人感染高致病性禽流感病毒H5N1核酸检测方法的建立及应用

目的 建立人感染高致病性禽流感病毒H5N1的核酸检测方法,用于人感染高致病性禽流感病毒疑似病例临床标本的检测.方法 针对甲型流感病毒保守基因M设计RT-PCR和real-time PCR引物检测是否为甲型流感病毒,同时针对H5N1禽流感病毒设计针对H5和N1基因的特异性RT-PCR和real-time PCR引物作亚型检测,建立禽流感H5N1病毒RT-PCR和real-time PCR检测方法.结果 本研究建立的RT-PCR和real-time PCR方法可以特异性地检测H5N1病毒,并且与人流感病毒H1、H3没有交叉反应.RT-PCR检测方法灵敏度可到1TCID50,real-time PCR灵敏度可达0.01TCID50.利用上述方法检测人感染高致病性禽流感病毒H5N1疑似病例临床标本,从42例不明原因肺炎病例中检测出阳性标本13例.结论 本研究建立的RT-PCR和real-time PCR方法可以用于人感染高致病性禽流感病毒H5N1临床标本的实验室检测.     

禽流感病毒H5N1 单克隆抗体的生物学特性鉴定

目的:探讨禽流感病毒单克隆抗体的生物学特性。方法:制备H5N1禽流感病毒的单克隆抗体,鉴定其亚型、效价、血凝抑制活性以及与其他亚型流感病毒的交叉反应性。免疫印迹方法验证单抗与H5N1抗原的结合,人体正常组织芯片的免疫组织化学染色法观察单抗的组织学反应。结果:发现两株禽流感病毒H5N1的单抗(H5-32和H5-35)分别与人体的肾、胰腺组织存在阳性结合。结论:禽流感病毒H5N1与人体组织间可能存在某种关联,这或许可为禽流感病毒感染和致病研究提供参考。     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

利用反向遗传学技术构建H5N9重组流感病毒株

目的 利用反向遗传学技术构建来源人感染禽流感病毒H5N1和H7N9 HA和NA基因的H5N9亚型禽流感病毒.方法 全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,插入到pHW2000载体,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒.结果 核酸测序、HA和NA基因转录和表达检测、细胞病变分析确定利用该反向遗传学系统可以成功拯救H5N9病毒.重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法拯救H1N1病毒.结论 利用反向遗传学技术成功构建一株H5N9重组病毒.     

H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析

目的:预测H5N1亚型禽流感病毒血凝素Th和B细胞相关抗原表位,并初步分析其抗原性.方法:依据近年H5N1亚型禽流感病毒流行趋势,下载得到相关HA蛋白氨基酸序列.进行生物信息学综合分析预测,获得Th和B细胞相关抗原表位,并比较其保守性和特异性.通过BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清,初步鉴定候选表位抗原性.结果:综合多项预测及空间构象模拟结果,我们获得了三条候选Th和B细胞表位,分别为HA141～155、HA206～223、HA302～316.候选表位处于H5N1亚型禽流感HA1 蛋白序列上相对保守的区域内,且与目前流行的H5N1亚型禽流感病毒HA相应区域具有较好的一致性.而不同候选表位在BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清反应中显示了不同抗体结合能力,预示了其成为功能表位的可能.结论:所筛选的表位具有成为H5N1亚型禽流感病毒HA Th和B细胞相关抗原表位的可能.本研究为深入揭示流感病毒感染与免疫机制,H5N1亚型禽流感功能表位认知及表位疫苗研究奠定了基础.     

禽流感病毒血凝素H5特异性单克隆抗体的制备及ELISA捕获法的建立

目的 制备和鉴定禽流感病毒(H5N1)血凝素(H5)特异性单克隆抗体(mAb),建立H5抗原的双抗体夹心ELISA捕获法.方法 以H5血凝素和携带H5全长基因的质粒免疫Balb/c小鼠制备mAb,利用血凝抑制(HI)实验筛选和鉴定,通过竞争抑制试验分析抗体识别表位,并采用抗体配对试验筛选捕获抗体和检测抗体,建立测定H5抗原的双抗体夹心ELISA捕获法.结果 获得16株特异性针对H5的单克隆抗体,与A型流感病毒H1、H3、H7、H9和B型流感病毒的血凝素无HI交叉反应,对H5血凝素的血凝抑制效价为1:100～1:51 200;通过配对实验,建立以单克隆抗体H5M9为捕获抗体,辣根过氧化物酶标记单克隆抗体H5M11为检测抗体的双抗体夹心ELISA;检测多株H5N1病毒和H5血凝素的最低检出值为1/32血凝单位,检测A型流感病毒H1N1、H3N2、B型流感病毒以及H7、H9血凝素均为阴性.结论 建立了一种灵敏度高、特异性强的测定H5抗原的ELISA捕获法,可应用于禽流感病毒H5N1感染的实验室早期诊断.     

抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定

目的:获得具有中和活性、高特异性和稳定性的抗H5N1禽流感病毒血凝素蛋白(HA)的羊驼重链单域(VHH)抗体。方法:利用pET-22b表达载体诱导表达抗H5N1禽流感病毒HA VHH抗体蛋白,以包涵体形式表达的VHH抗体蛋白采用最优复性方法进行复性后,获得高纯度的VHH抗体,分别采用ELISA法鉴定VHH抗体的亲和力和热稳定性,采用血凝抑制实验鉴定抗体的特异性和体外中和活性。结果:经复性的抗H5N1禽流感HA VHH抗体对H5N1禽流感病毒HA具有良好的特异性。通过对三种不同复性方法比较,利用柱上复性的VHH23抗体具有较好的热稳定性,亲和力为9.1×10-7mol/L,同时对H5N1禽流感病毒HA具有良好的体外中和活性。结论:实验结果表明通过原核表达获得具有较好中和活性、特异性及稳定性的抗H5N1禽流感病毒VHH抗体,为进一步开展抗体的体内病毒中和试验奠定良好基础。     

感染H5N1病毒恒河猴大脑的病理学观察

目的H5N1禽流感病毒与1918年大流感病毒基因序列的高度相似表明该病毒对人类构成巨大威胁,本文主要观察H5N1禽流感病毒对恒河猴大脑影响。方法恒河猴5只,年龄2～3岁,经环甲膜穿刺接毒105TCID50/ml7ml,取大脑行苏木精-伊红(HE)染色和免疫组化观察。结果和对照组11号猴比较,实验组猴出现神经元萎缩,噬神经元现象,局部坏死和血管套现象。结论H5N1禽流感病毒导致恒河猴大脑损害,出现非化脓性脑炎,进一步证实了H5N1禽流感病毒对哺乳类日渐适应并且表现出越来越强的神经毒性。在今后的临床治疗中要重视H5N1病毒引起脑炎的预防和治疗。     

H9N2禽流感病毒反向遗传系统的构建和鉴定

目的 建立H9N2亚型禽流感病毒反向遗传系统,为人禽流感疫苗研制以及传播和致病机制等方面的研究提供技术平台.方法 使用RT-PCR方法获得禽流感H9N2亚型病毒A/Guangzhou/333/99(H9N2)的8条全长基因节段,然后克隆到双表达载体pCI-pol Ⅰ中,获得H9N2禽流感病毒的8个基因节段的8质粒系统.将构建好的8质粒共转染293T细胞后,收获上清接种鸡胚,然后对鸡胚尿囊液进行鉴定;对拯救的病毒进行鉴定.结果 8质粒系统转染293T细胞后可以成功拯救出H9N2禽流感病毒,血凝效价可达到29/50μl,生长特性与野生型病毒类似.结论 成功建立了H9N2禽流感病毒反向遗传系统.     

湖北省首例人感染H5N6禽流感病例流行病学调查

目的 对湖北省首例人感染H5N6禽流感病例的流行病学资料进行分析,探讨病例发现过程、调查处理措施、实验室检测方法,为防控人感染H5N6禽流感疫情提供依据.方法 采用描述性流行病学方法,分析湖北省首例人感染H5N6禽流感病例诊治过程、密切接触者信息,开展现场流行病学调查,并采集病例、密切接触者、活禽市场外环境标本进行实验室检测分析.结果 患者2016年4月9日发病,体温40℃,有活禽市场暴露史.发病早期采集的下呼吸道标本(痰液、气管分泌液)为H5N6禽流感病毒核酸阳性,而上呼吸道标本H5N6禽流感病毒核酸检测均为阴性.患者无外出史和H5N6禽流感病例接触史.患者经两个月的治疗痊愈出院.密切接触者58人均未出现发热和呼吸道感染症状.采集病例经常路过的两家活禽市场和一家土鸡专卖店外环境标本共36份,其中检出H5N6禽流感病毒核酸1 1份,阳性率为30.56％.结论 该病例为湖北省首例人感染H5N6禽流感病例,属本地感染的散发病例,未出现人传人.传播途径可能为:活禽市场通过禽-环境-人的途径传播.在病例诊断中,下呼吸道标本(尤其是痰液、气管分泌液)具有重要意义.另外,尽早应用奥司他韦对病例的成功救治起到了重要的作用.     

