAIV strains that possess a risk of human infection have crossed the Chinese border.


Assuntos
Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Influenza Humana/virologia , Substituição de Aminoácidos , Animais , China , Patos/virologia , Humanos , Vírus da Influenza A/genética , Filogenia , Vírus Reordenados , Análise de Sequência , Vietnã
16.
Transbound Emerg Dis ; 66(6): 2342-2352, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31293102

RESUMO

The first human case of zoonotic H7N9 avian influenza virus (AIV) infection was reported in March 2013 in China. This virus continues to circulate in poultry in China while mutating to highly pathogenic AIVs (HPAIVs). Through monitoring at airports in Japan, a novel H7N3 reassortant of the zoonotic H7N9 HPAIVs, A/duck/Japan/AQ-HE30-1/2018 (HE30-1), was detected in a poultry meat product illegally brought by a passenger from China into Japan. We analysed the genetic, pathogenic and antigenic characteristics of HE30-1 by comparing it with previous zoonotic H7N9 AIVs and their reassortants. Phylogenetic analysis of the entire HE30-1 genomic sequence revealed that it comprised at least three different sources; the HA (H7), PB1, PA, NP, M and NS segments of HE30-1 were directly derived from H7N9 AIVs, whereas the NA (N3) and PB2 segments of HE30-1 were unrelated to zoonotic H7N9. Experimental infection revealed that HE30-1 was lethal in chickens but not in domestic or mallard ducks. HE30-1 was shed from and replicated in domestic and mallard ducks and chickens, whereas previous zoonotic H7N9 AIVs have not adapted well to ducks. This finding suggests the possibility that HE30-1 may disseminate to remote area by wild bird migration once it establishes in wild bird population. A haemagglutination-inhibition assay indicated that antigenic drift has occurred among the reassortants of zoonotic H7N9 AIVs; HE30-1 showed similar antigenicity to some of those H7N9 AIVs, suggesting it might be prevented by the H5/H7 inactivated vaccine that was introduced in China in 2017. Our study reports the emergence of a new reassortant of zoonotic H7N9 AIVs with novel viral characteristics and warns of the challenge we still face to control the zoonotic H7N9 AIVs and their reassortants.


Assuntos
Patos/virologia , Vírus da Influenza A Subtipo H7N3/genética , Vírus da Influenza A Subtipo H7N3/patogenicidade , Vírus Reordenados , Animais , China , Genoma Viral , Influenza Aviária/virologia , Japão , Filogenia , Sequenciamento Completo do Genoma
17.
Influenza Other Respir Viruses ; 13(5): 496-503, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31187583

RESUMO

BACKGROUND: Highly pathogenic avian influenza (HPAI) A(H7N9) virus emerged and caused human infections during the 2016-2017 epidemic wave of influenza A(H7N9) viruses in China. We report a human infection with HPAI H7N9 virus and six environmental isolates in Fujian Province, China. METHODS: Environmental surveillance was conducted in live poultry markets and poultry farms in Fujian, China. Clinical and epidemiologic data and samples were collected. Real-time RT-PCRs were conducted for each sample, and H7-positive samples were isolated using embryonated chicken eggs. Full genomes of the isolates were obtained by next-generation sequencing. Phylogenetic analysis and antigenic analysis were conducted. RESULTS: A 59-year-old man who raised about 1000 ducks was identified as HPAI H7N9 infection. Six HPAI H7 viruses were isolated from environmental samples, including five H7N9 viruses and one H7N6 virus. Phylogenetic results showed the human and environmental viruses are highly genetically diverse and containing significantly different gene constellation from that of other HPAI H7N9 previously reported. The internal genes derived from H7N9/H9N2, H5N6, and the Eurasian wild-bird gene pool, indicating waterfowl-originated genotypes, have emerged in HPAI H7N9/N6 viruses and caused human infection. CONCLUSION: The new genotypes raise the concern that these HPAI H7 viruses might transmit back into migratory birds and spread to other countries as the HPAI H5Nx viruses. Considering their capability of causing severe infections in both human and poultry, the HPAI H7 viruses in this study pose a risk to public health and the poultry industry and highlight the importance of sustained surveillance of these viruses.


