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1.
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
2.
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
3.
Afr Health Sci ; 19(1): 1402-1410, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31148967

RESUMO

Background: Avian influence virus H5N1 causes serious public health concern with significant morbidity and mortality from poultry to humans. Interferon-induced transmembrane (IFITM) proteins usually protect cells from many virus infections by viral entry and replication. Objectives: The purpose of this study was to investigate whether H5N1 viral proteins involved in regulation IFITM1, IFITM2, and IFITM3 following H5N1 infection. Methods: NS1, M1, NP, PB2, HA and NA genes of H5N1 virus were generated by PCR and cloned into pcDNA3.1/myc-His (+) A vector for genes over-expression experiments. Gene expression levels was performed using Real-time PCR. Results: Research displayed that NS1, M1, NP, and PB2 proteins of H5N1 virus increased IFITM1, IFITM2, and IFITM3 expression in A549 cells, only IFITM1 was upregulated by M1 in HEK293T cells. However, our study did not find that HA and NA of H5N1 virus affected IFITM genes family or interferon genes expression. Conclusion: Taken together, our data suggested that IFITM1, IFITM2, and IFITM3 might be directly upregulated via NS1, M1, NP, and PB2 proteins during H5N1 avian influenza virus infection. This study provided new insights into the influence of NS1 and NP proteins on regulation of IFITM1, IFITM2, and IFITM3 expression following H5N1 infection.


Assuntos
Antígenos de Diferenciação , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/genética , Células A549 , Proteínas de Transporte , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas do Core Viral , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais , Internalização do Vírus
4.
PLoS One ; 14(5): e0216984, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125350

RESUMO

In the re-emergence of Highly Pathogenic Avian Influenza (HPAI), live bird markets have been identified to play a critical role. In this repeated cross-sectional study, we combined surveillance data collected monthly on Jakarta's live bird markets over a five-year period, with risk factors related to the structure and management of live bird markets, the trading and slaughtering of birds at these markets, and environmental and demographic conditions in the areas where the markets were located. Over the study period 36.7% (95% CI: 35.1, 38.3) of samples (N = 1315) tested HPAI H5 virus positive. Using General Estimation Equation approaches to account for repeated observations over time, we explored the association between HPAI H5 virus prevalence and potential risk factors. Markets where only live birds and carcasses were sold, but no slaughtering was conducted at or at the vicinity of the markets, had a significantly reduced chance of being positive for H5 virus (OR = 0.2, 95% CI 0.1-0.5). Also, markets, that used display tables for poultry carcasses made from wood, had reduced odds of being H5 virus positive (OR = 0.7, 95% CI 0.5-1.0), while having at least one duck sample included in the pool of samples collected at the market increased the chance of being H5 virus positive (OR = 5.7, 95% CI 3.6-9.2). Markets where parent stock was traded, were more at risk of being H5 virus positive compared to markets where broilers were traded. Finally, the human population density in the district, the average distance between markets and origins of poultry sold at markets and the total rainfall per month were all positively associated with higher H5 virus prevalence. In summary, our results highlight that a combination of factors related to trading and marketing processes and environmental pressures need to be considered to reduce H5 virus infection risk for customers at urban live bird markets. In particular, the relocation of slaughter areas to well-managed separate locations should be considered.


Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Animais , Galinhas/virologia , Patos/virologia , Monitoramento Ambiental , Humanos , Indonésia/epidemiologia , Vírus da Influenza A , Influenza Aviária/transmissão , Influenza Aviária/virologia , Aves Domésticas/virologia , Doenças das Aves Domésticas/transmissão , Doenças das Aves Domésticas/virologia , Fatores de Risco
5.
Transbound Emerg Dis ; 66(3): 1306-1313, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30861310

