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2.
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
3.
Biosens Bioelectron ; 143: 111632, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31479987

RESUMO

We present a sunlight based handheld smartphone spectrometer. The device first gathers the sunlight to pass through the sample, and then the transmitted light illuminates on a grating to generate spectrum finally recorded by the smartphone monochrome camera. All the optical elements are assembled with the smartphone to integrate a handheld device with the size of 140.2 mm × 67.4 mm × 80.5 mm. Besides, a smartphone application is also developed for automatic spectral calibration, detection, analysis and display. Compared to the white light emitting diode and the halogen lamp, the sunlight has more uniform distribution covering the entire visible spectral range; and the proposed device also avoids the bulky sizes of those broadband light sources. Additionally, the monochrome camera is used instead of the color camera not only to pursue a high spectral resolution as 0.276 nm/pixel but also to avoid the color overlapping. We demonstrate the device capability on detecting avian influenza virus H7N9 and porcine circovirus type 2 antibodies, proving the device has rather high sensitivity similar to the commercial microplate reader. Considering its advantages as compact size, high spectral resolution and detecting sensitivity, it is believed the proposed sunlight based handheld smartphone spectrometer is potential to be broadly applied in on-site detections.


Assuntos
Técnicas Biossensoriais , Circovirus/isolamento & purificação , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Smartphone , Animais , Aves/virologia , Circovirus/patogenicidade , Colorimetria , Humanos , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Refratometria , Análise Espectral , Luz Solar , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia
4.
Colloids Surf B Biointerfaces ; 182: 110341, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284148

RESUMO

In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/diagnóstico , Nanopartículas Metálicas/química , Animais , Galinhas , Eletrodos , Ouro/química , Influenza Aviária/sangue , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Conformação de Ácido Nucleico , Ressonância de Plasmônio de Superfície , Compostos de Estanho/química
5.
Avian Pathol ; 48(5): 492-498, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31203638

RESUMO

An exogenous "armoured" PCR internal control (IC) short RNA was analyzed in conjunction with real-time RT-PCR method for diagnosis of avian influenza. The resistance to nucleases and increased physical stability of the IC was ensured using branched polyethyleneimine (PEI) which was in complex with IC-RNA. The option to add the IC directly to pathological material suspensions allows measurement of the nucleic acids extraction efficiency. Stability of armoured RNA-IC during storage and tissue suspension preparation was shown. The advantage of exogenous "armoured" IC was demonstrated in the experiment with AIV genome detection by qPCR in samples from different species of wild birds. The exogenous IC gave reproducible homogeneous Ct values in all tests.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Animais , Aves , Primers do DNA/genética , Vírus da Influenza A/genética , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sensibilidade e Especificidade
6.
Biosens Bioelectron ; 134: 123-129, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986614

RESUMO

We report a selection of a cognate pair of aptamers for whole avian influenza virus particles of H5N2 by using graphene-oxide based systemic evolution of ligands by exponential enrichment (GO-SELEX), and the application of a pair of sandwich-type binding aptamers on the lateral flow strips. The aptamers were characterized by GO-FRET assay, and Kd values of the selected aptamers were estimated to be from 6.913 × 105 to 1.27 × 106 EID50/ml (EID50/ml: 50% egg infective dose). Based on the evidence from confocal laser scanning microscope (CLSM), surface plasmon resonance (SPR), and circular dichroism (CD) spectrum analysis, the aptamers, J3APT and JH4APT, were found to be working as a cognate pair that binds to the target virus at the different sites simultaneously. This cognate pair of aptamers then was successfully applied on the lateral flow strips, clearly showing sandwich-type binding images with the presence of the certain numbers of H5N2 virus particles. On the newly developed lateral flow strips, the target virus was detectable down to 6 × 105 EID50/ml in the buffer and 1.2 × 106 EID50/ml in the duck's feces, respectively, by the naked eye. By using the ImageJ software, the LOD was found to be 1.27 × 105 EID50/ml in the buffer and 2.09 × 105 EID50/ml in the duck's feces, respectively. Interestingly, on the lateral flow strips, enhanced specificity towards the target virus (H5N2) appeared over other subtypes of H5Nx. To the best of our knowledge, this is the first report about the application of the cognate pair of aptamers for the detection of influenza virus on the lateral flow strips. This study shows the promising perspective of a cognate pair of aptamers for the on-site detection system which could be useful for rapid detection of avian influenza viruses for preventing the pandemic influenza viruses from spreading.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Grafite/química , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Influenza Aviária/virologia , Vírion/isolamento & purificação , Animais , Técnicas Biossensoriais/instrumentação , Patos/virologia , Desenho de Equipamento , Fezes/virologia , Influenza Aviária/diagnóstico , Limite de Detecção , Fitas Reagentes/análise , Técnica de Seleção de Aptâmeros
7.
Anal Biochem ; 572: 52-57, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844367

