1株H5N6亚型禽流感病毒分子特征分析

目的 对从某活禽市场环境中分离出的1株H5N6亚型禽流感病毒(A/environment/Zhenjiang/C13/2013,en/c13)进行全基因组序列测定,以揭示其基因起源和分子遗传特征。 方法 采用二代测序技术对该分离株进行全基因组测序,MEGA6.0软件进行序列比对和构建系统发生树。 结果 分离株NA基因与近年来中国禽类中流行的H6N6亚型禽流感的NA基因处于同一进化分支,其他7个基因(PB2、PB1、PA、HA、NP、M和NS)都与近年来东亚地区流行的H5N1亚型禽流感的相应基因处于同一进化分支,分离株en/c13的HA蛋白裂解位点为PLREKRRKR↓GLF,是高致病性禽流感病毒的典型分子特征序列。HA蛋白上关键受体结合位点226和228位(按照H3亚型的HA蛋白序列排序)氨基酸分别是Gln和Gly,是典型的禽流感病毒受体结合特征。 结论 本研究从环境中分离到一株新的基因重配禽流感病毒,其中NA基因来源于H6N6禽流感病毒,而其他7个基因则来源于H5N1禽流感病毒。结果提示我国禽流感病毒基因重配频繁,进化活跃,需要加强对禽流感病毒的持续监测。     

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。     

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。     

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.     

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HATh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。     

高致病性人禽流感H5N1病毒HA与M1蛋白共表达制备新型病毒样颗粒

目的 制备由H5N1亚型人禽流感病毒血凝素(HA)和基质蛋白1(M1)两种结构蛋白组装而成的H5亚型流感病毒样颗粒(viruslike particles,VLPs) 并检测其生物活性。 方法 构建同时包含A/Indonesia/05/2005(H5N1) HA和A/Anhui/01/2005(H5N1) M1基因的重组转移载体pFBD-M1-HA,筛选获得重组杆粒(重组杆状病毒质粒)rBacmid-M1-HA后,转染 Spodoptra frugiperda (Sf9)昆虫细胞,包装重组杆状病毒rBV-M1-HA;Western blot和间接免疫荧光鉴定HA和M1的表达;大量制备并纯化后,在透射电镜下观察VLPs的形态结构;血凝试验检测VLPs的生物活性。 结果 应用昆虫细胞-杆状病毒表达系统表达H5N1 HA和M1两种结构蛋白可以高效组装产生流感VLPs,纯化后的VLPs血凝效价为1024 HAU/50 μl。 结论 成功利用A/Indonesia/05/2005(H5N1)来源HA和A/Anhui/01/2005(H5N1)来源M1两种结构蛋白,在杆状病毒表达系统中制备有较好生物活性的人禽流感病毒H5亚型VLPs,为下一步研究人H5亚型禽流感VLPs疫苗奠定基础。     

抗H5N1禽流感病毒VHH抗体库的构建

目的:构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链可变区抗体库（VHH型抗体库）,为抗H5N1的VHH抗体筛选奠定基础。方法:利用H5N1灭活疫苗免疫小羊驼,一定免疫时间后测定小羊驼外周血清中抗体中和活性,分离其外周淋巴细胞,利用RT-PCR方法得到VHH抗体片段。通过优化连接和电转化方法,将足量VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1,获得VHH抗体基因库;检测基因库库容以及多样性,并采用血凝抑制试验对噬菌体抗体库进行初步功能性鉴定。结果:利用H5N1灭活疫苗免疫小羊驼四次后,其外周血清中抗体血清抑制效价可达1∶2 560,构建的VHH抗体基因库库容可达3×108,随机挑选14个抗体基因克隆进行测序鉴定,结果显示均为独立克隆,表明所建抗体库多样性好。上述基因库经辅助噬菌体拯救后,得到抗H5N1的噬菌体VHH型抗体初级库,对初级库进行血凝抑制试验,结果呈阳性,表明初级库中存在具有潜在中和活性的抗H5N1抗体。结论:结果表明,已成功构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链抗体库,为进一步筛选抗H5N1禽流感的重链抗体打下良好基础,并为H5N1的早期临床诊断和治疗提供新的手段。     

青海湖周边家禽相关环境标本中H5N1亚型禽流感病毒特征分析

目的 了解我国青海湖周边家禽中分离到的高致病性H5N1亚型禽流感病毒的抗原性和基因特性。 方法 将青海湖周边野鸟和家禽的环境标本采集处理后,接种SPF鸡胚分离病毒,将红细胞凝集阳性的标本通过real-time RT-PCR进行型别鉴定和亚型分析。将H5N1检测阳性的标本选取部分进行二代序列分析和抗原性分析。 结果 2013年1-9月间采集的家禽相关环境标本中共分离到19株高致病性H5N1亚型禽流感病毒,集中在1-3月。对7株病毒进行了全基因组序列分析和抗原性分析显示,血凝素基因进化树显示其中除A/Environment/Qinghai/XN02032/2013属于clade7.2分支病毒以外,其余6株属于clade2.3.2.1c分支。神经氨酸酶基因也显示该毒株与其他6株分属于两个不同的分支;抗原分析显示该毒株与所有参考血清低反应,另外6株病毒是A/Barn swallow/Hong Kong/D10-1161/2010的类似株。 结论 青海湖周边地区在2013年1-3月环境相关标本中检测到高致病性H5N1亚型禽流感病毒,属于clade2.3.2.1和clade7.2分支病毒。这种高致病性H5N1亚型禽流感病毒多个分支共同流行的趋势提示加强这一地区野鸟和家禽相关环境病原体监测的重要性。     