Assuntos
Patos/virologia , Genoma Viral , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/transmissão , Influenza Humana/epidemiologia , Animais , China/epidemiologia , Epidemias , Monitoramento Epidemiológico , Humanos , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Masculino , Pessoa de Meia-Idade , Filogenia , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , Saúde Pública
18.
Vet Microbiol ; 233: 1-4, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176393

RESUMO

Domestic ducks are considered as the interface between wild aquatic birds and terrestrial poultry and play an important role in the transmission and evolution of avian influenza viruses (AIVs). However, the infectivity of H9N2 AIVs in different domestic duck species has not been systematically evaluated. Here we investigated the infectivity of various genotypes of chicken H9N2 AIVs in Pekin duck (Anas Platyrhynchos), Mallard duck (Anas Platyrhynchos) and Muscovy duck (Cairina Moschata) through intranasal inoculation. We found that Pekin ducks and Mallard ducks were generally resistant to chicken H9N2 virus infection, while Muscovy ducks were relatively susceptible to H9N2 AIVs. All the tested viruses were isolated from oropharynx, trachea and lung tissues of Muscovy ducks. Additionally, genotype 57 (G57) H9N2 AIVs, which was predominant in chickens since 2010, showed increased virus replication in this duck species, indicating an improved interspecies transmission ability of recent H9N2 viruses from chickens to ducks. Our results demonstrated the role of Muscovy ducks in the ecology of H9N2 AIVs. More attentions should be paid to this host during viral surveillances. Additionally, inactivated H9N2 vaccine may be unnecessarily used in Pekin and Mallard ducks.


Assuntos
Patos/virologia , Influenza Aviária/transmissão , Doenças das Aves Domésticas/virologia , Replicação Viral , Animais , Galinhas/virologia , Suscetibilidade a Doenças , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Pulmão/virologia , Orofaringe/virologia , Traqueia/virologia
19.
Vet Microbiol ; 233: 85-92, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176417

RESUMO

Muscovy duck reovirus (MDRV) causes serious immunodeficiency in the intestinal mucosa, although the underlying histopathological mechanisms remain unclear. Thus, we investigated the impact of MDRV infection on intestinal morphology using hematoxylin and eosin staining. Immune-related cells were also quantified by staining with hematoxylin and eosin, toluidine blue, and periodic acid-Schiff stain, or by immunohistochemistry and cytochemistry for lectin. Similarly, CD4+ and CD8+ cells were quantified by flow cytometry, and the expression of several immune-related molecules was quantified by radioimmunoassay. We found that MDRV clearly damaged the intestinal mucosa, based on tissue morphology, villus length, villus width, intestinal thickness, villus height/crypt depth ratio, and villus surface area. MDRV also altered the density or distribution of lymphocytes, mastocytes, and goblet cells in the small intestinal mucosa, as well as microfold cells in Peyer's patches. In addition, MDRV markedly depleted CD4+ cells from the intestinal mucosa and lowered the CD4+:CD8+ ratio in peripheral blood. Moreover, MDRV diminished the levels of secretory IgA and mucosal addressin cell adhesion molecule-1 (p < 0.01), but elevated those of histamine and nitric oxide (p < 0.01 or p < 0.05). Finally, MDRV significantly suppressed IL-1ß, IL-4, IL-5, and IL-8 levels (p < 0.01 or p < 0.05) mid-infection. Collectively, our data suggest that MDRV severely damages the structure and function of the intestinal mucosa by modulating immune cells and immune-related factors, thus leading to local immunodeficiency. Our findings lay the foundation for further research on the pathogenesis of MDRV.


Assuntos
Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/virologia , Orthoreovirus Aviário/imunologia , Infecções por Reoviridae/imunologia , Fatores Etários , Animais , Contagem de Linfócito CD4 , Citocinas/imunologia , Patos/virologia , Duodeno , Fibroblastos/virologia , Histamina/análise , Imunoglobulina A Secretora/análise , Intestino Delgado/imunologia , Óxido Nítrico/análise , Orthoreovirus Aviário/patogenicidade , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/patologia , Carga Viral
20.
Artigo em Inglês | MEDLINE | ID: mdl-31174705

RESUMO

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections.


Assuntos
Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Aves Domésticas/virologia , Animais , Galinhas/virologia , Cianose/virologia , Patos/virologia , Egito/epidemiologia , Influenza Aviária/mortalidade , Doenças das Aves Domésticas/mortalidade , Reação em Cadeia da Polimerase em Tempo Real , Estudos Soroepidemiológicos , Perus/virologia , Virulência
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