RESUMO

Ducks are the "Trojan Horses" for Asian H5N1 avian influenza viruses (AIV) and attain carrier status without displaying overt infection. These birds help in the spread of the virus among the poultry and human population through direct or indirect contact. Preen oil is the secretion of preen gland of water birds such as ducks. In a process called preening, the water birds spread preen oil across their feather and body. Preen oil has been known to play a significant role in the accumulation of various pathogens including Highly Pathogenic Avian Influenza (HPAI) from water onto feathers. However, the studies are scarce on the role of preen oil in the survivability of HPAIV. We conducted a simulative study to analyse the effect of preen oil on the survivability of the HPAI virus (H5N1) on duck feathers. Duck feather samples along with relevant controls were spiked with the H5N1 virus at two different initial concentrations (104 EID50 and 106 EID50 ), stored at 37°C, 25°C and 10°C temperatures and tested at regular intervals for percent infectivity by egg culture method and qRT-PCR. The infectivity and viral load were significantly higher in naturally preened duck feathers in comparison to the three preen oil deficit controls at both low and high initial concentrations of virus (104 EID50 and 106 EID50 ). Maximum persistence was seen at 10°C in naturally preened duck feathers spiked with 106 EID50 concentration of viruses. It was also seen that depletion of preen oil from duck feathers reduced the persistence of the virus. These results demonstrate that preen oil plays a significant role in survivability and protection of HPAIV on duck feathers. This study herein will present new avenues in understanding one of the epidemiological niches of HPAIV.


Assuntos
Patos/virologia , Plumas/virologia , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/virologia , Animais , Aves , Asseio Animal , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Temperatura Ambiente , Carga Viral
6.
Bull Exp Biol Med ; 166(5): 637-640, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903504

RESUMO

In CBA mice infected with influenza viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05 in a dose of 10 MLD50, the mechanisms of death of pulmonary alveolocytes over 10 postinfection days were studied by light microscopy, immunohistochemistry, and morphometry. In mice infected with A/H1N1, alveolocytes died predominantly via necrosis, while apoptosis mostly employed the mitochondrial pathway. In mice infected with A/H5N1, apoptosis was the dominant mechanism of alveolocyte death proceeded via membrane receptor signaling followed by switching to FAS-mediated pathway via activation of FADD, the apoptotic signal transduction protein.


Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Pulmão/citologia , Alvéolos Pulmonares/virologia , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Infecções por Orthomyxoviridae/virologia , Alvéolos Pulmonares/citologia
7.
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
8.
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
9.
J Biol Chem ; 294(12): 4290-4303, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30737282

RESUMO

Most neutralizing antibodies against highly pathogenic avian influenza A virus H5N1 recognize the receptor-binding site (RBS) on the globular head domain and the stem of H5N1 hemagglutinin (HA). Through comprehensive analysis of multiple human protective antibodies, we previously identified four vulnerable sites (VS1-VS4) on the globular head domain. Among them, the VS1, occupying the opposite side of the RBS on the same HA, was defined by the epitope of antibody 65C6. In this study, we report the crystal structures of two additional human H5N1 antibodies isolated from H5N1-infected individuals, 3C11 and AVFluIgG01, bound to the head at 2.33- and 2.30-Å resolution, respectively. These two new antibody epitopes have large overlap with and extend beyond the original VS1. Site-directed mutagenesis experiments identified eight pivotal residues (Ser-126b, Lys-165, Arg-166, Ser-167, Tyr-168, Asn-169, Thr-171, and Asn-172) critical for 65C6-, 3C11-, and AVFluIgG01-binding and neutralization activities. These residues formed a unique "Y"-shaped surface on H5N1 globular head and are highly conserved among H5N1 viruses. Our results further support the existence of a vulnerable site distinct from the RBS and the stem region of H5N1 HA, and future design of immunogens should take this particular site into consideration.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Cristalografia por Raios X , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Mutagênese Sítio-Dirigida , Conformação Proteica , Reprodutibilidade dos Testes
11.
Bull Exp Biol Med ; 166(3): 358-363, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30627911

RESUMO

Changes in the kidney structure in outbred and inbred male BALB/c mice were analyzed in the acute period after infection with influenza viruses A/H5N1 (10 MLD50; 10 days) and A/H1N1 (1 MLD50; 30 days). Antibodies to influenza viruses of both strains were most often expressed by endothelial cells of the glomeruli and arterioles and were rarely expressed by mesangiocytes and tubule epithelial cells. In the kidney, destructive processes induced by viruses and by ischemia due to massive blood vessel thrombosis. Mesangiocytes expressed factors, indicating that they could be qualified as M1 and M2 macrophages. Kidney destruction was more significant after infection of mice with the A/H5N1 virus, but in both experiments cell infiltrates were actually absent, probably due to blood vessel thrombosis and limited possibility of migration of mononuclear phagocytes and lymphocytes to the kidney.


