共查询到20条相似文献，搜索用时 393 毫秒
1.
2.
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
3.
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
4.
采用RT-PCR对3株H9N2亚型禽流感病毒基因组进行扩增,测序后再利用DNAStar和MEGA 4对所得到的序列和部分具有代表性的参考序列进行遗传演化及分子特点分析,旨在从分子水平探讨华南地区H9N2亚型禽流感病毒的流行特点,为该病的综合防控提供依据。结果表明,3个分离株CK/GD/162/03、Francolin/GD/298/05与CK/GD/A8/10分别属于B34、B47和B54基因型;3个分离株HA基因未发生明显的变异,符合我国大陆H9N2亚型AIV HA基因的遗传进化特点,但内部基因出现不同程度重组且部分基因与人源高致病性H5N1亚型AIV同源性很高;CK/GD/162/03、Francolin/GD/298/05两株分离株具有宿主多样性。因此,对华南地区H9N2亚型AIV进行监控及分子流行病学调查具有重要意义。 相似文献
5.
根据GenBank中H6、N1亚型禽流感病毒(AIV)的HA、NA基因序列,设计2对特异性引物和2条用不同荧光基团标记的TaqMan探针.经反应条件优化,本试验建立了检测H6N1亚型AIV的二重荧光RT-PCR方法.该法特异性强,只对H6亚型和N1亚型AIV进行特异性扩增,对其他H亚型AIV、N亚型AIV及新城疫病毒、传染性支气管炎病毒等病原体的检测均为阴性;该法敏感性好,对H6N1亚型AIV的检测限为100拷贝/μL.本试验建立的H6N1亚型AIV的二重荧光RT-PCR方法,具有快速、敏感、特异的优点,为H6N1亚型AIV的防控提供技术支撑. 相似文献
6.
The H7N2 subtype of avian influenza virus (AIV) field isolate (H7N2/chicken/PA/3779-2/97), which caused the 1997-98 AIV outbreak in Pennsylvania, was evaluated for its infectivity, length of infection, and immune response in specific-pathogen-free (SPF) chickens. The composite findings of three clinical trials with various concentrations of virus indicated that this H7N2 subtype contained minimal pathogenicity for chickens. The concentration of the virus in the inoculum proved critical in the establishment of a productive infection in a chicken. Seven-day-old SPF chickens were not infected when inoculated with 10(0.7-2.0) mean embryo lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response to this virus was not detected by the hemagglutination-inhibition (HI) test. Nonetheless, chickens at ages of 5 and 23 wk old tested were successfully infected when exposed to 10(4.7-5.7) ELD50 of H7N2 infectious doses per bird by various routes of administration and also by direct contact. Infected birds started shedding virus as early as 2 days postinoculation, and the period of virus shedding occurred mostly within 1 or 2 wk postinoculation (WPI). This H7N2 subtype of AIV induced a measurable immune response in all birds within 2 wk after virus exposure. Antibody titers were associated with AIV infectious doses and age of exposure of birds. Challenge of these infected birds with the same H7N2 virus at 5 and 10 WPI indicated the infective virus was recoverable from cloacal swabs at 3 days postchallenge and disappeared thereafter. In these challenged birds, the antibody levels as measured by the HI test spiked within 1-2 wk. 相似文献
7.
LUO Si-si XIE Zhi-xun XIE Zhi-qin DENG Xian-wen LIU Jia-bo XIE Li-ji HUANG Li HUANG Jiao-ling ZENG Ting-ting ZHANG Yan-fang WANG Sheng 《中国畜牧兽医》2016,43(2):326-332
According to the sequences of HA and NA genes of H6 and N1 subtype avian influenza virus (AIV),two pairs of specific primers and two TaqMan probes with different fluorescence were designed.The duplex Real-time RT-PCR assay was developed and optimized to simultaneously detect H6 and N1 subtypes AIV in one reaction.The result showed that the specificity of this assay was high and only amplified H6 and N1 subtypes AIV,and was not cross-reactive with other H and N subtypes AIV,newcastle disease virus and infectious bronchitis virus.The detection limit of this assay was 100 copies/μL of H6N1 subtype AIV.This newly developed duplex Real-time RT-PCR assay was a rapid,specific and sensitive method for the detection of H6N1 subtype AIV,and it could provid a technical support to prevent and control H6N1 subtype AIV. 相似文献
8.
