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从湖南分离得到一株H5N1亚型禽流感病毒(AIV),首先设计合成两对HA和NA基因特异性引物,采用两步法RT-PCR,对鸭流感病毒株 A/Duck/HunanWugang/2/2004(H5N1)(简称DK/HNWG/2/04)的表面蛋白基因进行序列测定,并与国内外已经发表的H5N1亚型毒株表面蛋白基因进行序列分析和比较.结果表明,HA和NA基因全长分别约为1.7 kb和1.4 kb,分离株与14个参考毒株HA基因的同源性为95.9%～98.0%,NA基因的同源性为87.0%～98.4%.根据HA基因核苷酸序列推导HA裂解位点氨基酸,发现分离株的裂解位点包含多个碱性氨基酸,符合高致病性禽流感的特征. 相似文献
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Deng M Long L Xiao X Wu Z Zhang F Zhang Y Zheng X Xin X Wang Q Wu D 《Veterinary immunology and immunopathology》2011,141(3-4):183-189
Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step. 相似文献
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一株鸡源H6N1亚型禽流感病毒全基因的分子特征 总被引：2，自引：2，他引：0
2008年国家禽流感参考实验室在我国禽流感流行病学调查期间分离到1株鸡源H6N1亚型禽流感病毒(AIV)A/Chicken/ZheJiang/80/2008(H6N1)(简称为CK/ZJ/80/08),为了弄清该病毒的分子特征,我们对其8个基因片段分别进行扩增和序列测定,对每个基因进行BLAST分析,找出同源性最高的毒株。利用DNAStar中的Megalign功能进行进化分析。结果表明CK/ZJ/80/08的HA裂解位点附近的氨基酸序列为QIETR↓GLF,推测可能为一株低致病力AIV。其HA基因与日本北海道的A/duck/Hokkaido/228/2003(H6N8)和黑龙江的A/mallard/Heilongjiang/131/2006(H6N2)以及香港早期分离株A/chicken/HongKong/17/77(H6N1)等处于同一分支;NA基因在颈部没有缺失,与A/duck/Tsukuba/718/2005(H1N1)、A/goose/Guangdong/1/96(H5N1)等处于同一分支;M基因与A/duck/Hokkaido/W90/2007(H10N7)高度同源(同源性为99%);NS基因与A/duck/Denmark/65047/04(H5N2)和A/goose/Guangdong/1/96(H5N1)处于同一分支。NP、PA、PB1、PB2分别与贵州和江西分离的H5N2亚型AIV的相应基因关系密切,同源性分别为98%、97%、97%、97%。由此推测CK/ZJ/80/08可能是由H6N2、H1N1、H10N7、H5N2等多个亚型病毒重组而成。 相似文献
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2004初从正常鸭群中分离到一株鸭源禽流感病毒，命名为A／Duck／HN／4／2004(H6N2)。经对血凝素基因(HA)序列分析发现HA基因全长为1744bp，共编码566个氨基酸，在裂解位点仅含一个碱性氨基酸-精氨酸(R)，符合LPAIV的标准。将所得基因序列与已发表的同一亚型参考序列分析表明，与H6亚型流感HA基因同源性为89．2％-97．1％，经分子遗传演化分析表明本次分离株与香港分离株A／Duck／Hong Kong／3600／99(H6N2)、A／Duck／Hong Kong／3600／99(H6N2)最近。 相似文献
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为建立简便快速检测禽流感病毒(avian influenza virus,AIV)并同时区分出H9、N2亚型的方法,本试验根据基因库中H9亚型AIV的HA基因、N2亚型AIV的NA基因及AIV的M基因序列,分别设计了3对针对这3种基因保守序列的引物,建立了AIV H9N2亚型的三重PCR检测方法。应用该方法对H9N2亚型AIV模板进行PCR扩增,可得到3条与试验设计相符的目的条带,分别为313 bp (HA基因)、451 bp (NA基因)和667 bp(M基因);对非H9亚型的N2亚型AIV模板进行扩增,出现2条特异性扩增条带,即451 bp (NA基因)和667 bp(M基因);对非H9、N2亚型AIV模板进行扩增则只出现一条目的条带,即667 bp(M基因);对其他禽呼吸道病原体进行PCR扩增,结果均为阴性。敏感性试验结果显示此三重PCR方法最低检出限为10-2 ng/μL。应用所建立的三重PCR方法对120份临床病料进行检测的结果与病毒分离鉴定结果一致。各项试验结果均表明,该方法对于禽流感病毒尤其是H9、N2亚型禽流感病毒的检测具有快捷、特异、灵敏的特点。 相似文献
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Mar Costa-Hurtado Claudio L. Afonso Patti J. Miller Eric Shepherd Ra Mi Cha Diane Smith Erica Spackman Darrell R. Kapczynski David L. Suarez David E. Swayne Mary J. Pantin-Jackwood 《Veterinary research》2015,46(1)
Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (106.9 EID50) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (105.3–5.5 EID50) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-015-0237-5) contains supplementary material, which is available to authorized users. 相似文献9.