Assuntos
Células Endoteliais/patologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Glomérulos Renais/patologia , Macrófagos/patologia , Células Mesangiais/patologia , Infecções por Orthomyxoviridae/patologia , Animais , Animais não Endogâmicos , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Endoteliais/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Interleucina-16/genética , Interleucina-16/imunologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/virologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/virologia , Masculino , Células Mesangiais/virologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
Am J Physiol Lung Cell Mol Physiol ; 316(3): L525-L536, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30628490

RESUMO

The highly pathogenic H5N1 (HK483) viral infection causes a depressed hypercapnic ventilatory response (dHCVR, 20%↓) at 2 days postinfection (dpi) and death at 7 dpi in mice, but the relevant mechanisms are not fully understood. Glomus cells in the carotid body and catecholaminergic neurons in locus coeruleus (LC), neurokinin 1 receptor (NK1R)-expressing neurons in the retrotrapezoid nucleus (RTN), and serotonergic neurons in the raphe are chemosensitive and responsible for HCVR. We asked whether the dHCVR became worse over the infection period with viral replication in these cells/neurons. Mice intranasally inoculated with saline or the HK483 virus were exposed to hypercapnia for 5 min at 0, 2, 4, or 6 dpi, followed by immunohistochemistry to determine the expression of nucleoprotein of H5N1 influenza A (NP) alone and coupled with 1) tyrosine hydroxylase (TH) in the carotid body and LC, 2) NK1R in the RTN, and 3) tryptophan hydroxylase (TPH) in the raphe. HK483 viral infection blunted HCVR by ∼20, 50, and 65% at 2, 4, and 6 dpi. The NP was observed in the pontomedullary respiratory-related nuclei (but not in the carotid body) at 4 and 6 dpi, especially in 20% of RTN NK1R, 35% of LC TH, and ∼10% raphe TPH neurons. The infection significantly reduced the local NK1R or TPH immunoreactivity and population of neurons expressing NK1R or TPH. We conclude that the HK483 virus infects the pontomedullary respiratory nuclei, particularly chemosensitive neurons in the RTN, LC, and raphe, contributing to the severe depression of HCVR and respiratory failure at 6 dpi. NEW & NOTEWORTHY The H5N1 virus infection is lethal due to respiratory failure, but the relevant mechanisms remain unclear. In this study, we demonstrated a gradual diminution of hypercapnic ventilatory response to a degree, leading to respiratory failure over a 6-day infection. Death was associated with viral replication in the pontomedullary respiratory-related nuclei, especially the central chemosensitive neurons. These results not only provide insight into the mechanisms of the lethality of H5N1 viral infection but also offer clues in the development of corresponding treatments to minimize and prevent respiratory failure.


Assuntos
Hipercapnia/virologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Humana/virologia , Neurônios/virologia , Animais , Aves/virologia , Feminino , Humanos , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Camundongos Endogâmicos BALB C , Receptores da Neurocinina-1/metabolismo
13.
J Infect Dis ; 219(2): 186-196, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30085072