Pathogenesis of Infectious Disease of Mice Caused by H5N1 Avian Influenza Virus

The pathogenesis of a disease caused by Qinghai-like H5N1 influenza virus in BALB/c mice was studied. Clinical, morphological, and immunological characteristics of the experimental infection caused by highly pathogenic A/duck/Tuva/01/06/ (H5N1) virus are described. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 52–55, 2008     

Morphological Changes in Bird Viscera in Experimental Infection by Highly Pathogenic H5N1 Avian Influenza Virus

Intravenous infection of chicken with H5N1 avian influenza virus (strain A/Gs/Krasnoozerskoye/627/05) causes rapid lethal outcome. Pathomorphological study of bird viscera showed manifestations of disseminated intravascular coagulation syndrome, generalized inflammatory reaction, and wide-scale necrobiotic changes in tissues. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 56–59, 2008     

Genome-sequenee Analysis of the Pathogenic H5N1 Avian Influenza A Virus Isolated in China in 2004

Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000–2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000–2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.     

禽流感H5亚型压电免疫芯片的研究

目的研究一种新型、快速的检测禽流感H5亚型的压电免疫芯片。方法以AT切型、基频10MHz的石英晶体为材料,设计四通道芯片微阵列。采用聚乙烯亚胺黏附一戊二醛交联法将禽流感H5亚型单克隆抗体固定在石英晶体的金膜电极表面制备生物敏感膜,构建可用于检测禽流感H5亚型的压电免疫芯片。结果固定H5单克隆抗体的最佳稀释度为1：10,H5亚型抗原溶液的响应值与空白对照的噪声值之比大于2。结论本次实验构建的压电蛋白芯片可用于禽流感H5亚型的定性检测。     

Vaccination of gallinaceous poultry for H5N1 highly pathogenic avian influenza: Current questions and new technology

Vaccination of poultry for avian influenza virus (AIV) is a complex topic as there are numerous technical, logistic and regulatory aspects which must be considered. Historically, control of high pathogenicity (HP) AIV infection in poultry has been accomplished by eradication and stamping out when outbreaks occur locally. Since the H5N1 HPAIV from Asia has spread and become enzootic, vaccination has been used on a long-term basis by some countries to control the virus, other countries have used it temporarily to aid eradication efforts, while others have not used it at all. Currently, H5N1 HPAIV is considered enzootic in China, Egypt, Viet Nam, India, Bangladesh and Indonesia. All but Bangladesh and India have instituted vaccination programs for poultry. Importantly, the specifics of these programs differ to accommodate different situations, resources, and industry structure in each country. The current vaccines most commonly used are inactivated whole virus vaccines, but vectored vaccine use is increasing. Numerous technical improvements to these platforms and novel vaccine platforms for H5N1 vaccines have been reported, but most are not ready to be implemented in the field.     

Utility of the rapid antigen detection BinaxNOW Influenza A&B test for detection of novel influenza A (H1N1) virus

Nasopharyngeal aspirates, collected during outbreaks, of the novel influenza A (H1N1) virus in Barcelona, were used to compare the accuracy of a rapid antigen-based test (Binax) with the real-time RT-PCR assay developed by the CDC. The sensitivity, specificity and positive predictive value of the rapid test are higher in patients less than 18 years old and during the acute stage of the epidemic than in adult patients.     

Employing XIAP to Enhance the Duration of Antigen Expression and Immunity Against an Avian Influenza H5 DNA Vaccine

DNA vaccine represents a powerful approach for prevention of avian H5N1 influenza infection. Yet, DNA vaccine-induced immune responses might be limited by the short duration of antigen expression. As a strategy to enhance adaptive immune responses elicited by a hemagglutinin 5 (H5) DNA vaccine, we explored the effect of co-administration of a DNA encoding X-linked inhibitor of apoptosis protein (XIAP) as a modulator of apoptosis and a stimulator of inflammatory signaling. In cultured cells as early as 24?hours (h), we found that the DNA vaccine encoded H5 antigen was a potent stimulator of apoptosis, and the H5 pro-apoptotic activity was significantly suppressed by the co-expression of full-length XIAP or mutant XIAP (ΔRING). However, full-length XIAP showed a higher potency than mutant XIAP (ΔRING) in the inhibition of H5-induced apoptosis. We also compared the immunizing ability of transmembrane and secretory forms of H5. Mice vaccinated (twice with 3-week intervals) with the secretory form of H5 showed higher hemagglutination inhibition (HI) antibody titers than mice vaccinated with the transmembrane form of H5. Furthermore, co-administration of XIAP with the secretory form of H5 resulted into a stronger antibody response than the transmembrane form of H5. Our findings suggest that in the design of DNA vaccines for a given pro-apoptotic antigen, using an anti-apoptotic molecular adjuvant and the secretory form of antigen may be a greater stimulus to induce immune responses.