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1.
Biochemistry (Mosc) ; 84(11): 1221-1232, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760913

RESUMO

The review covers three independent blocks of research. The first one is discovery, isolation, and investigation of snake venom RNases and their use in studying RNA macrostructure. It has been established that snake venom RNases are not specific to the primary RNA structure but rather to the RNA helical conformation (double, single, or hybrid helix). Snake venom RNases hydrolyze RNA to short oligomers with the 5'-terminal phosphate. Analysis of the kinetics and products of tRNA hydrolysis exemplifies the use of snake venom RNases for deciphering RNA macrostructure. The second block is devoted to the principle formulated by the author for analyzing the primary structure of nucleic acids and describes the method of direct RNA sequencing that has been developed with author's participation. The third block describes the results of genotyping and etiologic control of epidemic influenza A viruses circulating in the Soviet Union in 1968 to 1992. The method for comparative analysis of genome sequences of viral isolates has made it possible to detect and characterize epidemic influenza virus strains that had emerged in the circulation as a result of reactivation of inactivated vaccines.


Assuntos
RNA/química , Humanos , Hidrólise , Vírus da Influenza A/genética , Cinética , Conformação de Ácido Nucleico , RNA/metabolismo , RNA/ultraestrutura , RNA Viral/química , RNA Viral/metabolismo , Ribonucleases/metabolismo , Venenos de Serpentes/metabolismo
2.
Ned Tijdschr Geneeskd ; 1632019 08 29.
Artigo em Holandês | MEDLINE | ID: mdl-31483584

RESUMO

An influenza epidemic can greatly increase the workload in primary care and the emergency department (ED) and can even disrupt the healthcare system. It is difficult to diagnose influenza by history taking and physical examination. A fast diagnosis usinginfluenza point-of-care tests (POCTs) could reduce unnecessary antibiotic prescriptions, diagnostic tests, consultations and hospital admissions. Moreover, length of stay on EDs and length of admission could be shortened. The analytical accuracy of antigen detection tests for influenza is relatively low compared to the well performing RT-PCR assays (sensitivity and specificity approximately 95%). Only 1 randomized controlled trial has shown the effect of a (combined) RT-PCR assay for influenza detection on clinically relevant outcome measures. Observational research suggests that introduction of RT-PCR assays for influenza detection reduces length of stay on the ED and decreased time from sample reception to result. For practical reasons, we should embrace the introduction of RT-PCR assays for influenza detection on EDs. Before POCTs can be implemented in primary care (family medicine) the analytical accuracy and time to receive results should be improved and effects of its clinical impact should be proven.


Assuntos
Serviço Hospitalar de Emergência , Medicina Geral , Influenza Humana/diagnóstico , Programas de Rastreamento/métodos , Testes Imediatos , Reação em Cadeia da Polimerase , Antibacterianos/uso terapêutico , Humanos , Vírus da Influenza A/genética , Influenza Humana/virologia , Tempo de Internação , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 904-910, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31484252

RESUMO

Objective: To analyze the etiologic and epidemiological characteristics of adult acute respiratory infections in Shanghai during 2015-2017. Methods: Data was collected from outpatients with acute respiratory infections who visited the Fever Clinics in three hospitals of different levels in three administrative regions of Shanghai, from 2015 to 2017. Basic information and nasopharyngeal swabs were collected from cases in line with the inclusion criteria. Multiplex RT-PCR and bacterial cultures were performed to detect the respiratory pathogens. Results: A total of 806 individuals were enrolled from 2015 to 2017. Respiratory pathogens were identified in 73.45% (592/806) of the cases, with the virus detection rate as 66.75% (538/806). It was found that the major respiratory pathogens for virus detection were influenza A in 326 (40.45%), influenza B in 116 (14.39%), rhinovirus/enterovirus in 39 (4.84%) of the cases. The overall detection rate of bacteria was 16.13% (130/806), including Klebsiella pneumoniae in 90 (11.17%) cases, Staphylococcus Aureus in 46 (5.71%) cases. Other kind of bacteria were not detected in our study. The detection rates on Mycoplasma pneumoniae was 5.33% (43/806) and on Chlamydia pneumonia was 0.37% (3/806). Co-infection with multiple pathogens was detected in 18.61% (150/806) of the cases, including 135 with double infection (accounting for 90.00%), 14 with triple infection and 1 with quadruple infection (accounted for 9.33% and 0.67%, respectively). Among the 150 cases with co-infections, the main identified pathogens were influenza A, Klebsiella pneumoniae, Staphylococcus aureus, and Mycoplasma pneumoniae. Pathogens of acute respiratory infections that identified among the outpatients from the Fever Clinics at different time, region or population, the characteristics were different (P<0.001). Conclusions: In 2015-2017, outpatients with acute respiratory infections in Shanghai were mainly caused by influenza virus or other viruses, however dynamically with its composition, time, region and characteristics of the population. It is necessary to strengthen and combine related medical and preventive services and to develop the appropriate strategies regarding clinical diagnosis and treatment.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Nasofaringe , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/etiologia , Viroses/diagnóstico , Vírus/isolamento & purificação , Doença Aguda , Adulto , Bactérias/genética , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , China/epidemiologia , Coinfecção/diagnóstico , Enterovirus/genética , Enterovirus/isolamento & purificação , Monitoramento Epidemiológico , Humanos , Incidência , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Mycoplasma pneumoniae , Nasofaringe/microbiologia , Nasofaringe/virologia , Vigilância da População , Infecções Respiratórias/diagnóstico , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Viroses/epidemiologia , Viroses/virologia , Vírus/genética
4.
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
5.
Int J Infect Dis ; 88: 113-119, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31401200

RESUMO

Here we review evidence for influenza A viruses (IAVs) moving from swine, avian, feline, equine, and canine species to infect humans. We review case reports, sero-epidemiological, archeo-epidemiological, environmental, and historical studies and consider trends in livestock farming. Although this focused review is not systematic, the aggregated data point to industrialized swine farming as the most likely source of future pandemic viruses, yet IAV surveillance on such farms is remarkably sparse. We recommend increased biosafety and biosecurity training for farm administrators and swine workers with One Health-oriented virus surveillance throughout industrialized farming and meat production lines. Collaborative partnerships with human medical researchers could aid in efforts to mitigate emerging virus threats by offering new surveillance and diagnostic technologies to livestock farming industries.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/veterinária , Animais , Aves , Gatos , Cães , Cavalos , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Suínos
6.
PLoS Pathog ; 15(8): e1007892, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31415678

RESUMO

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.


Assuntos
Aves/virologia , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Células A549 , Animais , Cães , Feminino , Cobaias , Humanos , Influenza Humana/genética , Influenza Humana/metabolismo , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Especificidade da Espécie , Proteínas da Matriz Viral/genética
7.
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
8.
Environ Health Prev Med ; 24(1): 53, 2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31421676

RESUMO

BACKGROUND: Influenza A viruses pose a significant risk to human health because of their wide host range and ability to reassort into novel viruses that can cause serious disease and pandemics. Since transmission of these viruses between humans and pigs can be associated with occupational and environmental exposures, we investigated the association between occupational exposure to pigs, occurrence of acute respiratory illness (ARI), and influenza A virus infection. METHODS: The study was conducted in Kiambu County, the county with the highest level of intensive small-scale pig farming in Kenya. Up to 3 participants (> 2 years old) per household from pig-keeping and non-pig-keeping households were randomly recruited and followed up in 2013 (Sept-Dec) and 2014 (Apr-Aug). Oropharyngeal (OP) and nasopharyngeal (NP) swabs were collected from participants with ARI at the time of study visit. For the animal study, nasal and oropharyngeal swabs, and serum samples were collected from pigs and poultry present in enrolled households. The human and animal swab samples were tested for viral nucleic acid by RT-PCR and sera by ELISA for antibodies. A Poisson generalized linear mixed-effects model was developed to assess the association between pig exposure and occurrence of ARI. RESULTS: Of 1137 human participants enrolled, 625 (55%) completed follow-up visits including 172 (27.5%) pig workers and 453 (72.5%) non-pig workers. Of 130 human NP/OP swabs tested, four (3.1%) were positive for influenza A virus, one pig worker, and three among non-pig workers. Whereas none of the 4462 swabs collected from pig and poultry tested positive for influenza A virus by RT-PCR, 265 of 4273 (6.2%) of the sera tested positive for virus antibodies by ELISA, including 11.6% (230/1990) of the pigs and 1.5% (35/2,283) of poultry. The cumulative incidence of ARI was 16.9% among pig workers and 26.9% among the non-pig workers. The adjusted risk ratio for the association between being a pig worker and experiencing an episode of ARI was 0.56 (95% CI [0.33, 0.93]), after adjusting for potential confounders. CONCLUSIONS: Our findings demonstrate moderate seropositivity for influenza A virus among pigs, suggesting the circulation of swine influenza virus and a potential for interspecies transmission.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/epidemiologia , Infecções por Orthomyxoviridae/epidemiologia , Zoonoses/epidemiologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/sangue , Criança , Feminino , Humanos , Incidência , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Influenza Humana/transmissão , Influenza Humana/virologia , Quênia/epidemiologia , Masculino , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/transmissão , Faringe/virologia , Aves Domésticas/virologia , RNA Viral/genética , Fatores de Risco , Estudos Soroepidemiológicos , Suínos/virologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , Adulto Jovem , Zoonoses/transmissão
9.
J Biomed Sci ; 26(1): 55, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366399

RESUMO

BACKGROUND: Mutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter. METHODS: Using entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses. RESULTS: We found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses. CONCLUSIONS: Our results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.


Assuntos
Vírus da Influenza A/fisiologia , Proteínas Virais/genética , Replicação Viral/genética , Adaptação Fisiológica/genética , Animais , Cães , Células HEK293 , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Mutação/genética , Proteínas Virais/metabolismo , Virulência/genética
10.
Nat Commun ; 10(1): 3396, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363119

RESUMO

Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.


Assuntos
Vírus da Influenza A/enzimologia , Influenza Aviária/genética , Processamento de RNA , Proteínas de Ligação a RNA/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Aves , Galinhas , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Influenza Aviária/metabolismo , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral
11.
Rev Soc Bras Med Trop ; 52: e20180249, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365620

RESUMO

INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.


Assuntos
Influenza Humana/epidemiologia , Infecções por Paramyxoviridae/epidemiologia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções Respiratórias/virologia , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Vírus da Influenza A/genética , Irã (Geográfico)/epidemiologia , Masculino , Metapneumovirus/genética , Nasofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/epidemiologia
12.
BMC Vet Res ; 15(1): 253, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324180

RESUMO

BACKGROUND: Avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) are important avian pathogens that can cause enormous economic loss on the poultry industry. Different respiratory etiological agents may induce similar clinical signs that make differential diagnosis difficult. Importantly, AIV brings about severe threat to human public health. Therefore, a novel method that can distinguish these viruses quickly and simultaneously is urgently needed. RESULTS: In this study, an oligonucleotide microarray system was developed. AIV, including H5, H7, and H9 subtypes; NDV; and IBV were simultaneously detected and differentiated on a microarray. Three probes specific for AIV, NDV, and IBV, as well as three other probes for differentiating H5, H7, and H9 of AIV, were first designed and jet-printed to predetermined locations of initiator-integrated poly(dimethylsiloxane) for the synchronous detection of the six pathogens. The marked multiplex reverse transcription polymerase chain reaction (PCR) products were hybridized with the specific probes, and the results of hybridization were read directly with the naked eyes. No cross-reaction was observed with 10 other subtypes of AIV and infectious bursal disease virus, indicating that the oligonucleotide microarray assay was highly specific. The sensitivity of the method was at least 100 times higher than that of the conventional PCR, and the detection limit of NDV, AIV, H5, H7, and H9 can reach 0.1 EID50 (50% egg infective dose), except that of IBV, which was 1 EID50 per reaction. In the validation of 93 field samples, AIV, IBV, and NDV were detected in 53 (56.99%) samples by oligonucleotide microarray and virus isolation and in 50 (53.76%) samples by conventional PCR. CONCLUSIONS: We have successfully developed an approach to differentiate AIV, NDV, IBV, H5, H7, and H9 subtypes of AIV using oligonucleotide microarray. The microarray is an accurate, high-throughput, and relatively simple method for the rapid detection of avian respiratory viral diseases. It can be used for the epidemiological surveillance and diagnosis of AIV, IBV, and NDV.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Influenza A/genética , Vírus da Doença de Newcastle/genética , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Doenças das Aves Domésticas/virologia , Animais , Infecções por Coronavirus/virologia , Vírus da Doença Infecciosa da Bursa/genética , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária , Doença de Newcastle/virologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Aves Domésticas , Sensibilidade e Especificidade
13.
Nat Microbiol ; 4(10): 1671-1679, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31263181

RESUMO

Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1-3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1 (NXF1-NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1-NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1-NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1-NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.


Assuntos
Núcleo Celular/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/genética
14.
Virology ; 534: 132-142, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31255797

RESUMO

The chicken upper respiratory tract is the portal of entry for respiratory pathogens including avian influenza virus (AIV). There is a paucity of information about the role of airway epithelial cells in the induction of antiviral responses in the chicken trachea. A better understanding of the role of these cells in the initiation of innate responses may improve prophylactic or therapeutic strategies for control of viral infections. The present study aimed to characterize antiviral innate responses in chicken tracheal epithelial cells (cTECs) induced by TLR ligands. The results demonstrated that stimulation of cTECs with TLR ligands induced antiviral responses, and subsequently reduced the replication of AIV in cTECs. Additionally, stimulated cTECs were able to influence the function of other cells such as macrophages. Overall, these results provided evidence that cTECs mount antiviral responses after stimulation with TLR ligands through IRF7 and NF-κB signaling pathways, leading to activation of other cells, such as macrophages.


Assuntos
Células Epiteliais/imunologia , Vírus da Influenza A/fisiologia , Influenza Aviária/imunologia , Macrófagos/imunologia , Doenças das Aves Domésticas/imunologia , Traqueia/virologia , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/imunologia , Galinhas , Células Epiteliais/virologia , Imunidade Inata , Vírus da Influenza A/genética , Influenza Aviária/genética , Influenza Aviária/virologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Ligantes , Macrófagos/virologia , Poli I-C/imunologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Traqueia/citologia , Traqueia/imunologia
15.
Virology ; 534: 36-44, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176062

RESUMO

Bangladesh has reported repeated outbreaks of highly pathogenic avian influenza (HPAI) A(H5) viruses in poultry since 2007. Because of the large number of live poultry markets (LPM) relative to the population density of poultry throughout the country, these markets can serve as sentinel sites for HPAI A(H5) detection. Through active LPM surveillance during June 2016-June 2017, HPAI A(H5N6) viruses along with 14 other subtypes of influenza A viruses were detected. The HPAI A(H5N6) viruses belonged to clade 2.3.4.4 and were likely introduced into Bangladesh around March 2016. Human infections with influenza clade 2.3.4.4 viruses in Bangladesh have not been identified, but the viruses had several molecular markers associated with potential human infection. Vigilant surveillance at the animal-human interface is essential to identify emerging avian influenza viruses with the potential to threaten public and animal health.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Animais , Bangladesh/epidemiologia , Surtos de Doenças , Patos , Evolução Molecular , Gansos/virologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Aviária/epidemiologia , Filogenia , Doenças das Aves Domésticas/epidemiologia
16.
J Microbiol Biotechnol ; 29(8): 1184-1192, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154753

RESUMO

The influenza A virus is a highly infectious respiratory pathogen that sickens many people with respiratory disease annually. To prevent outbreaks of this viral infection, an understanding of the characteristics of virus-host interaction and development of an anti-viral agent is urgently needed. The influenza A virus can infect mammalian species including humans, pigs, horses and seals. Furthermore, this virus can switch hosts and form a novel lineage. This so-called zoonotic infection provides an opportunity for virus adaptation to the new host and leads to pandemics. Most influenza A viruses express proteins that antagonize the antiviral defense of the host cell. The non-structural protein 1 (NS1) of the influenza A virus is the most important viral regulatory factor controlling cellular processes to modulate host cell gene expression and double-stranded RNA (dsRNA)-mediated antiviral response. This review focuses on the influenza A virus NS1 protein and outlines current issues including the life cycle of the influenza A virus, structural characterization of the influenza A virus NS1, interaction between NS1 and host immune response factor, and design of inhibitors resistant to the influenza A virus.


Assuntos
Vírus da Influenza A/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/prevenção & controle , Conformação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
17.
Int J Mol Sci ; 20(11)2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151205

RESUMO

This study aimed at assessing the frequency and the distribution of influenza virus types/subtypes in 172 laboratory-confirmed influenza-positive patients admitted to intensive care units (ICUs) during the 2017-2018 season in the Lombardy region (Northern Italy), and to investigate the presence of molecular pathogenicity markers. A total of 102/172 (59.3%) patients had influenza A infections (83 A/H1N1pdm09, 2 H3N2 and 17 were untyped), while the remaining 70/172 (40.7%) patients had influenza B infections. The 222G/N mutation in the hemagglutinin gene was identified in 33.3% (3/9) of A/H1N1pdm09 strains detected in the lower respiratory tract (LRT) samples and was also associated with more severe infections, whereas no peculiar mutations were observed for influenza B strains. A single-point evolution was observed in site 222 of A/H1N1pdm09 viruses, which might advantage viral evolution by favouring virus binding and replication in the lungs. Data from 17 paired upper respiratory tract (URT) and LRT samples showed that viral load in LRT samples was mostly higher than that detected in URT samples. Of note, influenza viruses were undetectable in 35% of paired URT samples. In conclusion, LRT samples appear to provide more accurate clinical information than URT samples, thus ensuring correct diagnosis and appropriate treatment of patients with severe respiratory infections requiring ICU admission.


Assuntos
Cuidados Críticos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/epidemiologia , Influenza Humana/virologia , Unidades de Terapia Intensiva , Admissão do Paciente , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Cuidados Críticos/métodos , Cuidados Críticos/estatística & dados numéricos , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , História do Século XXI , Humanos , Lactente , Recém-Nascido , Influenza Humana/história , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Filogenia , Vigilância em Saúde Pública , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/história , Infecções Respiratórias/virologia , Estações do Ano , Carga Viral , Adulto Jovem
18.
Virol J ; 16(1): 85, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31242907

RESUMO

BACKGROUND: Type A influenza viruses (IAVs) cause significant infections in humans and multiple species of animals including pigs, horses, birds, dogs and some marine animals. They are of complicated phylogenetic diversity and distribution, and analysis of their phylogenetic diversity and distribution from a panorama view has not been updated for multiple years. METHODS: 139,872 protein sequences of IAVs from GenBank were selected, and they were aligned and phylogenetically analyzed using the software tool MEGA 7.0. Lineages and subordinate lineages were classified according to the topology of the phylogenetic trees and the host, temporal and spatial distribution of the viruses, and designated using a novel universal nomenclature system. RESULTS: Large phylogenetic trees of the two external viral genes (HA and NA) and six internal genes (PB2, PB1, PA, NP, MP and NS) were constructed, and the diversity and the host, temporal and spatial distribution of these genes were calculated and statistically analyzed. Various features regarding the diversity and distribution of IAVs were confirmed, revised or added through this study, as compared with previous reports. Lineages and subordinate lineages were classified and designated for each of the genes based on the updated panorama views. CONCLUSIONS: The panorama views of phylogenetic diversity and distribution of IAVs and their nomenclature system were updated and assumed to be of significance for studies and communication of IAVs.


Assuntos
Evolução Molecular , Variação Genética , Vírus da Influenza A/genética , Filogenia , Sequência de Aminoácidos , Animais , Aves/virologia , Quirópteros/virologia , Cães/virologia , Genes Virais , Cavalos/virologia , Humanos , Suínos/virologia
19.
Transbound Emerg Dis ; 66(5): 2188-2195, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31241237

RESUMO

The 1918 Spanish flu virus has claimed more than 50 million lives. However, the mechanism of its high pathogenicity remains elusive; and the origin of the virus is controversial. The matrix (M) segment regulates the replication of influenza A virus, thereby affecting its virulence and pathogenicity. This study found that the M segment of the Spanish flu virus is a recombinant chimera originating from avian influenza virus and human influenza virus. The unique mosaic M segment might confer the virus high replication capacity, showing that the recombination might play an important role in inducing high pathogenicity of the virus. In addition, this study also suggested that the NA and NS segments of the virus were generated by reassortment between mammalian and avian viruses. Direct phylogenetic evidence was also provided for its avian origin.


Assuntos
Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Vírus Reordenados/genética , Animais , Galinhas , Humanos , Vírus da Influenza A/patogenicidade , Influenza Pandêmica, 1918-1919 , Filogenia , Vírus Reordenados/patogenicidade
20.
Virology ; 534: 80-86, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31220651

RESUMO

The immunopathological mechanisms as well as the role played by influenza A virus infection of human leukocytes and induction of apoptosis have not been fully elucidated. We confirm here that the percentage of cells that are infected is less than the percent of apoptotic cells. Depletion of monocytes/macrophages and depletion of cells expressing influenza neuraminidase from the cultures after exposure to virus decreased lymphocyte apoptosis. Treatment of virus-exposed leukocyte cultures with anti-neuraminidase antibodies but not with anti-hemagglutinin antibodies, reduced lymphocyte production of active caspase-3 and induction of apoptosis. Different strains of virus induced different levels of apoptosis. Variations in induction of apoptosis correlated with production and expression of viral neuraminidase by infected leukocytes. The data suggest that cell surface expression of neuraminidase plays an important role in the induction of apoptosis in human lymphocytes. The benefit, or cost, to the host of lymphocyte apoptosis warrants continued investigation.


Assuntos
Apoptose , Membrana Celular/virologia , Vírus da Influenza A/enzimologia , Influenza Humana/virologia , Linfócitos/citologia , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/enzimologia , Influenza Humana/genética , Influenza Humana/fisiopatologia , Linfócitos/virologia , Neuraminidase/genética , Proteínas Virais/genética
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