RESUMO

Background: Highly pathogenic avian influenza viruses can cause severe forms of acute lung injury (ALI) in humans, where pulmonary flooding leads to respiratory failure. The therapeutic benefits of bone marrow mesenchymal stromal cells (MSCs) have been demonstrated in a model of ALI due to influenza A(H5N1) virus. However, clinical translation is impractical and limited by a decline in efficacy as the age of the donor increases. Umbilical cord MSCs (UC-MSCs) are easier to obtain by comparison, and their primitive source may offer more-potent therapeutic effects. Methods: Here we investigate the therapeutic efficacy of UC-MSCs on the mechanisms of pulmonary edema formation and alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs were also tested in a mouse model of influenza ALI. Results: We found that UC-MSCs were effective in restoring impaired alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs consistently outperformed bone marrow MSCs, partly because of greater growth factor secretion of angiopoietin 1 and hepatocyte growth factor. Conditioned UC-MSC medium and UC-MSC exosomes were also able to recapitulate these effects. However, UC-MSCs only slightly improved survival of A(H5N1)-infected mice. Conclusions: Our results suggest that UC-MSCs are effective in restoring alveolar fluid clearance and protein permeability in A(H5N1)-associated ALI and confer functional in addition to practical advantages over conventional bone marrow MSCs.


Assuntos
Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/prevenção & controle , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Humana/terapia , Células-Tronco Mesenquimais/fisiologia , Cordão Umbilical , Células Epiteliais Alveolares , Angiopoietina-1/metabolismo , Animais , Líquidos Corporais/fisiologia , Medula Óssea , Modelos Animais de Doenças , Exossomos , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Influenza Humana/complicações , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/terapia , Permeabilidade , Edema Pulmonar
14.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30381488

RESUMO

The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.


Assuntos
Vírus da Influenza A/patogenicidade , Mutação , Vírus Reordenados/patogenicidade , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , Animais , Embrião de Galinha , Galinhas , Cães , Células HEK293 , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H7N1/genética , Vírus da Influenza A Subtipo H7N1/patogenicidade , Vírus da Influenza A/genética , Influenza Aviária/virologia , Células Madin Darby de Rim Canino , Vírus Reordenados/genética
15.
Vet Microbiol ; 228: 101-111, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593354

RESUMO

Ducks are the reservoir host of influenza A viruses, and are permissive for replication of most strains, yet can elicit robust innate immune responses to highly pathogenic strains. Tissue tropism and viral amino acid differences affect virulence, but we have limited knowledge about how viral differences influence the host innate immune response. Here we compare the innate immune response in Pekin ducks to a recombinant highly-pathogenic avian influenza (HPAI) H5N1 virus and a naturally arising attenuated variant of this strain that differs at one amino acid in polymerase A (T515A), as well as ducks infected with two different H5 strains of low pathogenic avian influenza (LPAI). Using qPCR we examined the relative abundance of transcripts for RIG-I and interferon-beta (IFNß), and downstream interferon stimulated genes (ISGs). The polymerase PA (T515A) mutation did not significantly affect replication in vivo but greatly attenuated host interferon responses. ISG induction was robust for both H5N1 strains, but was three times lower for the PA mutant strain. Low pathogenic viruses elicited detectable induction of RIG-I, IFNß and ISGs in lung and intestine tissues that correlated with the recovery of viruses from tracheal or cloacal swabs. Several genes in the MAVS signaling pathway were also upregulated by H5N1, which contributed to further amplification of the signal. We also examined hematoxylin-eosin stained tissue sections and observe evidence of lung pathology and splenocyte depletion with both H5N1 viruses at 3 dpi, and recovery by 6 dpi. However, for both H5N1 strains we observed inflammation around neurons in brain, with increased cytokine expression in some individuals. Our findings reveal HPAI H5N1 viruses induced stronger innate immune responses to the infection, while LPAI viruses elicit a milder response.


Assuntos
Patos/virologia , Imunidade Inata , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Citocinas/imunologia , Feminino , Regulação da Expressão Gênica , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/virologia , Interferon beta/imunologia , Masculino , Doenças das Aves Domésticas/virologia
16.
PLoS One ; 13(12): e0208197, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30566454