为研究H9N2亚型禽流感病毒(AIV)在哺乳动物间的传播能力,本研究以豚鼠为模型评价了5株H9N2亚型AIV在豚鼠体内的复制能力和水平传播能力,并分析了5株病毒血凝素(HA)蛋白的分子特征。结果表明,5株病毒均属于CK/Beijing谱系,其中2株病毒的HA具有人样受体特征(Lys226),2株病毒具有禽样受体特征(Gln226),而A/Chicken/JN/Li-2/2010(H9N2)株在该位点的氨基酸残基为苯丙氨酸(Phe226)。裂解位点分析表明,5株病毒均具有低致病性AIV特征。个别病毒的潜在糖基化位点存在增加或缺失现象。感染试验表明,5株病毒均能够在豚鼠呼吸道复制。并且在鼻甲骨处复制稳定,平均病毒滴度为2.01 Log EID50/mL～4.5 Log EID50/mL。传播试验表明,所有病毒株的人工接种豚鼠的鼻洗液中均能够检测到病毒,最长排毒期为接毒后第8 d,而接触组豚鼠鼻洗液中未检测到病毒。本研究表明,5株H9N2亚型AIV均属于CK/Beijing谱系,部分病毒株的HA蛋白已具备人样受体结合特征,并且关键氨基酸位点(226位)处出现新的突变。5株病毒均能够在豚鼠呼吸道复制并通过上呼吸道排毒,但不能在豚鼠间同群传播。 相似文献
9.
为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10-4至2.56×10-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。 相似文献
10.
为建立H5N1亚型禽流感病毒感染海兰白鸡模型,本研究选取1株鹅源H5N1高致病性禽流感病毒A/goose/guangdong/1/96(H5N1)(简称GD1/96),测定其对4周龄海兰白鸡的半数致死量.感染模型试验中,将30只4周龄海兰白鸡随机分成3组,每组10只,5只直接感染,5只同居,试验组设置一个重复,将病毒液稀释至104.5EID50,滴鼻、点眼各0.1 mL,对照组接种PBS,感染后24 h放入同居鸡;感染后连续观察14 d,记录死亡时间,每天采集咽喉拭子和泄殖腔拭子;感染组和同居组第3、5 天各剖解3只鸡,采集气管、肺脏、脑、脾脏、肾脏和十二指肠,进行病毒分离;qRT-PCR法分析感染组和同居组第3、5 天鸡肺组织中IFN-α和TNF-α的相对表达量.结果显示,GD1/96株的鸡胚半数感染量(EID50)为10-8.167/0.1 mL,对4周龄海兰白鸡的半数致死量为104.5 EID50.感染模型试验结果显示,以104.5EID50的攻毒剂量感染海兰白鸡,感染组鸡在感染后8 d全部死亡;在感染和同居3 d后,各组鸡的咽喉拭子和泄殖腔拭子均可检测到病毒;感染和同居后第3、5 天,各组鸡的6种组织中均可分离到高滴度的病毒;IFN-α和TNF-α在感染组和同居组的鸡肺脏组织中的表达量均显著增加(P <0.05).本试验建立了海兰白鸡的H5N1亚型禽流感病毒感染模型,为H5N1亚型禽流感病毒的致病机理及表达抗流感基因转基因鸡的研究奠定了基础. 相似文献
11.
This is the first reported isolation of avian influenza virus (AIV) from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic AIV (LPAIV), according to OIE standards. It was named A/Emu/HeNen/14/2004(H9N2)(Emu/HN/2004). The HA gene (1683bp) was amplified by RT-PCR and it was compared with other animal H9N2 AIV sequences in GenBank, the US National Institutes of Health genetic sequence database. The results suggested that Emu/HN/2004 may have come from an avian influenza virus (H9N2) from Southern China. 相似文献
12.
13.
为了解两株高致病性禽流感病毒(HPAIV)的分子特征及其对不同宿主的致病性,本研究对DK/HuN/4/08和CK/GX/2/09进行全基因组序列的测定,分析结果表明:两个病毒分离株HA基因的裂解位点均具有HPAIV特有的基序(341RRR(R)KR345/346),并且均属于Clade2.3.2分支,基因组同源性在97.4%～98.3%之间。致病性试验显示,两个病毒分离株均能够以106EID50感染量在3 d内引起鸡全部死亡,并且各脏器均检测到高滴度的病毒含量;两者在SPF鸭中呈现不同的致病性,CK/GX/2/09在4 d内可以使感染鸭100%死亡,而DK/HuN/4/08只引起25%的死亡率;同样在小鼠试验中,两者致病力差异与在鸭体中的反应一致,其MLD50分别为1.63 log10EID50和6.2 log10EID50。本研究表明,这两株遗传背景相似的HPAIV在鸡、鸭和小鼠中的致病性不同,为进一步利用反向遗传技术研究这两株病毒对水禽和哺乳动物致病力差异的分子机制奠定了基础。 相似文献
14.
LI DAN XIE Zhi-xun SONG De-gui LI Meng XIE Zhi-qin LUO Si-si XIE Li-ji HUANG Li FAN Qing HUANG Jiao-ling 《中国畜牧兽医》2016,43(12):3101-3106
This experiment was aimed to develop a method for simultaneous detection of H9 and H6 subtype avian influenza virus (AIV).Two pairs of specific primers were designed according to the conserved regions sequences of H6 and H9 AIV HA gene,a duplex RT-PCR simultaneous detection of H9 and H6 subtype AIV was developed by optimizing the PCR system such as the concentration of different primers and annealing temperature.It showed that all samples could be amplified specific bands from H9 subtype AIV single infection samples or H6 subtype AIV single infection samples,and the samples mix infection these two subtypes AIV.No specific bands of the same sizes were amplified from genomic materials of other avian pathogens.The detection limit of the duplex RT-PCR was 5×104 copies/μL. It suggested that this duplex RT-PCR assay was a specific,sensitive,stable and repeatable method for detection of H9 and H6 subtype of AIV,and could provide technical support for the monitoring of H9 and H6 subtype AIV. 相似文献
15.
试验旨在建立可同时鉴别检测H9和H6亚型禽流感病毒（avian influenza virus,AIV）的二重RT-PCR方法。根据GenBank中H9和H6亚型AIV的HA基因保守序列,分别设计2对特异性引物,优化引物浓度与退火温度等条件,建立了可同时鉴别检测H9和H6亚型AIV的二重RT-PCR检测方法。用该法对H9和H6亚型AIV混合感染样品、H9亚型AIV单一感染样品和H6亚型AIV单一感染样品进行扩增,结果均得到对应的目的条带,而对其余亚型AIV及其他禽病病原体均未扩增出特异性条带。该法对H9和H6亚型AIV的检测下限均为5×104拷贝/μL。本研究建立的二重RT-PCR检测方法特异性强、敏感性高、稳定性和重复性良好,可同时鉴别检测H9与H6两种亚型AIV,为H9与H6亚型AIV的监测提供技术支撑。 相似文献
16.
为模拟哺乳动物感染H5亚型高致病性禽流感病毒(HPAIV)的发病进程,本研究采用对哺乳动物高度致病的H5N1亚型HPAIV株A/bar-headed goose/Qinghai/3/05 (BHG/3/05),以低剂量鼻腔接种小鼠,观察发病、存活、病毒复制及组织病理损伤情况.结果显示,100.4 EID50即能够100％感染小鼠,但发病表现缓慢,死亡延迟至8d以后,存活达60％;体内病毒复制可持续10 d以上,感染后前3d病毒的增殖限于呼吸道,随后扩散至脑、脾、肾等其他器官;组织病理学观察肺脏早期表现出渗出性炎症,第10d发展为典型的间质性肺炎.本研究结果为探讨人禽流感的病理发生机制提供了具有价值的模型. 相似文献
17.
