高致病性禽流感H5N1病毒研究进展

1997年8月发现首例人禽流感病例以来,不断有人禽流感病例发生,截至2007年3月1日,WHO报道的经实验室确证的H5N1禽流感感染者共277例,死亡167例,死亡率超过50%。目前WHO对禽流感的预警是3级,即是一种新的流感亚型,已经在人类中引起严重疾病,但还没有在人类中持续传播流行。本文就目前H5N1禽流感病毒来源,跨越物种传播的机制,致病力决定因素及人间传播能力等几个关键的基础问题研究进展进行综述。     

高致病性禽流感H5N1病毒研究进展

1997年8月发现首例人禽流感病例以来,不断有人禽流感病例发生,截至2007年3月1日,WHO报道的经实验室确证的H5N1禽流感感染者共277例,死亡167例,死亡率超过50％。目前WHO对禽流感的预警是3级,即是一种新的流感亚型,已经在人类中引起严重疾病,但还没有在人类中持续传播流行。本文就目前H5N1禽流感病毒来源,跨越物种传播的机制,致病力决定因素及人间传播能力等几个关键的基础问题研究进展进行综述。     

中国大陆首例人感染高致病性禽流感病毒(H5N1)病例的实验室诊断

目的 对2005年10月湖南省湘潭市湘潭县发生的一名不明原因肺炎病例进行实验室检测,以确定导致该病例的主要病因.方法 采集病例呼吸道标本以及血清标本,对呼吸道标本利用分子鉴别诊断技术以及RT-PCR和实时荧光定量RT-PCR检测病毒核酸;通过血凝抑制试验以及微量中和试验检测血清中的特异性抗体.结果 该病例所有的呼吸道标本H5N1病毒特异性核酸及病毒分离均为阴性.红细胞凝集抑制及微量中和实验显示,恢复期血清较急性期血清H5N1特异性抗体阳转并且有4倍以上增高.结论 通过实验室检测结果分析,该病例为中国大陆第一例人感染高致病性禽流感病毒（H5N1）实验室确诊病例.     

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。     

我国分离人H5N1禽流感病毒血凝素基因特性的研究

目的 分析我国分离的人高致病性禽流感H5N1病毒的抗原性以及基因特性.方法 对所分离的H5N1病毒的血凝素基因和神经氨酸酶基因进行序列测定.结果 对血凝素基因的序列测定结果表明从我国分离的H5N1病毒具有较高的同源性,但是与越南、泰国等国家分离的H5N1病毒明显不同.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 目前我国分离的H5N1病毒属于同一个组,与泰国、越南分离毒株不同,并且发现病毒的特性仍然是禽源的,没有发现从禽到人的重组或者重配。     

广东地区人禽流感H5N1毒株的核蛋白基因变异和进化

目的通过对人禽流感H5N1毒株核蛋白（NP）基因序列的变异分析，揭示毒株NP基因变异与进化。方法检测广东地区人禽流感H5N1毒株NP基因核苷酸序列，同时检索全球人禽流感H5N1毒株NP基因序列，采用DNAStar5．0软件对检索的人禽流感H5N1毒株NP基因核苷酸序列进行比对和分析；并结合流行病学资料对变异毒株进行进化速度分析。结果1997--2006年45株毒株胛基因核苷酸序列同源性分成3类，1997--1998年毒株为第1类，2004--2005年毒株为第2类，2003年毒株和2006年毒株为第3类；NP基因35个氨基酸位点全部置换，占7．03％（35／498）；2003—2006年H5N1毒株通过氨基酸第430位（1〈430T）位点的置换，增加一个糖基化位点（NGT430-432；GD—01—06毒株第370位发生N3加S变异；增加一个糖基化位点（NES368.370）。同义变异中，NP基因Ks（同义变异速度）值为2．03×10^-5～2．55×10^-5核苷酸／d，Ka（错义变异速度）值为1．58×10^-6～3．10×10^-6核苷酸／d，检验发现进化无明显选择性压力存在。结论1997--1998年毒株、2004--2005年毒株、2003年毒株和2006年毒株NP基因核苷酸有差异；2003--2006年人禽流感H5N1毒株NP基因增加一个糖蛋白位点、GD-01—06毒株再增加一个糖蛋白位点可能改变毒株抗原性。人禽流感H5N1毒株在自然界变异频繁，随着其自然进化，H5N1毒株具有人．人传播能力的概率较大。     

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。     

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.     

PDCA模式在收治甲型H1N1流感管理中的应用

目的为控制甲型H1N1流感疫情,探讨了收治甲型H1N1流感的管理模式。方法应用计划、执行、检查、行动（Plan,Do,Check,Action,PDCA）循环法建立收治甲型H1N1流感管理制度,根据疫情收集资料、分析原因、制定计划、组织实施、检查管理。结果依照我国卫生部政策,完善甲型H1N1流感管理制度,做好消毒隔离,保证医疗质量,人性化管理隔离病人,有效控制了疫情流行。结论导入PDCA循环法,坚持计划、实施、检查、处理,循序渐进,是收治新发传染病管理行之有效的方法。     

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。     

Application of single radial hemolysis technique for diagnosis of influenza A (H5N1)]

BACKGROUND: To understand the optimal condition of single radial hemolysis (SRH) for diagnosis of avian influenza A (H5N1) virus in order that SRH could be performed in general laboratories. METHODS: The effect of different concentration of virus and species of red blood cells, as well as kind and concentration of agarose on testing sensitivity of SRH was determined. Meanwhile the sensitivity and specificity of this method were compared with those of micro-neutralization test. RESULTS: The optimal condition of SRH included the viral concentration of 1000 HA units per 0.1 ml packed chicken red blood cells, the agarose concentration of 1.0%, the compliment added into agarose-virus-rbc slides after diffusion of sera. The sensitivity and specificity of SRH were very similar to those of micro-neutralization test. Meanwhile, no cross reaction between antibodies, especially antibodies against N1 antigens, H5N1 and H1N1 viruses was detected. CONCLUSION: The sensitivity and specificity of SRH were very similar to those of micro-neutralization assay. SRH could be performed in normal laboratories and be used for testing large scale serum samples.     