RESUMO

In the past two decades, avian influenzas have posed an increasing international threat to human and livestock health. In particular, highly pathogenic avian influenza H5N1 has spread across Asia, Africa, and Europe, leading to the deaths of millions of poultry and hundreds of people. The two main means of international spread are through migratory birds and the live poultry trade. We focus on the role played by the live poultry trade in the spread of H5N1 across three regions widely infected by the disease, which also correspond to three major trade blocs: the European Union (EU), the Economic Community of West African States (ECOWAS), and the Association of Southeast Asian Nations (ASEAN). Across all three regions, we found per-capita GDP (a proxy for modernization, general biosecurity, and value-at-risk) to be risk reducing. A more specific biosecurity measure-general surveillance-was also found to be mitigating at the all-regions level. However, there were important inter-regional differences. For the EU and ASEAN, intra-bloc live poultry imports were risk reducing while extra-bloc imports were risk increasing; for ECOWAS the reverse was true. This is likely due to the fact that while the EU and ASEAN have long-standing biosecurity standards and stringent enforcement (pursuant to the World Trade Organization's Agreement on the Application of Sanitary and Phytosanitary Measures), ECOWAS suffered from a lack of uniform standards and lax enforcement.


Assuntos
Comércio/normas , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Pandemias/estatística & dados numéricos , Zoonoses/epidemiologia , África Ocidental/epidemiologia , Criação de Animais Domésticos/normas , Animais , Ásia Sudeste/epidemiologia , Comércio/estatística & dados numéricos , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Humanos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/prevenção & controle , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Pandemias/economia , Pandemias/prevenção & controle , Aves Domésticas/virologia , Zoonoses/prevenção & controle , Zoonoses/virologia
17.
PLoS One ; 13(11): e0207431, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30440054

RESUMO

Outbreaks of the highly pathogenic avian influenza H5N1 and H7N9 viruses have spurred an unprecedented research effort to develop antivirals and vaccines against influenza. Standardized methods for vaccine evaluation are critical for facilitating vaccine development. Compared with hemagglutination inhibition assays, mounting evidence suggest that microneutralization tests (MNTs) is a better choice for the evaluation of candidate pandemic influenza vaccines because they measure neutralizing antibody activity in cell cultures and are more sensitive in detecting H5 and H7. Here, we report a MNT measuring neuraminidase activity as the read-out (NA-MNT) for quantitative analysis of neutralizing antibodies against avian influenza viruses. Compared to the conventional microneutralization assay (ELISA-MNT), the NA-MNT is faster with lower intra- and inter-assay variations, while no difference in geometric mean titers was found between these two assays for the evaluation of H5N1 and H7N9 vaccines. These results suggest that NA-MNT is a reliable and high throughput method which could facilitate the development of candidate pandemic influenza vaccine.


Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Aves/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/imunologia , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Neuraminidase/genética , Neuraminidase/imunologia , Pandemias , Vacinas de Produtos Inativados/imunologia
18.
BMC Microbiol ; 18(1): 138, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30340527

RESUMO

BACKGROUND: Endothelial cells play a major role in highly pathogenic avian influenza (HPAI) virus pathogenesis in gallinaceous poultry species (e.g. chicken, turkey and quail). Upon infection of gallinaceous poultry with HPAI viruses, endothelial cells throughout the body become rapidly infected, leading to systemic dissemination of the virus, disseminated intravascular coagulation, oedema and haemorrhaging. In contrast, the pathogenesis of HPAI viruses in most wild bird species (e.g. duck, goose and gull species) is not associated with endothelial tropism. Indeed, viral antigen is not found in the endothelial cells of most wild bird species following infection with HPAI viruses. This differential endothelial cell tropism in avian species is poorly understood, mainly due to the absence of appropriate cell culture systems. RESULTS: Here, we describe the isolation and purification of primary duck endothelial cells from the aorta or bone marrow of Pekin duck embryos. Cells were differentiated in the presence of vascular endothelial growth factor and, if needed, enriched via fluorescent-activated cell sorting based on the uptake of acetylated low-density lipoprotein. The expression of von Willebrand factor, a key marker of endothelial cells, was confirmed by polymerase chain reaction. Monocultures of duck endothelial cells, either derived from the aorta or the bone marrow, were susceptible to infection with an H5N1 HPAI virus but to a much lesser extent than chicken endothelial cells. CONCLUSIONS: The methods described herein to isolate and purify duck endothelial cells from the aorta or bone marrow could also be applied to obtain microvascular endothelial cells from other tissues and organs, such as the lung or the intestine, and represent a valuable tool to study the pathogenesis of avian viruses.