2015年,从安徽合肥某养鸡场分离出一株H9N2亚型禽流感病毒（AIV）,命名为HF株。该毒株鸡胚半数感染量（EID50）为109.17/0.1 mL,最小致死量的平均死亡时间（MDT）为87 h。对其HA基因分析发现,其氨基酸裂解位点为RSSR↓GLF,符合低致病性AIV特征;HA基因的遗传进化分析结果表明,该分离株属于h9.4.2.5谱系,符合当前毒株流行趋势。将HF株与2006-2018年分离自全国各地的10株H9N2亚型AIV分离株同时制备灭活疫苗,免疫SPF鸡,制备阳性血清,通过交叉血凝抑制试验分析病毒抗原性,结果显示HF株与2014年之前毒株抗原相关性介于0.50~0.56之间,与2014年及之后毒株抗原相关性介于0.89~1.00之间,表明该分离株与2014年之后的流行毒株具有良好的抗原相关性。用0.2%甲醛灭活HF株病毒液,其HA效价在灭活前后未发生变化;用灭活抗原制备油乳剂灭活疫苗免疫SPF鸡,免疫后21 d HI抗体效价几何平均值达到9.0log2以上,可使免疫鸡完全抵抗H9亚型AIV的感染,提供100%的攻毒保护。研究结果表明,HF株具有良好的免疫原性,可作为疫苗候选株用于H9N2亚型禽流感疫苗的研制。 相似文献
18.
CHENG Yi TIAN Hui ZHANG Ya XUE Jingjing SHENG Dongbei LIU Wujie WANG Wanbing ZHANG Pantao TIAN Kegong 《中国畜牧兽医》2007,47(9):2945-2952
In 2015,an H9N2 subtype avian influenza virus (AIV) strain was isolated from a chicken farm in Hefei,Anhui,and named HF strain.The results of the chicken embryo proliferation characteristics study showed that the half infection rate of chicken embryo (EID50) was 109.17/0.1 mL,and the mean time to death for minimum lethal dose(MDT) was 87 h.The analysis result of HA gene showed that its amino acid cleavage site was located in RSSR↓GLF,which accorded with the characteristics of low pathogenic avian influenza.The genetic evolution analysis of HA gene revealed that the isolate belonged to the h9.4.2.5 lineage,which accorded with the current virus strain epidemic characteristics.The HF strain was prepared with 10 H9N2 subtype AIV isolates which isolated from all over the country from 2006 to 2018 to prepare inactivated vaccines,immunize SPF chickens,prepare positive sera,and analyze the virus antigenicity by cross hemagglutination inhibition test.The results showed that the correlation between the HF strain and the virus antigens before 2014 and was between 0.50-0.56,and the virus antigen correlation after 2014 was 0.89-1.00.This showed that the isolate had good antigenic correlation with epidemic strains in recent years.Inactivate HF strain virus solution with 0.2% formaldehydel,and its HA titer did not change before and after inactivation.After the inactivated virus solution was prepared into an oil emulsion inactivated vaccine to immunize SPF chickens,21 days after immunization,the average value of the HI antibody titer reached 9.0log2.It could make immune chicken completely resistant to H9 subtype AIV infection and provide 100% protection from challenge.The above research results showed that the HF strain had good immunogenicity and could be used as a vaccine candidate strain for the prevention of H9N2 subtype AIV. 相似文献
19.
H9N2亚型禽流感病毒HA蛋白S145N变异株致病性及抗原特性 总被引：1，自引：0，他引：1
为确定近年来H9N2亚型禽流感病毒(AIV) HA蛋白S145N点突变对病毒毒力变化和抗原性变异的影响,笔者对从全国不同地区分离的12株H9N2亚型AIV HA蛋白S145N变异株和HP疫苗参考株进行了半数鸡胚感染量(EID50)、半数鸡胚致死量(ELD50)、平均鸡胚致死时间(MDT)、雏鸡脑内致病指数(ICPI)、鸡静脉致病指数(IVPI)和8周龄SPF鸡感染排毒试验,并与抗H9N2亚型AIV HP参考株HA蛋白单抗2A4和F6的血凝抑制(HI)和中和反应特性进行测定.结果发现,H9N2亚型AIV HA蛋白S145N变异株毒力偏强,能引起部分SPF鸡发病和死亡,感染8周龄SPF鸡排毒时间更早,排毒期更长.单抗2A4和F6不能抑制H9N2亚型AIV HA蛋白S145N变异株的血凝特性,也不能中和病毒感染CEF细胞.研究结果表明,H9N2亚型AIV呈现变异趋势,有毒力增强和抗原性变异毒株出现.S145为H9N2亚型AIV HA蛋白的1个抗原位点,是血凝抑制抗体结合的位点,但有该位点漂变导致抗原变异毒株出现,并可逃避免疫作用.这提示该病的防控面临着新的挑战. 相似文献