贵州省新型甲型HIN1(2009)流感病毒HA-1基因分析

目的 建立并完善贵州省新型甲型H1N1流感病毒基因分析检测技术平台,了解贵州省新型甲型H1N1(2009)流感病毒HA1片断与WHO公布的流行株有否发生变异.方法 收集16株新型甲型H1N1流感病毒毒株,提取病毒核酸RNA,通过RT-PCR扩增HA1片断,用电泳观察PCR产物目的条带大小,经过纯化、测序,用Biodeit、MEGA4相关软件进行序列比对,同源性及差异性分析,构建系统发育树.结果 贵州省(2009)代表株与中俄代表株A/Habarovak/01/2009 (H1N1)和墨西哥第一株甲型H1N1流感A/Mexio City/001/2009(H1N1)同属一个谱系,亲缘关系较近,同源性在93.2％～95.8％之间.结论 贵州省(2009)代表株与WHO公布的流行株在核苷酸水平上有一定程度的变异.     

某区甲型H1N1流感病毒分离鉴定及同源性的分析

目的检测并分离甲型H1N1流感病毒,对开封地区首次分离到的病毒株进行全基因组序列测定及同源性分析,为研究流感病毒的流行及变异规律提供科学依据。方法采用Real-time RT-PCR方法检测,筛选确定出甲型H1N1流感病毒阳性标本;利用狗肾传代细胞分离得到甲型H1N1流感病毒株A/Kaifeng/01/2009(H1N1);测定并分析其全基因组序列;利用序列比对进行了同源性分析。结果从1828份流感样病例中检出甲型H1N1流感病毒阳性标本286份,阳性率15.6%。在开封地区首次获得甲型H1N1流感病毒株及全基因组序列。基因组序列分析表明:该毒株与2009年大流行株高度同源,为同一进化分支。与以往流行的猪流感病毒株对比发现,HA基因有12个碱基发生了点突变。结论 MDCK细胞对甲型H1N1流感病毒具有较高敏感性;开封地区首例甲型H1N1流感病例分离病毒株与北美流行株高度同源;相对于以往古典型猪流感代表株出现了HA蛋白抗原性漂移;为今后进一步开展甲型H1N1流感病毒分子生物学研究奠定基础。     

一清胶囊抗甲型H1N1流感病毒作用

目的 研究一清胶囊抗甲型H1N1流感病毒作用。方法 采用小鼠气管内注入甲型流感病毒鼠肺适应株A/PR/8/34(H1N1)病毒液建立病毒感染小鼠模型,以肺指数、肺指数抑制率、小鼠累积死亡率为指标,评价一清胶囊体内抗甲型H1N1流感病毒作用。结果 7.3 g·kg^-1一清胶囊(原生药)可降低病毒感染小鼠的肺脏指数(P<0.05)和小鼠感染病毒后7~9 d内的死亡率(P<0.01)。结论 一清胶囊具有明显的抗甲型H1N1流感病毒作用。     

甲型H5N1和甲型H9N2流感病毒的抗原性和遗传学特征以及可能用于人类疫苗的候选疫苗病毒的研制

此文概述了2010年2月17日-9月26日甲型H5N1和甲型H9N2流感疫情、甲型H5N1流感病毒的抗原性和遗传学特征以及可用于人类疫苗的病毒研制状况.     

甲型H1N1流感病毒疫苗接种的安全性分析

目的:探讨甲型H1N1流感病毒疫苗接种的安全性,为预防接种提供科学依据.方法:随机选取2009年11月～2010年2月在我县疾病预防控制中心接种甲型H1N1流感病毒疫苗的中小学生4 980名进行临床指标监测,对出现不良反应进行分析.结果:在4 980名接种者中51例出现不良反应,发生率1.02%,其中1级不良反应44例(86.27%),2级不良反应7例(13.73%),无3级及4级不良反应发生,不良反应均能在1～3 d自行缓解或治愈,未出现有临床意义的严重不良事件.结论:甲型H1N1流感病毒疫苗不良反应低,可以安全接种.     

