H7N9禽流感病毒HA2基因真核表达与免疫原性初步研究

目的利用真核表达载体构建H7N9禽流感病毒血凝素刺突茎部(HA2)的真核表达质粒,在293F细胞中表达HA2蛋白,并初步探讨其免疫原性。方法根据pMD18-T-HA中的序列设计H7N9禽流感病毒HA2扩增引物,在下游引物中引入胰酶酶切位点;目的基因经特异性酶切位点克隆入自身带有Fc标签的pFUSE-IgG1-Fc1载体,构建重组质粒HA2-Fc;将重组质粒转染293F细胞,通过间接免疫荧荧光(IFA)和免疫印迹法(WB)鉴定HA2蛋白的表达和免疫原性。结果成功构建H7N9禽流感病毒HA2基因真核表达质粒HA2-Fc,并在293F细胞中表达出分子量大约为50kDa的重组蛋白。IFA和WB显示该蛋白与抗H7N9病毒鼠多抗具有良好的免疫反应。结论成功构建表达HA2亚单位的真核表达系统,重组蛋白具有较高的免疫原性,为筛选H7N9广谱疫苗候选分子、广谱中和抗体,及深入研究其致病机理和免疫机制奠定基础。     

2015-2017年贵州省威宁13株H9N2亚型禽流感病毒HA基因序列分析

目的 了解2015-2017年贵州省威宁H9N2亚型禽流感病毒的分子特征,为禽流感病毒的研究与防控提供依据。方法 对选取的13株病毒进行核酸的提取、血凝素基因(hemagglutinin,HA)的扩增和测序,运用生物信息学软件对H9N2禽流感病毒(AIV)HA基因的同源性、遗传进化、受体结合关键位点、致病相关位点和糖基化位点变异情况进行分析。结果 13株H9N2AIV的HA基因核苷酸和氨基酸同源性分别为96.1%~99.9%和95.7%~100%,均属于DK/HK/Y280/97分支。HA基因与致病性高低相关的裂解位点区域2株为PSRSNR↓GLF,11株为PSRSSR↓GLF,均属于低致病性毒株。与传播感染相关的受体结合位点均发生E198T和Q234L的突变,具有人样受体结合特征。13株毒株HA均含7个与毒力相关的糖基化位点,218位点均存在一个糖基化位点缺失,313位点均存在一个糖基化位点的增加。结论 贵州省威宁县H9N2AIVs属于DK/HK/Y280/97系,均为低致病性禽流感病毒,存在人易感位点,病毒一直处于不断的变异之中,故应加强其监测与防控。     

H5N1亚型禽流感病毒聚合酶酸性蛋白真核表达载体的构建与表达

目的构建甲型禽流感病毒H5N1亚型聚合酶酸性蛋白(PA)的真核表达载体,并表达其编码蛋白PA。方法采用RT-PCR法扩增PA基因,克隆至pMD18-T载体中构pMD18-T-PA质粒。双酶切pMD18-T-PA质粒与pXJ40-HA质粒后,胶回收并连接目的片段,构建真核表达载体pXJ40-HA-PA,鉴定后转染293T细胞。采用免疫印迹法(Western blot)鉴定重组PA蛋白的表达。结果成功构建了禽流感病毒H5N1亚型PA基因的真核表达载体,并在真核细胞中成功表达出分子量为75kD的重组蛋白。结论成功构建禽流感病毒H5N1亚型PA基因的真核表达载体,为后期进一步研究PA蛋白的功能奠定了基础。     

2016年6月至2017年4月辽宁省人感染H7N9禽流感病毒HA和NA基因特征分析

目的 本文对2016-2017年辽宁省人感染H7N9禽流感病毒血凝素(HA)和神经氨酸酶(NA)基因遗传进化特征分析。方法 使用逆转录聚合酶链反应(RT-PCR)对选取的4株H7N9禽流感病毒株HA和NA基因进行扩增并测序,用生物信息学软件分析其基因进化变异特征。结果 2016年6月至2017年4月辽宁省4株毒株的HA和NA基因与WHO最新推荐的A/Hunan/02650/2016疫苗株的同源性最高,其HA基因的核苷酸和蛋白氨基酸序列同源性分别为99.3%~99.6%,99.8%~100%;NA基因的核苷酸和蛋白氨基酸序列同源性分别为97.7%~99.7%;98.3%~99.6%。本省所有毒株均属长江三角洲谱系,但主要聚集在两个次分支。4株病毒HA蛋白裂解位点333-342氨基酸序列均为PEIPKGR↓GLF,在338-339位点无连续的多个氨基酸插入(KRTA),属于低致病性禽流感病毒分子特征。HA受体结合位点均出现S138A、G186V、Q226L、T221P氨基酸的突变,NA茎部区均出现“QISNT”缺失,且NA蛋白上相关耐药位点上的氨基酸并未发生突变,3株毒株NA蛋白上糖基化位点第42位发生氨基酸序列NCSH→NCTH突变。结论 2016-2017年辽宁省人感染H7N9禽流感病毒HA和NA基因关键位点氨基酸发生突变,使病毒具有双受体结合特性以及毒力增强。     

