H9亚型禽流感病毒对不同动物红细胞血凝反应谱的研究

目的研究不同时间分离的H9N2亚型禽流感病毒对不同动物红细胞的吸附作用,探讨该病毒对人存在的感染风险。方法利用在不同时期不同地区分离到的H9N2亚型禽流感病毒的不同毒株分别对鸡、鸭、鹅、鸽子、鹌鹑、猪、羊、牛、马、人O型红细胞、兔、BalB/c小鼠、ICR小鼠、大鼠等14种不同动物的红细胞进行血凝试验,并描绘其血凝反应谱,以探讨不同病毒毒株的特性。结果不同H9N2禽流感病毒对动物红细胞的血凝谱存在差异,对禽类的血凝效价较高,对人的O型红细胞的平均血凝效价第二高。结论 H9N2亚型禽流感病毒不仅对禽类有着较高的致病性,对人也存在感染风险。     

血凝和血凝抑制试验常见问题及解决方法

随着社会经济文化的不断进步与发展，国家综合国力日益提升，在科学技术日渐更新和网络信息化时代的基础上，医学卫生事业也取得显著的进步。在医学发展进程中，血凝和血凝抑制试验是当前我国医疗卫生发展的重要项目，同时也是我国各地区域开展疫病诊断和疫病调查的重要手段之一。在血凝和血凝抑制试验中涉及到了关于红细胞和抗原体的反应，因此试验过程容易受诸多因素的影响，而造成试验结果的偏差。本篇文章主要对血凝和血凝抑制试验常见的问题进行分析，同时着重探讨血凝和血凝抑制试验常见问题的解决措施。     

单克隆抗体反向被动血凝抑制试验与间接血凝抑制试验在诊断乙脑中的比较

本文用乙脑单克隆抗体致敏羊血球的反向被动凝集抑制试验(RPHI),对临床诊断为乙脑患者的124份双份血清进行检测,并与常用的间接血凝抑制试验HI作比较,RPHI的阳性率为83.1%(103/124),HI法的阳性率为79.0%(98/124)。经统计学检验,P>0.05,两法的阳性率无明显差别。再进一步将检测血清按滴度的不同水平分组,进行相关分析,结果表明,两种方法检测的结果存在正相关,且恢复期血清检测结果的相关度高于急性期检测结果的相关度。因此,证明了简便快速的RPHI法可作为诊断乙脑的检测手段。     

麻疹血凝抑制试验试剂盒的研制与应用

为了解决麻疹血凝抑制(HI)试验中猴红细胞的保存与人血清中非特异性凝集物质吸收的难点,推广麻疹HI试验方法,研制了麻疹HI试验试剂盒,并用该试剂盒与经典HI试验方法平行检测353份待检血清,结果有322份血清两法检测的效价完全一致(91.2%),其余31份两法检测效价的差异为±2倍,即在HI试验允许误差范围内.两法检测效价的差异无显著的统计学意义 (P＞0.05).而且试剂盒不必用猴红细胞去吸收血清中的非特异性凝集物质,还可在4℃保存4个月以上.该试剂盒的研制与使用,可大大方便与促进麻疹血清流行病学工作的开展.     

应用被动血凝试验与血凝抑制试验检测麻疹抗体的比较

麻疹抗体的检测目前常规使用血凝抑制试验(HI)，该试验虽具有特异、快速的优点，但必须使用新鲜猴血球，而新鲜猴血球保存期限短，运输不方便，因而不便推广应用．目前随着麻疹活疫苗的广泛使用，为了解人群免疫水平和考核疫苗免疫效果，需要一个既简便又特异、敏感的测定麻疹抗体的方法。我们最近对被动血球凝集试验(PHA)和HI实验这种方法作了对比试验。现将结果报道如下：     

反向间接血凝抑制试验测定麻疹抗体

目的建立采用反向间接血凝抑制试验测定麻疹抗体的方法,并与传统的血凝抑制方法进行比较。方法采用麻疹单克隆抗体致敏人“O”型红细胞的反向间接血凝抑制法,检测麻疹抗体。结果两种方法检测麻疹疫苗免疫后抗体阳转率均为100％（11／11）;52份免疫后血清RPHI的GMT为26．55,HI的GMT为39．08,经统计学处理两种方法未见显著性差异,并具有很好的平行关系。应用该方法对其他相关病毒抗原和抗体的检测,结果无交叉反应。结论该法有较好的实际应用价值。     

狂犬病反相被动血凝抑制试验的建立和应用

狂犬病反相被动血凝抑制试验的建立和应用浙江省卫生防疫站（杭州３１０００９）翁景清李敏红姚苹苹姜理平陆群英唐汉英朱智勇（指导狂犬病是一种病死率非常高的病毒性传染病。目前，常有因被狂犬咬伤而得病死亡的病人。狂犬病因无特效药治疗，唯一有效的办法就是通过接种...     