甲型H5N1和甲型H9N2流感病毒的抗原性和遗传学特征以及可能用于人类疫苗的候选疫苗病毒的研制

此文概述了2010年2月17日-9月26日甲型H5N1和甲型H9N2流感疫情、甲型H5N1流感病毒的抗原性和遗传学特征以及可用于人类疫苗的病毒研制状况.     

重症甲型H1N1流感1例临床分析

甲型H1N1流感为一种新型呼吸道传染病,其病原菌为新甲型H1N1流感病毒株,病毒基因中包含有猪流感、禽流感和人流感3种流感病毒的基因片段。2009年12月我院收治了重症甲型H1N1流感患儿1例,现将该病的救治与药学监护报道如下。     

重症甲型H1N1流感影像学表现初步分析

甲型H1N1流感是一种新型的甲型H1N1流感病毒引起的急性呼吸道传染病,目前已波及全球各大洲.我国已将甲型H1N1流感纳入<中华人民共和国传染病防治法>规定的乙类传染病,并采取甲类传染病的预防、控制措施[1].到目前为止,我国内地有关该病影像学表现的报道尚少.笔者收集有完整临床和影像学资料的17例甲型H1N1流感重症患者进行总结,重点探讨其影像学特点.     

FDA批准首个非鸡胚培养的重组疫苗Flublok

<正>2013年1月16日,美国FDA批准世界上首个完全未使用流感病毒与鸡胚培养的重组疫苗Flu-blok上市,用于预防18～49岁人群的季节性流感。Flublok是一种新型的蛋白疫苗,含三个全长的重组血凝素(HA)蛋白,即甲型H1N1流感病毒HA蛋白、甲型H3N2流感病毒HA蛋白和乙型流感病毒     

上海地区人群甲型H1N1流感抗体水平监测分析

目的 了解上海地区不同年龄组人群甲型H1N1流感血清抗体水平,对人群中甲型H1N1与2008-2009年季节性流感疫苗株A/Brisbane/59/2007(H1N1)的血清抗体水平进行分析比较.方法 应用常规微量血凝抑制试验(micro-hemagglutination inhibition test,HI)对上海地区不同年龄组(0～5月龄、6月龄～4岁、5～24岁、25～59岁和≥60岁)人群进行甲型H1N1流感抗体检测.各年龄组抗体阳性率的比较采用Pearson X2检验和Fisher确切概率法进行分析.结果 上海地区人群甲型H1N1流感血清抗体总阳性率为9.2%(37/404).不同年龄组中以老年人组(≥60岁)的血清抗体阳性率最高,为25.0%(21/84);其次是成年人组(25～59岁),血清抗体阳性率为10.0%(8/80);其他年龄组血清抗体阳性率较低.Pearson X2检验结果显示不同年龄组之间抗体阳性率差异有统计学意义.在2008-2009年季节性流感疫苗株A/Brisbane/59/2007(H1N1)抗体阳性的329份血清中有31份甲型H1N1流感抗体阳性,季节性疫苗株抗体阴性的75份血清中有6份甲型H1N1流感抗体阳性.经Pearson X2检验分析,这两层人群甲型H1N1流感抗体阳性率差异没有统计学意义.结论 上海地区人群血清中甲犁H1N1流感抗体水平普遍偏低,其中老年人组抗体水平最高,人群对甲型H1N1流感病毒普遍高度易感;季节性疫苗株A/Brisbane/59/2007(H1N1)HI抗体阳性人群血清不能对甲型H1N1流感病毒产生交叉保护抑制.     

The 2008-2009 H1N1 influenza virus exhibits reduced susceptibility to antibody inhibition: Implications for the prevalence of oseltamivir resistant variant viruses

A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008-2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.     