Assuntos
Técnicas de Cultura de Células , Células Endoteliais/virologia , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/patogenicidade , Animais , Antígenos Virais , Aorta/citologia , Aorta/virologia , Células da Medula Óssea/virologia , Células Cultivadas , Patos/virologia , Citometria de Fluxo , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
19.
Sci Adv ; 4(9): eaas9930, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30221194

RESUMO

Adjuvants are key to shaping the immune response to vaccination, but to date, no adjuvant suitable for human use has been developed for intradermal vaccines. These vaccines could be self-administered and sent through the mail as they do not require long needles or technical expertise in immunization. In the event of a pandemic outbreak, this approach could alleviate the congregation of patients in health centers and thus reduce the potential of these centers to enhance the spread of lethal infection. A reliable and potent vaccine system for self-administration would provide an effective countermeasure for delivery through existing product distribution infrastructure. We report results from preclinical and clinical trials that demonstrate the feasibility of an adjuvanted, intradermal vaccine that induced single shot protection in ferrets and seroprotection in humans against one of the more lethal strains of pandemic flu, Indonesia H5N1. In the human trial, the vaccine was safe and clinical responses were above approvable endpoints for a protective flu vaccine. Inclusion of a modern TLR4 (Toll-like receptor 4) agonist-based adjuvant was critical to the development of the response in the intradermal groups. In humans, this is the first report of a safe and effective intradermal adjuvant, GLA-AF (aqueous formulation of glucopyranosyl lipid adjuvant), and provides a future path for developing a vaccine-device combination for distribution by mail and self-administration in case of a pandemic.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/imunologia , Adjuvantes Imunológicos/farmacologia , Vacinas contra Influenza/farmacologia , Lipídeo A/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/efeitos adversos , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adulto , Animais , Combinação de Medicamentos , Feminino , Furões , Cobaias , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Injeções Intradérmicas , Lipídeo A/efeitos adversos , Lipídeo A/imunologia , Lipídeo A/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/agonistas
20.
Emerg Microbes Infect ; 7(1): 147, 2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-30131494

RESUMO

The highly pathogenic avian influenza (HPAI) A(H5N1) virus is endemic in Indonesian poultry and has caused sporadic human infection in Indonesia since 2005. Surveillance of H5N1 viruses in live bird markets (LBMs) during 2012 and 2013 was carried out to provide epidemiologic and virologic information regarding viral circulation and the risk of human exposure. Real-time RT-PCR of avian cloacal swabs and environmental samples revealed influenza A-positive specimens, which were then subjected to virus isolation and genomic sequencing. Genetic analysis of specimens collected at multiple LBMs in Indonesia identified both low pathogenicity avian influenza (LPAI) A(H3N8) and HPAI A(H5N1) viruses belonging to clade 2.1.3.2a. Comparison of internal gene segments among the LPAI and HPAI viruses revealed that the latter had acquired the PB2, PB1, and NS genes from LPAI progenitors and other viruses containing a wild type (wt) genomic constellation. Comparison of murine infectivity of the LPAI A(H3N8), wt HPAI A(H5N1) and reassortant HPAI A(H5N1) viruses showed that the acquisition of LPAI internal genes attenuated the reassortant HPAI virus, producing a mouse infectivity/virulence phenotype comparable to that of the LPAI virus. Comparison of molecular markers in each viral gene segment suggested that mutations in PB2 and NS1 may facilitate attenuation. The discovery of an attenuated HPAI A(H5N1) virus in mice that resulted from reassortment may have implications for the capability of these viruses to transmit and cause disease. In addition, surveillance suggests that LBMs in Indonesia may play a role in the generation of reassortant A(H5) viruses and should be monitored.


Assuntos
Vírus da Influenza A Subtipo H3N8/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Doenças das Aves Domésticas/virologia , Recombinação Genética , Animais , Galinhas , Criança , Pré-Escolar , Feminino , Humanos , Indonésia , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Vírus da Influenza A Subtipo H3N8/patogenicidade , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Virulência
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