RESUMO

Since 2013, the H7 subtype avian influenza virus (AIV-H7) has seriously endangered human life and health, and has had a serious impact on the poultry industry in China. A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects the antibody for AIV-H7 was developed, basing on a monoclonal antibody (mAb) against the neutralizing epitopes on hemagglutinin (HA)gene. Twelve hybridoma cell lines were screened by cell fusion. Hemagglutination inhibition (HI) assay and indirect ELISA were used to identify the competitive effect of the mAbs. High-affinity mAb 1H11 was selected as a competitive antibody. The reaction conditions for the C-ELISA were optimized for AIV-H7 antibody detection. The cross-reactivity of the C-ELISA was determined by AIV-(H1H15), NDV, IBV and IBDV positive serum. A total of 1294 field samples (chicken (462), duck (318), goose (219), quail (203) and pigeon (92) were simultaneously detected by C-ELISA and HI assay. The C-ELISA was found to have a high specificity of 93.23% and a sensitivity of 96.24%. These results reveal a positive coincidence between C-ELISA and HI assay at a coincidence rate of 97.52%. In addition, It confirmed that this method can be used for the diagnosis of AIV-H7 antibodies from chicken, ducks, goose, quail and pigeons.


Assuntos
Anticorpos Antivirais/análise , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Influenza A/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Galinhas , Patos , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/imunologia , Influenza Aviária/diagnóstico , Sensibilidade e Especificidade
8.
Arch Virol ; 164(4): 1111-1119, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790106

RESUMO

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.


Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/isolamento & purificação , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Galinhas , Patos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H5N2/classificação , Vírus da Influenza A Subtipo H5N2/genética , Influenza Aviária/diagnóstico , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/genética , Doenças das Aves Domésticas/diagnóstico , Sensibilidade e Especificidade
9.
J Virol Methods ; 265: 121-125, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633948

RESUMO

Rapid and accurate diagnosis of influenza virus infection is essential for quick responses for both human and animal health. The Alere™ i Influenza A&B is a novel isothermal nucleic acid amplification kit that can detect and differentiate between influenza A and B viruses in human specimens in approximately 15 min. In the present study, the performance of the Alere™ i Influenza A&B kit was evaluated for its ability to detect avian influenza virus in chickens. The kit was able to detect representative avian influenza virus strains (hemagglutinin subtypes H1-H16, including the recently isolated H5 and H7 highly pathogenic avian influenza viruses), and the detection limit of the kit for these viruses varied between 10-1.4-102.1 50% egg-infective dose per test, which is higher than the analytical sensitivity of the antigen detection immunochromatography kit ESPLINE® A INFLUENZA. In experimentally infected chickens inoculated with a highly pathogenic avian influenza virus strain A/chicken/Hokkaido/002/2016 (H5N6), viral RNA was detected in the tracheal and cloacal swabs. These results indicate that this kit has the potential to be used as a rapid screening test of influenza A virus infection in chickens.


Assuntos
Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Aviária/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Aves Domésticas/diagnóstico , Animais , Galinhas , Cloaca/virologia , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Sensibilidade e Especificidade , Traqueia/virologia
10.
ACS Nano ; 13(1): 812-820, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30596428

RESUMO

The thin film transistor (TFT) is a promising biosensor system with great sensitivity, label-free detection, and a quick response time. However, even though the TFT sensor has such advantageous characteristics, the disadvantages hamper the TFT sensor's application in the clinical field. The TFT is susceptible to light, noise, vibration, and limited usage, and this significantly limits its on-site potential as a practical biosensor. Herein, we developed a fully packaged, portable TFT electrochemical biosensor into a chip form, providing both portability through minimizing the laboratory equipment size and multiple safe usages by protecting the semiconductor sensor. Additionally, a safe environment that serves as a miniature probe station minimizes the previously mentioned disadvantages, while providing the means to properly link the TFT biosensor with a portable analyzer. The biosensor was taken into a biosafety level 3 (BSL-3) laboratory setting to analyze highly pathogenic avian influenza virus (HPAIV) samples. This virus quickly accumulates within a host, and therefore, early stage detection is critical to deterring the further spread of the deadly disease to other areas. However, current on-site methods have poor limits of detection (105-106 EID50/mL), and because the virus has low concentration in its early stages, it cannot be detected easily. We have compared the sample measurements from our device with virus concentration data obtained from a RT-PCR (virus range: 100-104 EID50/mL) and have identified an increasing voltage signal which corresponds to increasing virus concentration.