人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性研究

 目的   比较人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性,为该疫苗的类型选择提供初步依据。 方法   采用相同血凝素含量（5 μg）的H7N9全病毒灭活和裂解疫苗（含或不含氢氧化铝佐剂共4种类型）,分别对BALB/c小鼠进行1针或2针免疫。免疫后,用血凝抑制（hemagglutination inhibition,HI）试验检测血清抗体滴度,比较不同类型疫苗的免疫效果。 结果   小鼠免疫1针全病毒灭活疫苗后,全部血清阳转,HI抗体几何平均滴度（geometric mean titer,GMT）为149;免疫2针后,抗体GMT为243。小鼠免疫1针裂解疫苗后无抗体阳转;免疫2针后全部抗体阳转,GMT为139。两种疫苗添加铝佐剂后,诱导的HI抗体GMT仅略有增加。 结论   H7N9全病毒灭活疫苗在小鼠中的免疫原性较强。在同样类型和剂量的情况下,裂解疫苗需要免疫两次才能达到与全病毒疫苗相同的效果。铝佐剂对免疫原性提升不明显。     

抗H7N9流感病毒中和抗体快速检测方法的建立及应用

目的：对建立的抗H7N9流感病毒中和抗体快速检测方法进行方法学验证及初步应用。方法：分别采用不同代次细胞对高、中、低不同滴度的阳性血清进行多次平行检测,考察细胞代次对检验结果的影响;采用NIBSC提供的参考品对方法学的特异性进行验证;同时应用抗H7N9的阳性血清检测进一步评估方法的准确性和精密性;采用ELISA-MNT法和血凝抑制（hemagglutination inhibition,HI）试验分别接种H7N9灭活流感疫苗免疫的小鼠血清样本20份,分析两种方法检测结果的相关性。结果：采用ELISA-MNT中和法,使用不同代次MDCK细胞（25、30和35代）检测相同的血清样本的中和抗体滴度结果相同;该方法只对羊抗H7N9的血清具有较高保护力,对其他血清基本没有交叉反应;该方法准确性良好;该方法组间、组内精密性的平均变异系数分别为4%和11%。该方法测定H7N9型流感疫苗免疫后的小鼠血清抗体效价,其结果与HI抗体的相关系数为0.61,表明两种方法的检测结果之间呈良好的正相关性。结论：建立的微量病毒中和法能够满足H7N9流感病毒中和抗体效价检测的要求,可用于H7N9新型大流行流感疫苗的免疫评价。     

H9N2禽流感病毒血凝素和神经氨酸酶DNA疫苗对小鼠的免疫保护研究

目的 检测H9N2禽流感病毒血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)DNA疫苗保护小鼠抵抗致死量同源病毒感染的能力.方法 通过小鼠肺对肺传代,建立禽流感病毒A/chicken/Jiangsu/07/2002(H9N2)小鼠适应株.同时,构建病毒HA和NA DNA疫苗,以不同剂量电击法免疫小鼠1或2次,在初次免疫后4周或加强免疫后1周用致死量(40 LD50)鼠适应型病毒攻击小鼠.通过测定小鼠血清抗体滴度、小鼠存活率和肺部病毒滴度来评价疫占的效果.结果 HA或NADNA 10μg免疫1次或3μg免疫2次均可保护小鼠抵抗致死昔H9N2病毒的感染.结论 低剂量HA或NA DNA可为抗H9N2禽流感病毒感染提供有效的免疫保护.     