禽流感H5N1型病毒对NIH小鼠的致病敏感性研究

目的观察禽流感H5N1型病毒对NIH小鼠的致病性。方法将NIH小鼠随机分为接毒组（20只）和对照组（10只）,在负压感染实验室,接毒组于乙醚麻醉后给予AIVH5N1型病毒滴鼻,对照组予正常阴性尿囊液滴鼻,观察14d,记录小鼠的体温、体重、临床症状、死亡情况、病理变化、肺指数及抗体变化。结果NIH小鼠感染禽流感H5N1型病毒后第1天就出现精神不振,病程主要集中在第3—7天,临床症状主要表现为弓背、蜷缩、竖毛、颤抖、反应迟钝、活动减少、爱扎堆,体重（下降）出现减轻,体温降低,死亡率为40％;接毒组死亡小鼠肺指数为（2．21±0．40）,较对照组的（0．44±0．23）明显增高,差异有统计学意义（P〈0．01）;肺组织病理严重改变,第8天才能检出抗体（OD值等于0．231）。结论禽流感H5N1型病毒对NIH小鼠有一定的致病性。     

Avian influenza A virus H7N9 in China,a role reversal from reassortment receptor to the donator

Reassortment can introduce one or more gene segments of influenza A viruses (IAVs) into another, resulting in novel subtypes. Since 2013, a new outbreak of human highly pathogenic avian influenza has emerged in the Yangtze River Delta (YRD) and South-Central regions of China. In this study, using Anhui province as an example, we discuss the possible impact of H7N9 IAVs on future influenza epidemics through a series of gene reassortment events. Sixty-one human H7N9 isolates were obtained from five outbreaks in Anhui province from 2013 to 2019. Bioinformatics analyses revealed that all of them were characterized by low pathogenicity and high human or mammalian tropism and had introduced novel avian influenza A virus (AIV) subtypes such as H7N2, H7N6, H9N9, H5N6, H6N6, and H10N6 through gene reassortment. In reassortment events, Anhui isolates may donate one or more segments of HA, NA, and the six internal protein-coding genes for the novel subtype AIVs. Our study revealed that H7N9, H9N2, and H5N1 can serve as stable and persistent gene pools for AIVs in the YRD and South-Central regions of China. Novel AIV subtypes might be generated continuously by reassortment. These AIVs may have obtained human-type receptor-binding abilities from their donors and prefer binding to them, which can cause human epidemics through accidental spillover infections. Facing the continual threat of emerging avian influenza, constant monitoring of AIVs should be conducted closely for agricultural and public health.     

Cytometric microsphere array for subtyping avian influenza virus

Avian influenza is a highly contagious disease, and different subtypes of avian influenza virus (AIV) have different levels of pathogenicity. A microsphere-based fluorescent assay was initially established for subtyping AIV. DNA fragments were amplified with biotinylated primers. AIV subtype-specific DNA probes with an amino-linker at the 5' end were covalently bound with carboxy-modified encoded beads. The modified beads and the denatured DNA fragments were mixed together for hybridization. Then, quantum dots-streptavidin (QDs-streptavidin) was added to conjugated biotinylated PCR products. The reaction products were screened by flow cytometry. AIV strains (such as H5N1 and H9N2) could be determined and subtyped according to their combination of encoded beads and fluorescent QDs. The method's combined sensitivity of the nucleic acids of H5N1 and H9N2 avian influenza virus at a threshold of 74 pg and 1 pg could be detected. This is a powerful method for detecting many pathogens or many types of a pathogen simultaneously.     