为了对供港猪群中的猪流感流行情况进行分析,从华南地区供港猪群中用无菌棉拭子采集鼻腔粘液样品,采用鸡胚接种方法,从供港猪群中分离出了2株不同亚型的猪流感病毒株,经国家流感中心鉴定分别为H1N1和H3N2亚型。本研究设计了猪流感常见亚型的HA和NA分型特异性引物,建立了猪流感型特异性RT-PCR检测方法;对分离鉴定的2株猪流感病毒和禽流感H5N1 HI检测抗原进行了RT-PCR检测,并对其部分HA和NA基因进行克隆测序分析。对供港猪群的血清检测结果表明:供港猪群中H1N1和H3N2亚型抗体阳性率分别为26.87%、38.26%,禽流感H5N1和H9N2亚型抗体阳性率均为0%。 相似文献
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为了开发用于南方水禽且适合血清学监测的H5亚型禽流感疫苗,作者通过反向遗传技术,删除A/mallard/Huadong/S/2005(H5N1,S株)病毒HA编码多碱性氨基酸序列,使之成为低致病特征,分别与A/duck/England/1/1956(H11N6,E株)和A/Chicken/Shanghi/F/98(H9N2,F株)的NA组合,再以本室禽源高产病毒F株的6个内部片段为骨架,构建全部基因都来自禽流感的疫苗株.成功拯救出2株重组病毒,分别命名为rH5N6/F和rH5N2/F,并引入分子标记N6和N2.重组病毒在鸡胚和MDCK细胞上均具有较好的繁殖能力,且rH5N6/F更适合在鸡胚中生产,对SPF鸡和鸡胚无致病性.重组病毒在clade2.3.4毒株中具有很好的抗原代表性,引入的分子标记有利于血清学监测的区分,为防控水禽H5亚型禽流感提供了良好的疫苗候选株. 相似文献
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Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum 总被引：1，自引：0，他引：1
D J King 《Avian diseases》1991,35(3):505-514
Viruses conveyed in shipments of eggs, viral diagnostic reagents, or avian serum samples are a potential hazard for susceptible poultry. Different methods of treatment of those materials to eliminate the hazard of virulent and avirulent strains of Newcastle disease virus (NDV) or avian influenza virus (AIV) were evaluated. The NDV strains tested were more thermostable than the AIV strains. The results suggest that standard pasteurization methods would not reliably inactivate the concentrations of NDV used. beta-Propiolactone (BPL) (greater than or equal to 0.025%) inactivated NDV or AIV in allantoic fluid, but higher concentrations were needed to inactivate virus diluted in serum. Hemagglutination (HA) of NDV and AIV and hemolysis (HL) activity of NDV were reduced or eliminated by 0.4% BPL. Formalin (greater than or equal to 0.04%) inactivated either virus but adversely affected HA and HL activity. NDV or AIV was inactivated by binary ethylenimine (BEI) (0.01 M) with no adverse effect on HA or HL. Heat (56 C) or BEI (0.01 M) had no apparent effect on hemagglutination-inhibition (HI) titers of NDV and AIV antisera, the effect of formalin (0.1%) was variable, and BPL (greater than or equal to 0.25%) depressed the HI titers of both antisera. The optimum method should achieve virus inactivation without harming the treated material. 相似文献
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CHEN Jun-xia SUN Peng XU Shu-zhen LI Si-fei SU Shuai ZHAO Peng CUI Zhi-zhong 《中国畜牧兽医》2015,42(11):2888-2894
To prepare the mono-specific serum to diagnose H9N2 avian influenza virus (AIV),this test extracted H9N2 subtype AIV RNA and then amplified the upper HA1 gene,the middle HA2 gene and the lower HA3 gene by RT-PCR,respectively.Then they were inserted into expression vector pET-32a(+) and transformed into BL21(Rosetta) expression strain.The expressed proteins were used to immune Kunming White mice to prepare antiserum.Recombinant fusion proteins of HA1 HA2 and HA3 were obtained successfully and they showed good immunogenicity.Indirect immunofluorescence assay (IFA) showed that the two serums obtained by the upper HA1 and the middle HA2 could react with the H9N2 subtype AIV,while that of the lower HA3 could not.Recombinant Marek's disease virus (MDV) MZC12 HA/NA also proved that the serums prepared by HA1 and HA2 could recognize the expression of HA gene.The mono-specific serum of H9N2 subtype AIV was prepared successfully,which could lay the foundation for the diagnosis and research of H9N2 subtype AIV. 相似文献
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Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore 总被引：1，自引：0，他引：1
Dawn Su-Yin Yeo Sock-Hoon Ng Chin-Wen Liaw Ley-Moy Ng Eugene Jing-Hui Wee Elizabeth Ai-Sim Lim Shirely Lay-Kheng Seah Wai-Kwan Wong Chee-Wee Lim Richard J. Sugrue Boon-Huan Tan 《Veterinary microbiology》2009,138(3-4):304-317
We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia. 相似文献
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为进一步分析禽流感病毒(AIV)H5N2分离株血凝素(HA)基因的特性,参照已发表H5亚型禽流感HA基因序列设计了1对引物,采用RT-PCR技术,以禽流感病毒A/Ostrich/Denmark/72420/96(D96)RNA为模板,扩增了HA全基因并进行核苷酸同源性比较,氨基酸编码分析,绘制系统发育进化树。结果表明,扩增片段长1737个核苷酸,包含了完整的HA基因的开放阅读框架,与Genbank已发表的H5N1和H5N2分离株的HA基因序列比较,发现与国内H5N1分离株同源性较低,只有80%左右,而与H5N2各株序列具有很高的同源性,最高达97.5%,印证了AIV基因组8个片段间频繁的重组及AIV高变异性的特点。推导的氨基酸序列分析表明,HA蛋白裂解位点上游丢失了4个连续碱性氨基酸(R-R-R-K),裂解位点处氨基酸序列为E-T-R,仅包含一个碱性氨基酸(R-)残基,符合低致病性毒株的特征,证明为低致病性毒株。其HA推导后氨基酸序列与H5N1AIV的同源性接近90%,以其研究的疫苗,可以有效抵御我国流行的H5亚型AIV病毒的感染,同时因为是弱毒株,以其研制的疫苗具有更好的安全性,也更符合公共卫生学的要求。 相似文献
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国家禽流感参考实验室在2006年,从来自青、藏两省(区)3种野鸟及辽宁省的2种死亡的野鸟体内共分离到14株H5N1亚型高致病力禽流感病毒(High pathogenic avian influenza virus,HPAIV),比较发现其HA、NA基因核苷酸的同源率在97.9%～99.9%之间。所有病毒的HA基因的裂解位点均具有HPAIV特有的连续碱性氨基酸-RRRKKR-,并具有近年来H5N1亚型流行株所特有的NA基因颈部49～68位20个氨基酸缺失及NS基因80～84位5个氨基酸的缺失;遗传进化分析表明2006年野鸟病毒由2005年野鸟病毒进化而来,并形成独立的进化分支;辽宁省近2年的4株野鸟病毒亲缘关系较近,说明引起2005年该省锦州地区H5N1 HPAI疫情的病毒在该地区的野鸟体内继续存在。 相似文献
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为了建立一种快速、简便的H5亚型、N6亚型禽流感病毒(AIV)的检测方法,根据GenBank中H5亚型、N6亚型AIV的HA和NA基因保守序列,分别设计了2对特异性引物,通过优化条件,建立了H5亚型和N6亚型AIV二重RT-PCR检测方法。特异性试验结果显示,该方法对H5N6亚型AIV特异性扩增出418bp和251bp目的片段,对H5Ny(y≠6)亚型AIV扩增出418bp目的片段,对HxN6(x≠5)亚型AIV扩增出251bp目的片段,对其他亚型和常见的禽病病原体均未扩增出目的片段;敏感性结果显示,该方法对H5亚型和N6亚型最低检测限为1.59×10-5ng/μL。本研究建立的H5亚型和N6亚型AIV检测方法,具有特异性强,灵敏度高的特点,为H5亚型和N6亚型AIV临床检测以及防控提供了有效方法。 相似文献
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鸭源H5N1亚型高致病性禽流感病毒的分离鉴定及其HA和NA基因的生物信息分析 总被引：1，自引：1，他引：0
分离到1株 H5N1亚型高致病性禽流感病毒, 经序列测定发现HA蛋白裂解位点上插入多个连续的碱性氨基酸（PQREIRRKKR*G）,从分子上证实是一株高致病性禽流感病毒。核酸序列比较分析结果表明,分离的流感病毒HA基因与A/duck/VietNam/Ncvd1/2002(H5N1)同源率最高,达到98.8%;NA基因与A/duck/VietNam/Ncvd1/2002(H5N1) 和A/chicken/Jiangsu/cz1/2002(H5N1)同源率最高,达到98.7%。氨基酸水平上,HA与A/duck/Viet Nam/Ncvd1/2002(H5N1)同源率最高,可达99.3%;NA与A/chicken/Jiangsu/cz1/2002(H5N1)同源率最高,达98.7%。HA与NA基因的潜在糖基化位点与作者所选参比毒株一致。通过遗传进化树分析结果表明,A/duck/VietNam/Ncvd1/2002(H5N1)可能是该毒株的来源株。 相似文献
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为建立一种同时检测H4、N2和所有亚型禽流感的方法,分别针对H4亚型禽流感病毒(AIV)HA基因、N2亚型AIV NA基因和所有亚型AIV M基因保守序列,设计筛选出3对特异性引物,优化引物之间的浓度,对三重反应体系进行特异性和敏感性验证,建立了H4、N2和所有亚型AIV三重RT-PCR检测方法,并用该法对临床样品进行检测。建立的方法能特异性扩增H4、N2和所有亚型AIV,与其他禽病病原体不发生交叉反应;对H4、N2和所有亚型AIV至少能检测到6 pg/μL。在185份临床样品的检测中,检出4份H4、10份N2和19份AIV阳性。所建立的三重RT-PCR方法特异性强、灵敏度高,为快速检测H4、N2和所有亚型AIV提供了有效的方法。 相似文献
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本研究针对AIV H5的血凝素(HA)基因和vNDV的融合蛋白(F)基因设计两对引物,建立了单管同时检测AIVH5和vNDV的双重RT-PCR(dRT-PCR),该方法能在单一反应管内同时检测AIVH5和vNDV,不与其它亚型AIV、弱毒NDV毒株以及其它病原体发生非特异性扩增;对含有AIV H5 HA基因和vNDV F基因重组质粒DNA的最低检测限分别为20.6和406fg,敏感性与单项RT-PCR相同;从样本处理到报告结果仅需5h。对24份临床疑似样本进行检测,AIVH5均为阴性,vNDV阳性18份,PCR产物测序证明为靶基因序列,其具有vNDV F基因的特征性序列,随机选9份vNDV阳性样本接种SPF鸡胚,分离出6株vNDV,病毒分离率为6/9;对100 ELD50的AIV H5N1和100 ELD50的vNDV人工同时感染5日龄SPF鸡的脑、肝脏、肺脏和泄殖腔棉拭子进行dRT-PCR检测,AIV H5的检测率为4/5,3/5,5/5,4/5,vNDV的检测率为3/5,5/5,4/5,3/5,而对H9N2和LaSota毒株实验同时感染样本及阴性对照样本的检测均为阴性。可见本研究建立的dRT-PCR为AIV H5和vNDV诊断、监测、检疫及分子流行病学调查提供了特异性强、操作简单、检测速度快、成本低廉的新方法。 相似文献
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利用反向遗传技术,通过基因重排方法,以A/chicken/shanghai/F/98（H9N2）禽流感病毒（Avian influenza virus,AIV）的6个内部基因为骨架,与A/Chicken/Guangdong/SS/94（H9N2）AIV的HA和NA基因组合,产生3株H9N2亚型重排AIVs。动物试验发现A/Chicken/Shanghai/F/98（H9N2）和A/Chicken/Guangdong/SS/94（H9N2）AIV主要在呼吸系统复制,A/chicken/shanghai/F/98（H9N2）株在气管和肺组织的复制能力明显强于A/Chicken/Guangdong/SS/94（H9N2）AIV株。3株H9N2亚型重排AIVs的动物试验发现HA和NA基因对H9N2亚型AIV在呼吸道的复制特性起主要作用。内部基因对H9N2亚型AIV在呼吸道的复制也有一定的作用。结果表明1994年中国首次分离到的H9N2亚型AIV经过4年的宿主适应和基因进化,加强了其在呼吸系统的复制能力,奠定了气溶胶传播的基础。 相似文献