对禽流感H5N1灭活疫苗不同免疫方式诱导的免疫应答及异亚型保护研究

目的 观察禽流感H5N1灭活疫苗以不同方式免疫后诱导的免疫应答及抗异亚型病毒攻击的保护作用.方法 将H5N1灭活疫苗分别通过腹腔和滴鼻方式免疫BALB/c小鼠,同时以PBS作为对照;免疫后分别以PR8和H9N2病毒攻击,观察小鼠的体重变化和生存情况.采用ELISA对各组小鼠攻毒后不同时间的血清IgG及其亚类水平进行动态检测;流式细胞仪检测脾淋巴细胞亚群情况.采用t检验对各组数据进行比较.结果 PR8和H9N2病毒攻击后,各组小鼠体重均下降,但疫苗组小鼠于后期体重恢复正常,存活率分别为100%和70%-80%,而PBS组小鼠则全部死亡.无论以何种方式免疫,疫苗组的特异性IgG及其亚类水平均明显升高,其中以IgG2a水平升高更为明显.攻毒后疫苗组小鼠脾CD4+与CD8+T淋巴细胞比值出现下降(t=6.8017,P<0.01);滴鼻免疫组与腹腔免疫组相比降低更明显(t=3.9701,P<0.05).结论 H5N1疫苗免疫原性良好,可诱导较高水平的特异性抗体产生,诱导的抗体亚类以IgG2a为主.两种免疫方式均可对小鼠提供很好的异亚型保护,而滴鼻免疫能够诱导更强的CD8+T细胞应答.

Abstract：

Objective To observe immune responses and heterosubtypic protection elicited by an inactivated influenza H5N1 vaccine with different immunization routes in mice. Methods BALB/c mice were intraperitoneally injected or intranasally immunized with the inactivated H5N1 vaccine, the mice administered with PBS were used as control. Weight loss and survival in mice were observed after PR8 or a H9N2 virus challenge. Serum specific IgG antibody and its subclasses were detected by ELISA kits. Ratios of CD4+ /CD8+ lymphocytes in spleens of mice were assayed. The t-test was used in the comparison of different groups.Results After challenge, weight loss was found in all groups, but the weight of mice in vaccination groups returned to normal later. The mice in PBS groups all died. The vaccinated mice were completely protected against PR8 and partly protected against H9N2 virus. The level of IgG antibody increased significantly after vaccination, and the increase magnitude of IgG2a was higher than that of IgG1. The ratios of CD4+ /CD8+ lymphocytes in spleens of vaccinated mice decreased after challenge (t=6. 8017,P<0. 01) , especially in the intranasal group (t = 3. 9701, P < 0. 05 ). Conclusions Both intraperitoneal injection and intranasal immunization can induce a high level of specific IgG, especially the level of IgG2a, and provide protection against heterosubtypic viruses. Intranasal immunization seems to induce a higher level of CD8+ T cell response.     

禽流感H5N1灭活疫苗与不同纳米化佐剂联合免疫研究

目的 观察禽流感H5N1灭活疫苗加不同佐剂以及纳米化佐剂免疫小鼠后产生的免疫应答的差异,同时观察免疫对异亚型病毒攻击后的保护情况.   方法 用H5N1灭活疫苗分别联合佐剂氢氧化铝、纳米化氢氧化铝、MF59和纳米化MF59通过腹腔注射方式免疫雌性BALB/c小鼠,同时分别以H5N1灭活疫苗免疫小鼠以及PBS腹腔注射处理小鼠作对照.采用ELISA方法分别对各组小鼠免疫后血清特异性IgG及其亚类IgG1、IgG2a水平进行检测,以PR8病毒鼻腔攻击后观察小鼠体重变化情况和生存率.采用t检验作组间比较.    结果 与PBS处理组相比,无论以何种方式免疫H5N1疫苗,免疫后特异性IgG及其亚类水平均明显升高(t=7.4004,P<0.01),以联合MF59后诱导的特异性抗体水平最高.其中疫苗单独免疫组和联合M59免疫组IgG2a水平升高明显,联合纳米化MF59免疫小鼠后IgG2a水平有所下降,IgG1水平有所升高;联合铝佐剂组以IgG1升高为主.攻毒后各组小鼠体重均出现下降,但疫苗单独免疫小鼠以及疫苗与佐剂联合免疫小鼠于攻毒后期体重恢复接近正常或者正常.PBS处理组小鼠攻毒后全部死亡,佐剂联合免疫组小鼠存活率100%,而疫苗单独免疫小鼠存活率为70%.   结论 H5N1疫苗无论是单独免疫或是联合不同佐剂免疫均可诱导较高水平的特异性抗体产生,有非常好的免疫原性.H5N1灭活疫苗诱导的抗体亚类以IgG2a为主,MF59以及纳米化MF59也以诱导IgG2a亚类为主,但纳米化MF59可诱导更均衡的免疫应答.联合铝佐剂或纳米化铝佐剂免疫小鼠后均可诱导以IgG1亚类为主的抗体应答.联合两种佐剂均可对小鼠产生很好的异亚型保护.     