Lack of evidence of avian-to-human transmission of avian influenza A (H5N1) virus among veterinarians,Guangdong, China, 2012

BackgroundCases of human infections with H5N1 avian influenza viruses have been reported all over the world with the reason of direct contact with sick or diseased poultry, which suggests the direct contact with poultry may be one of the major risk factors for human infection.ObjectivesIn this study, we estimated the seroprevalence of antibodies against avian influenza A (H5N1) virus in veterinarians with exposure to avians.Study designFrom May 21, 2011 through April 22, 2012, 406 veterinarians exposure to poultry in Guangdong province were interviewed a questionnaire. A serum specimen was collected from participants to test for H5N1 antibodies by HI and NT assay.ResultsNone of the 406 sera from occupationally exposed veterinarians was positive according to the HI test and the NT test with the H5N1 AIV.ConclusionOur seroepidemiologic survey suggests that the risk of avian-to-human transmission of the H5N1 AIV is very low based on the samples that we tested. However, prevention regarding the risk of H5N1 AIV transmission is essential and should be recommended as public health measures.     

Rapid pathotyping of recent H5N1 highly pathogenic avian influenza viruses and of H5 viruses with low pathogenicity by RT-PCR and restriction enzyme cleavage pattern (RECP)

Rapid and accurate diagnosis of avian influenza virus infections of poultry and humans comprises detection, subtyping, and, in case of subtypes H5 and H7, pathotyping of such viruses. Reliable methods for pathotyping of H5 avian influenza viruses (AIV) are based on determination of the intravenous pathogenicity index (IVPI) in specific pathogen free (SPF) chickens and on characterization of the hemagglutinin (HA) gene cleavage site by sequencing. The number of basic amino acids (arginine and lysine) at the cleavage site is an important indicator of pathogenicity. In this paper, a new rapid method for pathotyping of H5 subtype avian influenza viruses is described which is based on RT-PCR and restriction enzyme cleavage pattern (RECP) assay using the MboII restriction enzyme. Validation of the method using 28 H5 subtype reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity, especially when targeting recent highly pathogenic AIV (HPAIV) of subtype H5N1 and Asian origin. In addition, RECP results were validated by testing 47 field samples from different sources and by sequencing of their RT-PCR products. This approach for H5 AIV pathotyping proved to be fast, reliable, and comparatively sensitive and is suitable especially for laboratories lacking sequencing or in vivo pathotyping facilities.     

Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP

The worldwide outbreak of avian influenza among poultry species and humans is associated with the H5N1 subtype of avian influenza A virus (AIV). This highlighted the need to develop safe H5 AIV diagnostic methods. 7H10, an H5-specific monoclonal antibody (Mab), can be used for immunohistochemical (IHC) staining for formalin-fixed tissue. An assortment of H5N1 tissue specimens infected naturally in paraffin sections from Asia, between years 2002-2006, including one human specimen, were tested. 7H10 detected H5 infection in all of these tissue samples infected naturally. In addition, 24 different human H5N1 isolates from Indonesia, 5 avian H5 isolates and 3 non-H5 isolates from Asia were inoculated into BALB/C mice and chicken embryos. Among these influenza viruses, 7H10 detected 28 of the 29 H5 virus strains by immunohistochemical staining, while none of non-H5 strains used in this study could be detected by 7H10, confirming its specificity to H5. Further, the eight-residue-long linear epitope, "FFWTILKP", identified through epitope mapping, enables 7H10 to detect >98.3% of H5 subtype viruses reported worldwide before 2007. This study describes a specific H5 diagnostic system with minimal possibility of exposure to live virus based on immunochemical staining.     

The first lack of evidence of H7N9 avian influenza virus infections among pigs in Eastern China

In this study, we sought to examine whether evidence existed suggesting that pigs were being infected with the novel H7N9 avian influenza virus. From November 2012 to November 2013, blood was drawn from 1560 pigs from 100 large farms in 4 provinces of eastern China. Many of these pigs were in close proximity to wild birds or poultry. Swine sera were studied using hemagglutinin inhibition (HI) assays and enzyme-linked immunosorbent assays (ELISAs) against the H7 antigen derived from the emergent H7N9 avian influenza virus (AIV). Only 29 of the 1560 samples had HI titers of 1:20 when using the H7N9 AIV antigens, and none of the 29 (H7N9 AIV) HI-positive samples were positive when using ELISA, indicating that no samples were positive for H7N9. The negative results were also verified using a novel competitive HA-ELISA. As pigs have been shown to be infected with other avian influenza viruses and as the prevalence of novel influenza A viruses (e.g., H7N9 AIV) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be periodically conducted in the future.     

Development of an antigen-capture ELISA for detection of H7 subtype avian influenza from experimentally infected chickens

Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.