南宁市1株人感染高致病H7N9禽流感病毒遗传特性分析

目的通过对广西省南宁市2017年首例人感染高致病H7N9禽流感病毒株血凝素(HA)及神经氨酸酶(NA)基因测序,从分子水平分析毒株的溯源及遗传特性。方法通过RT-PCR扩增H7N9禽流感毒株HA基因和NA基因并测序,经NCBI数据库BLAST比对,利用MEGE 5.1等软件构建进化树及统计蛋白关键位点的变异情况。结果系统进化树表明中国H7N9毒株HA基因与NA基因主要为2个类群,长三角分支和珠三角地区分支,南宁毒株A/Nanning/01/2017(H7N9)HA和NA基因均在珠三角分支上,与广东毒株高度同源。病毒HA蛋白裂解位点插入4个氨基酸由PEIPKGR↓GLF突变为PEVPKRKRTAR↓GLF,含有5个碱性氨基酸,使其具有高致病性禽流感病毒的分子特征;毒力相关位点225由天冬氨酸(D)突变为甘氨酸(G)(D225G),毒力增强;受体结合186位点由甘氨酸(G)突变为缬氨酸(V)(G186V);飞沫传播关键氨基酸位点没有发生突变组合;糖基化位点高度保守。NA蛋白丢失5个氨基酸,毒力可能增强,耐药性位点、糖基化位点均相对保守,未发生突变。结论南宁市人感染H7N9禽流感病毒可能来源于广东省珠三角地区禽类的感染,人传人的可能性不大,对NA抑制剂药类敏感,但毒株已经具有高致病性禽流感病毒的分子特征,毒力增强。南宁市外环境已经检测到H7N9核酸阳性标本,提示需要加强监测有效防控传染源。     

高致病性禽流感病毒H5N1亚型核蛋白NP真核表达载体的构建、表达与鉴定

目的构建高致病性禽流感病毒H5N1亚型核蛋白(NP)的真核表达载体,并在哺乳动物细胞中表达、鉴定其编码重组蛋白NP。方法采用RT-PCR法扩增NP基因,T-A克隆到pMD18-T载体中构建pMD18-T-NP质粒。经PCR、双酶切鉴定后,双酶切阳性质粒与pXJ40-HA载体,电泳后胶回收,连接目的片段,构建pXJ40-HA-NP质粒,经PCR、双酶切、测序分析鉴定为阳性的质粒即为NP蛋白的真核表达载体。转染293T细胞后,采用免疫印迹法(Western blot)鉴定重组NP蛋白的表达。结果成功构建了高致病性禽流感病毒H5N1亚型NP基因的真核表达载体,并在293T细胞中成功表达出分子量为56kD的重组蛋白。结论成功构建的NP蛋白真核表达载体为进一步研究其功能,研发高致病性禽流感病毒H5N1亚型的诊断、治疗方法和疫苗奠定了基础。     

江苏地区7株H9N2亚型禽流感病毒HA基因的序列分析

目的了解江苏地区H9N2亚型禽流感病毒的遗传变异情况及分子生物学流行规律。方法采用逆转录聚合酶链反应(RT-PCR)技术对7株从江苏淮安地区分离的H9N2亚型禽流感病毒的血凝素(HA)基因进行扩增和测序,并对所获得的HA基因序列与该亚型的代表毒株进行同源性分析,构建系统进化树。结果 7个分离株核苷酸同源性达97%以上,与临近省份分离毒株同属国内常见的BJ94-like亚系。7株毒株HA裂解位点的氨基酸序列均为RSSR↓GLF,为低致病性禽流感病毒。结论 2014年江苏省H9N2禽流感病毒进化相对比较稳定,此7株毒株可能有着相同的起源。     