Transmission of influenza A/H5N1 viruses in mammals

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans and cause severe respiratory disease and fatalities. Currently, these viruses are not efficiently transmitted from person to person, although limited human-to-human transmission may have occurred. Nevertheless, further adaptation of avian H5N1 influenza A viruses to humans and/or reassortment with human influenza A viruses may result in aerosol transmissible viruses with pandemic potential. Although the full range of factors that modulate the transmission and replication of influenza A viruses in humans are not yet known, we are beginning to understand some of the molecular changes that may allow H5N1 influenza A viruses to transmit via aerosols or respiratory droplets among mammals. A better understanding of the biological basis and genetic determinants that confer transmissibility to H5N1 influenza A viruses in mammals is important to enhance our pandemic preparedness.     

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.     

Efficacy of inactivated vaccines against H5N1 avian influenza infection in ducks

The current Asian H5N1 highly pathogenic avian influenza virus has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenza viruses in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild water birds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use. In this report, a bivalent H5N9+H7N1 oil emulsion vaccine is compared, in ducks, to a monovalent H5N3 oil emulsion vaccine that has been derived by reverse genetics with an H5 from A/chicken/Vietnam/C58/04. While both vaccines protected against morbidity, the monovalent vaccine provided effective protection, with no evidence of shedding of the challenge virus and no serological response to the H5N1 challenge virus.     

Pandemic influenza A(H1N1)pdm09: risk of infection in primary healthcare workers

Background

Healthcare workers in primary care are at risk of infection during an influenza pandemic. The 2009 influenza pandemic provided an opportunity to assess this risk.

Aim

To measure the prevalence of seropositivity to influenza A(H1N1)pdm09 among primary healthcare workers in Canterbury, New Zealand, following the 2009 influenza pandemic, and to examine associations between seropositivity and participants’ sociodemographic characteristics, professional roles, work patterns, and seasonal influenza vaccination status.

Design and setting

An observational study involving a questionnaire and testing for influenza A(H1N1)pdm09 seropositivity in all primary healthcare workers in Canterbury, New Zealand between December 2009 and February 2010.

Method

Participants completed a questionnaire that recorded sociodemographic and professional data, symptoms of influenza-like illness, history of seasonal influenza vaccination, and work patterns. Serum samples were collected and haemagglutination inhibition antibody titres to influenza A(H1N1)pdm09 measured.

Results

Questionnaires and serum samples were received from 1027 participants, from a workforce of 1476 (response rate 70%). Seropositivity was detected in 224 participants (22%). Receipt of seasonal influenza vaccine (odds ratio [OR] = 2.0, 95% confidence interval [CI] = 1.2 to 3.3), recall of influenza (OR = 1.9, 95% CI = 1.3 to 2.8), and age ≤45 years (OR = 1.4, 95% CI = 1.0 to 1.9) were associated with seropositivity.

Conclusion

A total of 22% of primary care healthcare workers were seropositive. Younger participants, those who recalled having influenza, and those who had been vaccinated against seasonal influenza were more likely to be seropositive. Working in a dedicated influenza centre was not associated with an increased risk of seropositivity.     

The avian and mammalian host range of highly pathogenic avian H5N1 influenza

Highly pathogenic H5N1 influenza viruses have been isolated from a number of avian and mammalian species. Despite intensive control measures the number of human and animal cases continues to increase. A more complete understanding of susceptible species and of contributing environmental and molecular factors is crucial if we are to slow the rate of new cases. H5N1 is currently endemic in domestic poultry in only a handful of countries with sporadic and unpredictable spread to other countries. Close contact of terrestrial bird or mammalian species with infected poultry/waterfowl or their biological products is the major route for interspecies transmission. Intra-species transmission of H5N1 in mammals, including humans, has taken place on a limited scale though it remains to be seen if this will change; recent laboratory studies suggest that it is indeed possible. Here we review the avian and mammalian species that are naturally susceptible to H5N1 infection and the molecular factors associated with its expanded host range.     

Altered hemolysis in single radial hemolysis from a single serum sample as an indicator of recent primary rubella virus infection

The quality of hemolysis in the single radial hemolysis (SRH) test was observed to be altered in a small proportion (5.7%) of Sera (N = 9628) studied for rubella antibodies. Three different types of altered hemolysis were identified. Two of these types, the “soft margin” (SM) or the “soft zone” (SZ), occurred singly or in combination in 97% of paired sera (N = 321) taken 3–30 days after primary rubella infection (diagnostic seroconversion). For comparison, diagnostic inercases of rubella antibodies (N = 77), including both primary and reinfections, contained these types of altered hemolysis in 80% of the cases. Of the remaining 20% of the samples (N = 15) rubella antibodies of IgM class were looked for in seven cases but not found. After primary rubella, SZ was always present in the first serum sample and disappeared rapidly within 20 days while SM persisted longer. The predictive value of the different types of hemolysis was estimated from 96 primary sera, which had altered hemolysis. In this material, SZ was followed by a diagnostic increase of antibodies in 87% of the cases; the prognostic value of the SM was significant but lower. These data show that the appearance of SM or SZ in a single serum sample is a useful marker of recent primary rubella. On the other hand, a normal hemolysis in SRH can be used to exclude recent primary rubella, but not reinfection, with a high degree of probability.     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.