麻疹血凝抑制试验和酶联免疫吸附试验的应用比较

为了比较用于检测麻疹抗体的血凝抑制 (HI)试验和酶联免疫吸附试验 (ELISA) ,1998年河北省采用这两种方法平行检测了麻疹疫苗 (MV)强化免疫前后 1～ 2 4岁人群的血清 1317份。结果显示 :MV强化免疫前HI抗体和ELISAIgG抗体阳性率分别为 99 78%和 95 2 2 % ,抗体几何平均滴度 (GMT)分别为 1∶36 15和 1∶812 15 ;强化免疫后 ,HI抗体和ELISAIgG抗体阳性率分别为 10 0 0 0 %和 99 88% ,抗体GMT分别为 1∶91 6 5和 1∶2 15 8 17,强化免疫前后分别比较 ,差别均有非常显著的统计学意义。两种方法阳性符合率为 98 33% ,ELISAIgG抗体的GMT比HI抗体的GMT高 2 3 5 5倍 ,两种方法具有较密切的相关关系 ,相关系数r=0 73,95 %可信区间为 (0 70 ,0 75 ) ;所建回归方程为 :Log^Y =- 0 32 10 0 6 +0 6 70 75 6LogX0 。两种方法平行检测 5 8份强化免疫前后双份血清抗体≥ 4倍增长情况 ,符合率为 82 76 %。结果表明 ,两种检测方法用于麻疹抗体检测相关性较好 ,符合率较高。     

用新的实验方法检测A/H5禽流感病毒

2006—2—3，美国食品药品管理局（FDA）宣布批准检测流感病毒A／Hs（亚洲系列）的实时逆转录聚合酶链反应的（RT—PCR）引物以及探针装置，以及灭活了的病毒作为体外定量检测高致病性流感A/H5病毒（亚洲系列）的阳性RNA对照的来源。存在2个系列的A／H5流感病毒：欧亚（亚洲）和北美系列。CDC研制的引物及其探针装置用来检测亚洲系列的高致病性A/H5流感病毒，这些病毒与最近在东亚（土尔其和伊拉克）实验室确诊的人感染禽流感病例有关。     

鸽红细胞检测风疹血凝抑制抗体的探讨

随着风疹疫苗的广泛使用 ,对风疹血凝抑制抗体的测定显得更加重要。在风疹血凝抑制试验中 ,鹅、鸽、一日龄雏鸡的红细胞均可与风疹血凝素发生血凝现象[1 ] 。以往通常都用鹅红细胞作为实验材料。为了解鸽红细胞在风疹血凝抑制试验中的作用 ,我们用鸽红细胞检测风疹血凝抑制抗体 ,现将结果报告如下。材料与方法1　实验材料　 (1)血清标本来自德清县 15岁以下人群 ,计 387人。 (2 )风疹血凝素为中国药品生物制品检定所提供 (冻干制剂批号 0 0 0 5 0 2 )。 (3)鸽子红细胞。 (4)肝素 -氯化锰溶液 (M -H)。 (5 )葡萄糖 -明胶 -巴比妥缓冲液(DGV…     

广西禽流感H5N1亚型病毒HA基因序列分析

目的了解广西禽流感H5N1亚型病毒的基因特性.方法2011年在广西农贸市场采集污水、笼具涂抹、粪便标本,经H5亚型特异实时荧光定量PCR方法(Real-time fluorescence quantitative RT-PCR)检测,阳性样本进行病毒血凝素(hemagglutinin,HA)基因扩增后对产物直接测序,测序结果与已知参考毒株进行序列比对及系统进化分析.结果对阳性样本病毒HA基因测序获得6份HA序列,均分布在进化分支2.3.2的Ⅱ-1分支下.广西的6序列无论是氨基酸还是核苷酸的都是高度同源的,其核苷酸同源性在99.5%~100%,氨基酸同源性在99.5%~99.8%;序列测定的结果同时表明无论是受体特异性还是连接肽都是禽源的.结论2011年广西农贸市场流行的禽流感H5N1亚型病毒主要以进化分支2.3.2Ⅱ-1为主,均为禽源性的病毒.     

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.     

广州市2014-2019年H5亚型禽流感病毒流行与基因遗传特征分析

目的 分析广州市H5亚型禽流感病毒流行特点及基因进化和变异特征,为禽流感的防控提供依据。方法 对2014-2019年广州市禽类市场外环境标本进行禽流感病毒核酸检测并分析H5亚型流行情况,随机选取48份（46份来源于环境和2份来源于病例）H5阳性标本进行HA和NA基因测序,应用生物信息学软件分析分子遗传特征。结果 2014-2019年监测的52 284份环境标本中,检出H5亚型阳性标本1 094份,阳性率为2.09%。遗传进化分析显示,HA基因属于Clade 2.3.4.4.C分支,NA基因主要归属于欧亚谱系H6N6进化分支,HA和NA基因的进化以时间聚类为特点,相近年份流行株聚成一个进化分支。分子特征显示,48份毒株裂解位点呈现高致病性分子特点,受体结合位点仍为禽源受体,但普遍发生S123P、S133A和T156A倾向结合人源受体的位点突变。抗原位点变异主要出现在B、E区,并表现时间分布的特点,同一年份流行株常发生与上一年份流行株不同的抗原位点变异。2017年开始所有毒株出现由氨基酸缺失导致的糖基化位点的增加（140-NHT）。结论 广州市禽类市场外环境H5亚型禽流感病毒长期流行,且阳性率占比逐渐升高。测序分析提示,广州市H5亚型禽流感病毒为Clade 2.3.4.4.C分支H5N6高致病性病毒,并且持续进化和变异,特别是普遍出现与人源受体结合能力增强的突变,需加强监测。     

Lack of H5N1 avian influenza transmission to hospital employees, Hanoi, 2004

To establish whether human-to-human transmission of influenza A H5N1 occurred in the healthcare setting in Vietnam, we conducted a cross-sectional seroprevalence survey among hospital employees exposed to 4 confirmed and 1 probable H5N1 case-patients or their clinical specimens. Eighty-three (95.4%) of 87 eligible employees completed a questionnaire and provided a serum sample, which was tested for antibodies to influenza A H5N1. Ninety-five percent reported exposure to > or = 1 H5N1 case-patients; 59 (72.0%) reported symptoms, and 2 (2.4%) fulfilled the definition for a possible H5N1 secondary case-patient. No study participants had detectable antibodies to influenza A H5N1. The data suggest that the H5N1 viruses responsible for human cases in Vietnam in January 2004 are not readily transmitted from person to person. However, influenza viruses are genetically variable, and transmissibility is difficult to predict. Therefore, persons providing care for H5N1 patients should continue to take measures to protect themselves.     

500名中国和越南医科大学新生H9亚型禽流感病毒血清抗体调查

H9N2亚型禽流感病毒已在家禽中建立稳定的种系并广泛分布于世界各地[1]. 一般情况下,禽流感病毒不感染人类,但1997年以来,相继暴发禽流感病毒H5N1、H7N7和H9N2直接传染给人,特别是在中国内地和香港出现H9N2亚型流感病毒感染人[2,3],H9在人群中的隐性感染率比较高[4].中国广西和越南顺化医科大学新生来自全国多个省市,生活环境各不相同.为此,本研究选取这两所医科大学2010年新生进行禽流感病毒H9血清抗体调查.     

Atypical avian influenza (H5N1)

We report the first case of avian influenza in a patient with fever and diarrhea but no respiratory symptoms. Avian influenza should be included in the differential diagnosis for patients with predominantly gastrointestinal symptoms, particularly if they have a history of exposure to poultry.     

Novel reassortant highly pathogenic H5N6 avian influenza viruses in poultry in China

We characterized two novel highly pathogenic H5N6 influenza viruses isolated from Chinese poultry in 2013. Genomic analysis showed that both isolates were reassortants, and derived their genes from H5 and H6 subtype viruses found in poultry in China. The virulence of the two isolates was examined in chickens and mice, and both isolates were found to be highly pathogenic in chickens and only moderately virulent for mice. Our results show that continued circulation of these viruses could endanger both avian species and humans.     

云南省边境地区禽流感H5N1亚型病毒遗传多样性分析

目的 了解云南省边境地区禽流感H5N1亚型病毒遗传多样性.方法 2009-2011年7月在云南省边境地区采集境外家禽和野生鸟类棉拭子样品,经H5/Nl亚型特异性多重RT-PCR检测,阳性样品进行病毒HA基因扩增,克隆至pMD 18-T载体测序,并与已知参考毒株进行序列比对及系统发育分析.结果 36份阳性样品病毒HA基因测序获得15种HA序列,存在2个不同进化分支(2.3.2、2.3.4),2.3.2进一步可划分为3个进化小分支(Ⅱ-1～Ⅱ-3),2.3.4进一步可划分为2个进化小分支(Ⅰ-1和Ⅰ-2).2.3.2Ⅱ-1、Ⅱ-2毒株是新出现的H5N1亚型病毒变异株.结论 2009- 2011年7月云南省边境地区H5N1亚型病毒具有遗传多样性,病毒经历了多分支(2.3.2、2.3.4)至单一支(2.3.2)的进化过程.     

A recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (H7N1)

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 cause fatal disease in poultry (fowl plague) but also have zoonotic potential. Currently commercially available vaccines often do not provide sufficient protection and do not allow easy discrimination between vaccinated and infected birds. Therefore, vaccination of domestic poultry against H5 and H7 HPAIV is not allowed in many countries, or is only possible after special permission has been provided. We generated a recombinant marker vaccine based on non-transmissible vesicular stomatitis virus (VSV) expressing the HA antigen of HPAIV A/FPV/Rostock/34 (H7N1) in place of the VSV G gene. This virus, VSV*ΔG(HA), was propagated on a helper cell line providing VSV G in trans. Since no progeny virus was produced after infection of non-complementing cells, the vector was classified as biosafety level 1 organism (“safe”). Chickens were immunized via the intramuscular route. Following booster vaccination with the same replicons high titers of serum antibodies were induced, which neutralized avian influenza viruses of subtypes H7N1 and H7N7 but not H5N2. Vaccinated chickens were protected against a lethal dose of heterologous HPAIV A/chicken/Italy/445/99 (H7N1). Secretion of challenge virus was short-term and significantly reduced. Finally, it was possible to discriminate vaccinated chickens from infected ones by a simple ELISA assay. We propose that VSV replicons have the potential to be developed to high-quality vaccines for protection of poultry against different subtypes of avian influenza viruses.     

湖南省2006--2009年人感染高致病性禽流感病毒(H5N1)分子特征研究

目的 分析湖南省2006-2009年4例人感染高致病性禽流感病例感染病毒的可能来源、基因重配情况以及分子特征.方法 鸡胚分离核酸检测H5N1病毒阳性标本,获得高致病性H5N1病毒,对病毒进行全基因组序列测定,采用BLAST和MEGA4.0进行同源性比对、基因进化分析和各基因分子特征分析.结果 4株病毒的基因片段均为禽源,并未发现与人季节性流感病毒之间发生重配,且与当地禽类中分离的病毒高度同源.全基因组进化树分析显示,4株病毒在分支2.3.4中,2株为基因型V、2株为新的基因型.分子特征分析显示,4株病毒的血凝素(HA)分子裂解位点均为PLRERRKR/G,均出现A160T位点突变,神经氨酸酶(NA)分子49～68位均出现20个氨基酸(aa)缺失,非结构蛋白1(NSI)分子80～84位均出现5个aa的缺失.在HA分子大部分位点,4株病毒仍然表现出与禽类受体的亲和性,HN/1/09和HN/2/09出现可能使病毒对α-2,6连接的唾液酸人类受体的亲和性增强的T192I突变.HN/1/08的PB2基因出现增加小鼠致病力的D701N改变.耐药性基因片段分析显示,4株病毒对金刚烷胺和奥司他韦均敏感.结论 2006-2009年湖南省4株人感染高致病性禽流感病毒(H5N1)为禽源,但存在多种基因型,而且发生了部分位点的突变.

Abstract：

Objective To understand the possible origins,genetic re-assortment and molecular characterization of 4 highly pathogenic avian influenza A(H5N1)viruses isolated from humans in Hunan province,between 2006 and 2009,Methods H5N1 PCR test-positive specimens were inoculated in embryonated eggs while H5N1 virus was isolated and genomes sequenced.Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 4.0.Results All gene segments of the 4 viruses were avian in origin.No re-assortment was found between avian influenza A(H5N1)viruses and human seasonal influenza viruses.Virnses that isolated from domestic poultry shared high similarity with the 4 human viruses in gene homology.Data from the whole genome phylogenetic analysis showed that the 4 viruses were in clade 2.3.4,while 2 viruses belonged to genotype V,and another 2 were new genotypes.Results from molecular characterization showed that amino acid sequences of HA cleavage site of the 4 viruses were PLRERRKR/G.All 4 viruses had A160T mutation in HA,a 20 amino acid deletion in the neuraminidase(NA)stalk at position 49-68,and a 5 amino acid deletion in the non-structural protein 1(NS1).Most sites in the HA molecules showed that the viruses preferentially bound to avian influenza virus receptor.However,T192I mutation that might enhance the α2,6-linked sialic acid human influenza receptor binding had emerged in HN/1/09 and HN/2/09.D701N mutation of PB2 that increased the virulence in mice was found in HN/1/08.Analysis on drug resistance gene amino acid showed that all 4 viruses were sensitive to amantadine and oseltamivir.Conclusion Highly pathogenic avian influenza A(H5N1)viruses isolated from humans in Hunan province from 2006 to 2009 were avian in origin,and the 4 viruses belonged to different genotypes.Some mutations that related to virulence and receptor binding positions had emerged in some of the strains.