Assuntos
Técnicas Biossensoriais/métodos , Influenza Aviária/virologia , Técnicas de Diagnóstico Molecular/veterinária , Transistores Eletrônicos/normas , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/veterinária , Patos/virologia , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Influenza Aviária/diagnóstico , Miniaturização , Técnicas de Diagnóstico Molecular/instrumentação , Sensibilidade e Especificidade
11.
Transbound Emerg Dis ; 66(1): 341-348, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30267611

RESUMO

Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro-amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro-amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G-coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real-time reverse transcription polymerase chain reaction (rRT-PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50 ml of phosphate-buffered saline (PBS) and 1 ml of 10%-25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30 min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro-amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.


Assuntos
Criação de Animais Domésticos/métodos , Patos , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Doenças das Aves Domésticas/diagnóstico , Vírion/fisiologia , Virologia/métodos , Animais , Fezes/virologia , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia
12.
Poult Sci ; 98(3): 1494-1499, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30476286

RESUMO

Using Luminex xMAP (x = analyte, MAP = multi-analyte profiling) technology, a serological method for the simultaneous detection of antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV) was established. Nano-magnetic beads coated with purified NDV protein and AIV nucleoprotein were incubated with serum samples. Using biotinylated rabbit anti-chicken IgY and streptavidin-R-phycoerythrin, the optical signals measured by a Luminex 200 detection system indicated the quantification of NDV or AIV antibodies in the serum. Specific pathogen-free (SPF) chicken serum was used as a negative control. The Luminex xMAP assay developed in this study demonstrated high specificity as there was no cross-reaction with antibodies to infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus, avian leukosis virus, and Marek's disease virus. The results from reproducibility experiments showed that intra-coefficients of variation were 3.36 and 9.23% and inter-coefficients of variation were 6.50 and 7.66% for NDV and AIV, respectively. The results also indicated that the Luminex xMAP assay was 16 times more sensitive for NDV antibody detection and 1,024 times more sensitive for AIV antibody detection compared to the enzyme-linked immunosorbent assay (ELISA). A total of 300 chicken serum samples were subjected to both Luminex xMAP assay and ELISA, showing the coincidence rates of 98.67 and 98% for NDV and AIV antibody detection, respectively. This study provides a new method for the simultaneous detection NDV and AIV antibodies in the serum with high specificity and sensitivity.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Influenza A/imunologia , Influenza Aviária/diagnóstico , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Galinhas , Imunoensaio/métodos , Imunoensaio/veterinária , Microesferas , Doenças das Aves Domésticas/virologia , Reprodutibilidade dos Testes
13.
Transbound Emerg Dis ; 66(2): 696-704, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30390413

RESUMO

A detailed veterinary and laboratory investigation revealed an unusual case of concurrent avian avulavirus type 1 (AAvV-1, formerly called avian paramyxovirus type 1) and low pathogenicity avian influenza (LPAI) virus infections of chickens during March 2010 in a mixed poultry and livestock farm in Great Britain. Respiratory signs and daily mortality of 5-6 birds in a broiler flock 8-weeks of age prompted submission of two carcasses to an Animal and Plant Health Agency (APHA) regional laboratory. Infectious bronchitis virus infection was suspected initially and virus isolation in SPF embryonated fowls' eggs was attempted at APHA-Weybridge. Avirulent AAvV-1 was detected in the first sampling. Both in vitro nucleotide sequencing of the fusion gene and in vivo pathotyping by intracerebral pathogenicity index revealed an avirulent AAvV-1 not definitively ascribed to licensed vaccine. Upon initial detection of the AAvV-1 virus, statutory restrictions were placed on the farm, an official veterinary visit was performed and further samples were submitted to APHA-Weybridge for official statutory disease investigation. An H2N3 LPAI virus was subsequently isolated from tissue samples and swabs submitted from the follow-up statutory investigation. The subtype was confirmed by haemagglutination inhibition test (HAIT) and neuraminidase inhibition (NI) tests on egg-amplified virus. As neither virus was notifiable according to the internationally recognized EU and OIE standards, and/or definitions of disease, statutory farm restrictions were lifted. Veterinary investigations identified the broiler flock to be free-range, next to a river and duck pen, reinforcing the suspicion of wild bird origin for both viruses which may have been co-circulating in ducks. It could not, however, be established as to whether there were separate introductions of the two viruses or whether there had been a single co-introduction of the viruses. The described case highlights the value of integrated surveillance and laboratory approaches, including veterinary field investigations, international standards and definitions of notifiable avian disease, validated RRT-PCR assays, and virus isolation in achieving rapid and accurate diagnostic results.