A/H5N1 prepandemic influenza vaccine (whole virion, vero cell-derived, inactivated) [Vepacel®

The influenza A subtype H5N1 virus is a likely causative agent for the next human influenza pandemic. Pandemic influenza vaccine production can begin only after a novel pandemic virus emerges. Cell-based vaccine production has advantages over conventional egg-based methods, allowing more rapid large-scale vaccine production. A reliable Vero cell culture system is available for pandemic and prepandemic influenza vaccine production. Prepandemic influenza vaccines are an important component of influenza pandemic preparedness plans, as their targeted use in the pandemic alert period or early in a pandemic is likely to mitigate the consequences of an influenza outbreak. Vepacel? is a prepandemic influenza vaccine (whole virion, Vero cell-derived, inactivated) containing antigen of H5N1 strain A/Vietnam/1203/2004 and is approved for use in the EU. Clinical immunogenicity studies with the vaccine have demonstrated good rates of functional neutralizing antibody responses against the vaccine strain (A/Vietnam/1203/2004), meeting established immunogenicity criteria for seasonal influenza vaccines, and cross-reactivity against H5N1 strains from other clades. In phase I/II and III studies, a heterologous (A/Indonesia/05/2005) booster vaccine administered to healthy adult and elderly volunteers 6-24 months after the two-dose priming vaccine (A/Vietnam/1203/2004) regimen induced good immunogenic responses against both H5N1 strains, demonstrating strong immunological memory. Broadly similar, albeit less robust, responses were observed in two special risk cohorts of immunocompromised and chronically ill patients. In general, adverse events observed in clinical immunogenicity studies with H5N1 vaccine (A/Vietnam/1203/2004) were similar to those reported with non-adjuvanted, inactivated, seasonal influenza vaccines.     

Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector

H5N1 influenza viruses have caused significant disease and deaths in various parts of the world in several species, including humans. Vaccination combined with culling can provide an attractive method for outbreak containment. Using synthesized oligos and overlapping extension PCR techniques, we constructed an H5 HA gene, optiHA, containing chicken biased codons based on the HA amino acid sequence of the highly pathogenic H5N1 virus A/goose/Guangdong/1/96 (GS/GD/96). The optiHA and wild-type HA genes were inserted into plasmids pCI or pCAGGS, and designated as pCIoptiHA, pCAGGoptiHA, pCIHA and pCAGGHA, respectively. To evaluate vaccine efficacy, groups of 3-week-old specific pathogen free (SPF) chickens were intramuscularly injected with the four plasmids. Sera were collected on a weekly basis post-vaccination (p.v.) for hemagglutination inhibition (HI) assays and neutralization (NT) antibody detection. All chickens receiving pCAGGoptiHA and pCAGGHA developed high levels of HI and NT antibodies at 3 weeks p.v., and were completely protected from lethal H5 virus challenge, while only partial protection was induced by inoculation with the other two plasmids. A second experiment was conducted to evaluate if a lower dose of the pCAGGoptiHA vaccine could be effective, results indicated that two doses of 10 microg of pCAGGoptiHA could induce complete protection in chickens against H5 lethal virus challenge. Based on our results, we conclude that construction optimization could dramatically increase the H5 HA gene DNA vaccine efficacy in chickens, and therefore, greatly decrease the dose necessary for inducing complete protection in chickens.     

Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1

EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice.