H5亚型禽流感病毒单抗-生物素捕获ELISA的建立

目的建立一种单克隆抗体介导的、经生物素—亲和素系统放大的H5亚型禽流感病毒捕获ELISA检测方法,为进一步研究检测试剂盒提供基础。方法用亲和层析法纯化抗禽流感病毒H5亚型血凝素单克隆抗体,包被微量反应板,用于捕获病毒抗原,再用生物素标记的单抗和酶标亲和素来检测病毒血凝素抗原,经方阵试验优化ELISA反应体系。用该方法检测H1—H15亚型AIV标准毒株和H5、H7、H9亚型AIV分离株,并与血凝和血凝抑制试验比较,评价其敏感性和特异性。结果纯化后的单抗具有良好的反应活性,生物素标记单抗工作浓度为1∶5000;ELISA对H5亚型AIV的检出限为025个血凝单位。该ELISA反应体系能检出H5N3标准株和所有20株国内H5亚型AIV分离株,而与其他14个血凝素亚型的AIV标准株、15个H9亚型AIV分离株和2个H7亚型AIV分离株均无交叉反应。结论初步建立了检测H5亚型禽流感病毒的单抗—生物素捕获ELISA方法,为研制试剂盒和进一步应用试验提供了基础。     

一株抗H5亚型禽流感病毒血凝素蛋白单克隆抗体的广谱中和活性

以H5N1禽流感病毒株Ck/HK/Yu22/02作为抗原,应用常规杂交瘤技术和血凝抑制实验筛选出抗H5亚型禽流感病毒血凝素蛋白的单抗8H5,单抗8H5经免疫荧光鉴定具有很好的H5特异性.选择33株2002～2006年不同地域,不同宿主中分离的不同遗传变异亚系的H5N1病毒代表株,对单抗8H5分别进行血凝抑制实验及中和试验分析,结果显示单抗8H5对所有H5亚型病毒均有较强反应,而对非H5亚型标准病毒株均不反应,说明8H5是一株广谱性抗H5特异性中和单抗,并提示单抗8H5的HA识别表位可能是一个相当保守的中和表位.并且单抗8H5双抗夹心系统的初步评价显示了其在诊断应用上的前景.     

新型H7N9亚型禽流感病毒多重荧光RT-PCR快速检测方法的建立

为研制新型H7N9亚型禽流感病毒快速检测试剂盒,根据GenBank中公布的新型H7N9亚型(2013年)禽流感病毒血凝素抗原(HA)和神经氨酸酶抗原(NA)的基因序列,设计两套特异性的引物和探针,建立基于TaqMan探针的多重荧光RT-PCR快速检测新型H7N9亚型禽流感病毒方法。实验结果表明,该方法灵敏度高、特异性好,能够检测到最低浓度为10拷贝/μL数量级的阳性标准品,不但能够将禽流感病毒H7亚型与H1、H3、H5、H6和H9亚型区分开,而且可将新型N9亚型禽流感病毒与原有的N9亚型区分开。采用该方法对珠海市2 700份样品的检测结果与预期结果一致,证实该方法具有较好的推广应用前景。     

抗H5N1亚型禽流感病毒血凝素单克隆抗体的制备及鉴定

目的建立稳定分泌抗H5N1亚型禽流感病毒血凝素单克隆抗体的杂交瘤细胞系,为进一步研究禽流感诊断技术奠定基础。方法以纯化的H5亚型禽流感病毒按常规方法免疫BALBc小鼠,最后一次免疫后第3天取其脾细胞与SP20细胞在聚乙二醇作用下融合,用选择性培养、有限稀释法克隆和血凝抑制试验进行筛选,对获得阳性克隆株用ELISA方法进行亚型鉴定,并用37株H5、H7、H9亚型AIV测定其特异性、覆盖性。结果最后获得了3株分泌特异性抗体的杂交瘤细胞,命名为1E5、4A4、4B1,经长期体外培养和冻存后复苏能稳定地分泌抗体。经鉴定,其亚型均为IgG1、kappa链。腹水HI效价1∶210～1∶216,细胞培养上清HI效价1∶26～1∶28。3株杂交瘤所分泌的单克隆抗体均能与本中心保存的全部20株H5亚型禽流感病毒分离株发生反应,而与15株H9亚型禽流感病毒分离株、2株H7亚型禽流感病毒分离株以及H1H4、H6H15亚型禽流感病毒标准毒株均不反应,与鸡新城疫病毒、鹅新城疫病毒、鹅腺病毒和鸡产蛋下降综合征病毒等均无交叉反应。结论所获3株单克隆抗体可用于禽流感病毒特异性诊断试剂的研制。     

禽流感病毒A型和H5亚型RT-PCR检测试剂盒研究

目的　检测和鉴定A型、H5亚型禽流感病毒 (AIV) ,研发一种高效实用的检测手段。方法　根据Ming ShiuhLee报道的文献设计、合成引物 ,采用反转录和PCR一步法对A型、H5亚型禽流感病毒cDNA进行扩增和电泳鉴定 ,组装成禽流感病毒RT PCR试剂盒 ,对H1～ 15亚型AIV参考株、38份AIV国内分离株进行检测试验。结果　建立了A型、H5亚型禽流感病毒RT PCR检测方法 ,并在此基础上组装试剂盒 ,用A型试剂盒检测时 ,全部AIV毒株均为阳性 ,能检测 1 10 2 4血凝单位禽流感病毒 ;用H5亚型试剂盒检测时 ,仅有H5亚型AIV参考株和 19株H5亚型AIV分离株呈阳性 ,其余H1～H4、H6～H15参考株和H7、H9分离株以及 1株H5分株均为阴性 ,能检测1 6 4血凝单位禽流感病毒。 2种试剂盒对实验感染鸡病料检出率均为 10 0 %。结论　研制的AIVA型、H5亚型RT PCR试剂盒具有特异性强、敏感性高、稳定性和重复性好的特点。     

高致病性H5亚型禽流行性感冒病毒血凝素单克隆抗体的制备与初步应用

以禽流感病毒株Ck/HK/Yu22/02（H5N1）作为免疫原,利用常规杂交瘤技术和血凝抑制试验法成功地筛选出6株稳定分泌抗高致病性H5亚型禽流感病毒血凝素的单克隆抗体（单抗）,分别命名为2F2、3C8、3FC1、7C6、10HD4和13G4.经血凝抑制试验法分析,结果发现这6株单抗具有特异性高、反应性强、识别谱宽且互补等特点.基于单抗2F2,初步建立了三种H5N1病毒诊断方法,经评估证实均具有很好的特异性.由此说明,研究制备的抗H5亚型禽流感病毒血凝素单抗可适用于H5N1病毒的诊断.     

H5N1禽流感病毒血凝素抗原快速检测试剂对不同现场标本的检测比较

利用H5亚型禽流感病毒血凝素抗原快速检测试剂“H5-HA(Ag)Dot-ELISA(H5-Dot)”对来自陆禽、水禽的484份气管拭子、泄殖腔拭子和粪便拭子标本进行检测,结果:①不同采集方式的H5N1阳性标本的检出率高低有别,气管拭子的检出率最高,泄殖腔拭子次之,粪便拭子最低(P<0·05),因此建议现场采样时应尽可能采集气管拭子标本;②陆禽标本检出率显著高于水禽标本(P<0·05),对病毒培养阳性的陆禽气管拭子检出率达80%(95%CI:70·6%~87·8%),对水禽气管拭子标本的检出率为38%(95%CI:26·9%~49·4%),可能与标本中病毒滴度高低有关;③有症状与无症状陆禽标本的检出率无显著差异。另外,H5-Dot试剂对333份非H5病毒气管拭子标本的特异性为99·4%(95%CI:97·9%~99·9%)。这些结果表明,H5-Dot是一种较为可靠的H5亚型禽流感病毒早期快速检测方法,在缺乏仪器设施和高素质专业技术人员的H5N1禽流感病毒防控第一线具有重要推广价值。     

H5N1禽流感病毒环介导等温扩增快速检测方法的建立

目的：建立H5N1禽流感病毒环介导等温扩增（LAMP）快速检测方法。方法：从GenBank中获得H5N1禽流感病毒血凝素（HA）基因序列,应用DNAStar软件MegAlign程序分析其序列,利用PrimerExplorerV3软件在序列保守区域设计LAMP引物,即外引物、内引物和环引物,同时以所克隆的阳性质粒为模板,对试验中的几个重要参数进行优化。结果：LAMP检测方法对H5N1禽流感病毒的灵敏度达到4～6个拷贝,其引物对于H1、H9亚型禽流感病毒和新城疫病毒无非特异性扩增,表现出良好的H5亚型特异性。结论：建立的H5N1禽流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测禽流感病毒提供了新方法和新思路。     

高致病性禽流感病毒H5N1中和抗体的单链抗体构建与活性鉴定

在本实验室研制出的多株针对H5N1血凝素的鼠单抗中,10F7对34株H5N1病毒株都有血凝抑制和中和活性,具有特异性高、反应性强、识别谱广的特点。通过基因工程构建10F7单链抗体(scFv)表达重组质粒,在大肠杆菌中表达并纯化scFv,经血凝抑制实验及中和实验检测其活性。结果在针对3株病毒的血凝抑制实验中,10F7scFv蛋白对其中2株H5N1病毒均显示出结合活性,而对H9毒株没有反应。在针对7株H5N1病毒的中和实验中,10F7scFv对5株病毒具有较好的中和能力。H5N1广谱中和抗体10F7的单链抗体构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础。     

抗禽流感病毒H5N1亚型单克隆抗体制备初报

目的制备禽流感H5N1亚型病毒的单克隆抗体,为相关研究提供工具。方法以禽流感H5N1亚型病毒免疫BALBc小鼠,取其脾细胞和SP20细胞融合,用血凝抑制试验(HI)和酶联免疫反应(ELISA)检测培养上清,并将阳性融合细胞稀释克隆化3次直至100%孔均为阳性,筛选阳性克隆株,运用免疫荧光法评估单克隆抗体检测病毒感染的犬肾细胞(MDCK)。结果得到三株稳定分泌抗体的细胞并命名为F8、F9、G11,抗体亚型鉴定结果分别为IgG1、IgG2a和IgG2b;在免疫荧光法单克隆抗体能够检测出感染MDCK细胞的病毒。结论建立了3株抗禽流感H5N1亚型病毒的单克隆抗体细胞株,其产生的一株高特异性的McAbG11能够用于H5N1亚型禽流感病毒感染诊断,并可能应用于禽流感病毒H5N1亚型感染的防治。     

Development of an immunochromatographic kit for rapid diagnosis of H5 avian influenza virus infection

Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.     

应用跨膜区置换的血凝素蛋白建立H7N9禽流感病毒抗体间接ELISA检测方法

【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r0.5,P0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。     

一种更灵敏的特异性检测H9亚型禽流感抗体的间接ELISA方法的建立

H9亚型禽流感在全球范围内广泛流行,控制其传播需监测H9亚型禽流感病毒的感染情况及疫苗的免疫效果。为了建立便于检测且灵敏特异的H9亚型禽流感抗体间接ELISA方法,本实验利用不同亚型之间变异较大的H9亚型禽流感病毒HA蛋白的头部球状区作为包被抗原,确定了最佳复合封闭液和抗体稀释液,提高了其特异性。结果显示建立的ELISA方法灵敏度高于血凝抑制试验(HI),且与H3N2、H5N2、H7N9亚型流感病毒及新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)、鸡传染性法氏囊病毒(IBDV)和产蛋下降综合征病毒(EDSV)的阳性血清均无交叉反应。另外,利用该方法及HI试验对200份临床鸡血清样本进行检测,两种检测方法的符合率达97%,且存在较高的相关性(R2=0.981 1)。     

A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses

Background

Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met.

Methodology/Principal Findings

Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA.

Conclusion/Significance

The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.     

A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.     

2017–2019年我国南方地区高致病性H5N6亚型禽流感病毒血凝素蛋白分子特征分析

【背景】自2014年以来,H5N6禽流感病毒在我国家禽和活禽市场持续进化,成为人类和动物健康的重大威胁。【目的】对2017-2019年中国南方地区93株高致病性H5N6禽流感病毒的HA基因进行分子进化分析。【方法】接种9-11日龄鸡胚分离核酸检测阳性的H5N6标本,运用下一代测序平台对病毒分离物进行全基因组测序,从NCBI和GISAID数据库下载参考序列,利用BLAST、MEGA6.1及Clustal X等软件进行序列分析。【结果】2017-2019年,从189份江苏省H5亚型禽类/环境标本和1名H5N6患者咽拭子标本中共分离到43株病毒,完成了33株H5N6病毒的全基因组测序。下载网上同时期中国其他地区流行的H5N6毒株序列,对总计93株H5N6病毒的HA基因进行分子进化分析。93株H5N6病毒中有78株属于Clade 2.3.4.4h,9株病毒属于Clade 2.3.4.4e,4株H5N6病毒属于Clade 2.3.4.4b,1株属于Clade 2.3.4.4f,1株属于Clade 2.3.4.4g。所有93株病毒HA蛋白的裂解位点含有多个碱性氨基酸,表明它们都属于高致病性禽流感病毒。所有93株病毒HA蛋白的Q222和G224位氨基酸没有发生突变,保留了禽类受体α2-3半乳糖苷唾液酸(SAα2-3Gal)结合特性;158位点丧失糖基化,同时124位出现一个新的潜在糖基化位点。【结论】2017-2019年间中国南方地区H5N6病毒进化活跃,具有明显的基因多样性,需要加强对病毒分子进化的监测。     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong

The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of "internal" virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.     

H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析

血凝素(hemagglutinin,HA)蛋白是禽流感病毒(avian influenza virus,AIV)的一个重要表面抗原性蛋白,在疾病诊断和防治上有重要意义。本研究为了探讨一种更为简便有效的HA重组蛋白表达途径,利用生物信息学软件,对H5N1亚型AIVHA基因编码的氨基酸序列进行分析,在分析其在大肠杆菌中的密码子偏好性、稀有密码子分布情况及有关蛋白的抗原性等重要特性后,构建了HA抗原表位重组表达质粒pET-32a(+)-HA。经测试,该重组质粒在1mmol/LIPTG诱导剂作用下诱导过夜,能在大肠杆菌Rosetta-gami B(DE3)中高效表达,并得到48.1kD大小的目的重组表达蛋白。重组蛋白用6×His-tagged protein纯化试剂盒纯化后,与福氏佐剂等量混合制备成抗原,以200μg/鸡的剂量皮下注射2月龄SPF鸡3次,采血分离血清。Western-Blot试验结果表明,该重组表达蛋白能分别与所制备的高免鸡血清及H5N1亚型AIV阳性血清发生特异性反应,在硝酸纤维素膜上出现特异性杂交带。说明本试验研究的HA抗原重组表达蛋白具有良好的免疫原性和反应原性,保留了HA蛋白的抗原活性,提示该重组蛋白在H5亚型AIV的防治技术研究中具有重要的实际应用价值。