Assuntos
Coinfecção/veterinária , Influenza Aviária/diagnóstico , Influenza Aviária/epidemiologia , Doença de Newcastle/diagnóstico , Doença de Newcastle/epidemiologia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/epidemiologia , Animais , Animais Selvagens , Galinhas , Coinfecção/diagnóstico , Coinfecção/epidemiologia , Coinfecção/virologia , Patos , Monitoramento Epidemiológico/veterinária , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/isolamento & purificação , Doenças das Aves Domésticas/virologia , Perus , Reino Unido/epidemiologia , Virulência
14.
Transbound Emerg Dis ; 66(1): 546-551, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30403438

RESUMO

H9N2 avian influenza viruses (AIVs) have been detected from wild birds and domestic poultry worldwide. Serious diseases combined with secondary infection have caused high mortality and great economic losses to poultry industry. Therefore, simple, rapid, sensitive and accurate methods suitable for field detection of H9N2 AIVs are crucial to efficiently control virus infection and spread in time. In this study, an isothermal reverse transcription recombinase polymerase amplification with lateral-flow dipstick (RT-RPA-LFD) assay for detection of hemagglutinin (HA) gene of H9 subtype influenza viruses was developed. The optimal forward and reverse primers targeting HA gene of H9 subtype influenza viruses were labeled with fluorescein isothiocyanate (FITC) and biotin at the 5'-end, respectively. The amplification reaction could be finished in 20 min at a wide temperature range of 30-42°C, and then the products could be visualized with naked eyes. The developed H9 RT-RPA-LFD was able to detect 0.15 pg of H9N2 AIV RNA, which was 10 times more sensitive than that of conventional RT-PCR. The H9 RT-RPA-LFD assay did not detect nucleic acids extracted from H9 negative samples or from other poultry respiratory pathogens. The clinical performance of H9 RT-RPA-LFD was determined by testing 120 cloacal samples collected from chickens with respiratory syndromes. The coincidence rate of the detection results between RT-RPA-LFD and conventional RT-PCR was 95.8%. Therefore, the developed RT-RPA-LFD assay provides a rapid, reliable and sensitive method for field diagnosis of H9 subtype AIVs.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/veterinária , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transcrição Reversa , Animais , Bioensaio , Galinhas , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/virologia , Técnicas de Diagnóstico Molecular/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Aves Domésticas , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade
15.
J Virol Methods ; 263: 38-43, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30355516

RESUMO

H9N2 avian influenza virus is threatening animals and public health systems. Effective diagnosis is imperative to control the disease. Thus, we developed a panel of monoclonal antibodies (Mabs) against the H9N2 avian influenza virus (AIV) and implemented a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to detect the H9 viral antigen. Hybridomas 4D10 and 5G2 were screened to secrete immunoglobulin G (IgG) and IgA, respectively. Antibody 4D10 was used as the capture antibodies and HRP labeled 5G2 as the detector antibody. The specificity of the optimized DAS-ELISA was evaluated by using AIV subtypes H1, H3, H5, H9 and H10. Specimens containing AIV H9 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limit of the DAS-ELISA is 10-2.3 TCID50 (50% Tissue culture infective doses). Negative-positive threshold was 0.211 (OD630). In comparison with virus isolation the sensitivity and specificity of DAS-ELISA were found to be 98.9% and 98.1% respectively. Taken together, the newly developed Mab-based DAS-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H9 subtype AIV.


Assuntos
Ensaio de Imunoadsorção Enzimática , Vírus da Influenza A Subtipo H9N2/imunologia , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/diagnóstico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Embrião de Galinha , Galinhas , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Influenza Aviária/virologia , Células Madin Darby de Rim Canino , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
16.
Prep Biochem Biotechnol ; 48(10): 930-939, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30388960

RESUMO

Antibodies play an important role in combating and controlling viral diseases such as influenza. Immunoglobulin Y (IgY) antibodies have several advantages such as a less invasive manufacturing process, ease of isolation, higher affinity compared with IgG antibodies, and cost-effectiveness. To date, although specific IgY production has been performed for different strains of influenza A, to the best of our knowledge, an IgY against the M2e peptide has not been produced. In the current study, IgY antibodies are produced, purified, and characterized using the M2e peptide sequence for the first time with the intent to apply them for the diagnosis of influenza A virus. Anti-M2e IgY antibodies are obtained from eggs using a two-step purification method. The activity and characterization of the antibodies are determined using an enzyme-linked immunosorbent assay, a nano-spectrophotometer, an SDS-Page assay, and a Western Blot analysis. Finally, anti-M2e IgY antibodies are conjugated to the latex nanoparticles, and the reaction between the influenza A virus and the nanoparticles is demonstrated using light microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy. In conclusion, this study shows that anti-M2e IgY antibodies can contribute to the diagnosis, treatment, and prevention of the influenza A virus.


Assuntos
Anticorpos Antivirais , Galinhas/imunologia , Imunoglobulinas , Vírus da Influenza A Subtipo H1N1 , Influenza Aviária/diagnóstico , Nanopartículas/química , Peptídeos/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Imunoglobulinas/química , Imunoglobulinas/imunologia , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Aviária/imunologia , Peptídeos/química , Proteínas Virais/química
17.
Prev Vet Med ; 159: 99-105, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30314797

RESUMO

Avian influenza virus subtype H9N2 (AIV-H9N2) has become established in domestic poultry in Asia and Africa. AIV-H9N2 has not been reported previously in Indonesia. Here we describe the presence of AIV-H9N2 in chicken farms in Indonesia. Ninety-nine cases were observed in various provinces in Indonesia. Clinical signs, pathologic lesions and egg production were recorded. Confirmation was made using virus isolation, reverse transcriptase PCR (RT-PCR), and sequencing. To construct hemaglutinin (HA) phylogeny, the secondary data of Eurasian lineages were downloaded from GenBank. For neuraminidase, five sequences with the highest similarities with every sequence found in this study were downloaded. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package. Forty-nine AIV-H9N2-positive cases were observed, of which 35 were tested positive for AIV-H9N2 only. The age of the infected chickens was 43.17 ± 16.56 weeks, and their egg production was 35.85 ± 17.80% lower than before outbreak. BLAST search revealed that the nucleotide sequence of the HA-encoding gene identified in this study shared 98% sequence identity with that of A/Muscovy duck/Vietnam/LBM719/2014(H9N2), while its neuraminidase-encoding gene sequences shared 94%, 98%, and 100% identities with three different influenza viruses. The phylogeny shows that the HA of AIV-H9N2 found in this study forms distinct cluster with some Vietnam and China's sequence data. The NA sequence data form three distinct clusters. We conclude that AIV-H9N2 is widespread in many provinces in Indonesia. To lessen economic losses to the poultry industry, flock biosecurity and vaccination against this virus subtype should be implemented rapidly. Thorough and rigid AIV surveillance is paramount to prevent further veterinary and public health consequences of the circulation of this virus in Indonesia.


Assuntos
Galinhas , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Animais , Surtos de Doenças/veterinária , Indonésia/epidemiologia , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologia , RNA Viral/análise , Análise de Sequência de RNA/veterinária
18.
Arch Razi Inst ; 73(3): 177-182, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30280837

RESUMO

Avian influenza H9N2 subtype viruses have had a great impact on Iranian industrial poultry production economy since introduction in the country. To approach Rapid and precise identification of this viruses as control measures in poultry industry, a real time probe base assay was developed to directly detect a specific influenza virus of H9N2 subtype -instead of general detection of Influenza A viruses- which has been endemic over two last decades in the country. An Iranian avian influenza virus strain of A/Iran/chicken/772/1998 H9N2 subtype were selected as reference strain for of primers and probe designing. The high agreement value of 99% indicated that the devolved real time assay for detection of H9 subtype viruses could easily replace the conventional method of virus isolation particularly in investigation of viruses like national surveillance plan. The limit of detection was almost one EID50 which was the least real infectious unit could be detected. So it can be said that this sensitive assay provided a powerful tool to not to miss any significant viral biological activity neither in the host body nor in the environment. A high level of correlation coefficient (R2 = 0.998) also indicated a good correlation between Ct values and viral concentrations. , it can be conclude that the real time RT-PCR could be easily replace virus isolation in detection of H9N2 influenza viruses especially in large monitoring program. The ability in quantifying of the virus concentration extends usage of test in more accurate studies.


Assuntos
Galinhas , Testes Diagnósticos de Rotina/veterinária , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/diagnóstico , Animais , Embrião de Galinha , Testes Diagnósticos de Rotina/métodos , Influenza Aviária/virologia , Irã (Geográfico) , Reação em Cadeia da Polimerase/métodos , Doenças das Aves Domésticas/virologia , Sensibilidade e Especificidade
19.
Avian Pathol ; 47(6): 607-615, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30207746

RESUMO

Avian influenza viruses have been isolated from many bird species; however, little is known about the susceptibility of pet birds to low pathogenic avian influenza (LPAI) viruses. To address this research gap, domestic canaries (Serinus canaria forma domestica) were experimentally infected with H5 and H7 LPAI viruses to determine susceptibility and to evaluate samples for diagnostic purposes. Clinical evidence of infection (e.g. ruffled plumage and apathy) and mortality were noted for the canaries inoculated with chicken-adapted LPAI viruses. Real-time reverse transcription-polymerase chain reaction (RRT-PCR) demonstrated higher viral RNA levels in buccal compared to faecal samples. No clinical signs or mortality were observed in canaries inoculated with LPAI virus originating from wild birds; however, the canaries in this group did have evidence of viral RNA in buccal and faecal samples. Overall, this study showed that domestic canaries are susceptible to LPAI virus infections and that they can shed large amounts of viral RNA, primarily through the respiratory route. Thus, buccal swabs might be better samples than faeces for efficient detection of some LPAI virus infections in these birds. Although canaries have not been identified as a significant reservoir for LPAI viruses, they may be infected by LPAI viruses. Thus, the importance of the control of domestic canaries for detection of LPAI viruses should not be underestimated, especially in the contexts of international commercial exchange and outbreaks. RESEARCH HIGHLIGHTS Canaries are susceptible to infection with H5/H7 LPAI viruses. Canaries inoculated with LPAI viruses excrete large amounts of viral RNA. Buccal swabs may be appropriate specimens for AI virus detection in canaries. The control of canaries for LPAI virus detection should not be overlooked.


Assuntos
Canários/virologia , Surtos de Doenças/veterinária , Vírus da Influenza A/patogenicidade , Influenza Aviária/diagnóstico , Animais , Animais Domésticos , Suscetibilidade a Doenças/veterinária , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , RNA Viral/análise , RNA Viral/genética , Virulência
20.
PLoS Comput Biol ; 14(9): e1006439, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30212472

RESUMO

In Bangladesh, the poultry industry is an economically and socially important sector, but it is persistently threatened by the effects of H5N1 highly pathogenic avian influenza. Thus, identifying the optimal control policy in response to an emerging disease outbreak is a key challenge for policy-makers. To inform this aim, a common approach is to carry out simulation studies comparing plausible strategies, while accounting for known capacity restrictions. In this study we perform simulations of a previously developed H5N1 influenza transmission model framework, fitted to two separate historical outbreaks, to assess specific control objectives related to the burden or duration of H5N1 outbreaks among poultry farms in the Dhaka division of Bangladesh. In particular, we explore the optimal implementation of ring culling, ring vaccination and active surveillance measures when presuming disease transmission predominately occurs from premises-to-premises, versus a setting requiring the inclusion of external factors. Additionally, we determine the sensitivity of the management actions under consideration to differing levels of capacity constraints and outbreaks with disparate transmission dynamics. While we find that reactive culling and vaccination policies should pay close attention to these factors to ensure intervention targeting is optimised, across multiple settings the top performing control action amongst those under consideration were targeted proactive surveillance schemes. Our findings may advise the type of control measure, plus its intensity, that could potentially be applied in the event of a developing outbreak of H5N1 amongst originally H5N1 virus-free commercially-reared poultry in the Dhaka division of Bangladesh.


Assuntos
Galinhas/virologia , Surtos de Doenças/veterinária , Virus da Influenza A Subtipo H5N1 , Influenza Aviária/epidemiologia , Influenza Aviária/prevenção & controle , Aves Domésticas/virologia , Animais , Bangladesh/epidemiologia , Controle de Doenças Transmissíveis , Simulação por Computador , Geografia , Política de Saúde , Influenza Aviária/diagnóstico , Modelos Teóricos
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