甘肃省人感染和外环境来源的H9N2禽流感病毒基因特征分析

目的 分析甘肃省发现的人感染H9N2禽流感病毒分离株全基因组特征。方法 对甘肃省2016年流感样病例中发现并确诊的1例人感染H9N2禽流感病毒病例进行病原学分析,并使用MEGA 7.0等软件解析该毒株全基因组特征。结果 该毒株HA、NA、MP、NP、NS、PA、PB1和PB2各个基因片段与甘肃省2014-2019年外环境中分离获得的H9N2禽流感病毒各基因片段高度相似,且均>90%;其HA基因属于BJ/94-like支系,PB2和MP属于G1/97-like支系,PB1、PA、NS和NP基因属于F/98-like支系,MP和PB2与H7N9、H10N8和H5N6亲缘关系较近;氨基酸序列比对发现HA裂解位点呈PSRSSR↓GLF排列,发生H183N和Q226L突变,有7个HA糖基化位点;NA茎部62～64位均缺失ITE 3个氨基酸;M2-31N、NS1-42S、PA-356R和PA-409N突变。结论 人感染H9N2禽流感病毒为偶发感染,但甘肃省外环境中分离的H9N2禽流感病毒具有一系列哺乳动物适应性分子标记,提示人群感染风险较高,需多部门加强监测,共同应对流感大流行。     

甲型流感病毒H1N1亚型NS1蛋白真核表达载体的构建与表达

[目的]构建甲型流感病毒H1N1亚型NS1蛋白真核表达载体,并在293T细胞中表达.[方法]采用RT-PCR技术,从甲型流感病毒H1N1毒株提取的病毒总RNA中,扩增NS1全长基因,将其克隆至pMD18-T Vector中构建pMD18-T-NS1质粒,双酶切pMD18-T-NS1与PXJ40-HA后,构建真核表达载体PXJ40-HA-NS1,经酶切及测序鉴定后将质粒转染到293T细胞中,通过免疫印迹法鉴定NS1蛋白的表达.[结果]酶切、测序证明重组真核表达载体PXJ40-HA-NS1构建成功,免疫印迹法可见NS1基因编码蛋白表达.[结论]成功构建甲型流感病毒H1N1亚型NS1蛋白真核表达载体PXJ40-HA-NS1,并在293T细胞中传染表达,该表达载体的构建为后期建立稳定表达NS1蛋白的细胞模型和NS1蛋白功能研究提供了材料.     

甲型流感病毒SwH1N1血凝素蛋白HA1真核表达载体的构建与表达

目的构建甲型流感病毒SwH1N1血凝素蛋白(HA1)的真核表达载体,并表达其编码蛋白HA1。方法利用RT-PCR技术扩增HA1基因,克隆至pMD18-T Simple Vector中构建pMD18-T-HA1质粒。双酶切pMD18-T-HA1与PXJ40后回收并连接回收片段,构建PXJ40-HA1真核表达载体,鉴定后转染293T细胞,用免疫印迹法(western-bolt)鉴定重组HA1蛋白的表达。结果实验成功构建HA1基因的真核表达载体,并在真核细胞中表达出分子量为40kD的重组蛋白。结论成功构建的甲型流感病毒SwH1N1HA1基因的真核表达载体,可为后期的流感快速检测及基因工程疫苗的制备奠定良好的基础。     

Induction of protective immunity in swine by immunization with live attenuated recombinant pseudorabies virus expressing the capsid precursor encoding regions of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) causes morbidity to livestock and serious economic consequences to its associated industry and therefore it is necessary to develop a safe and efficient vaccine to prevent or control this disease. A recombinant live attenuated virus vaccine, designated PRV-P1, was generated by insertion of an expression cassette containing CMV promoter, FMDV P1 gene and SV 40 poly-A into the gG gene region of a live attenuated pseudorabies virus vaccine strain (TK⁻/gG⁻/LacZ⁺). To determine the induction of protective immunity, 16 FMDV and PRV seronegative white swine were randomly divided into four groups and immunized intramuscularly. The parental virus (TK⁻/gG⁻/LacZ⁺) was injected into three pigs, the recombinant virus PRV-P1 into five pigs and commercial FMD-inactivated vaccine into five pigs, with PBS (negative control) into three pigs. All animals were immunized again 4 weeks later to boost the immune response and challenged with virulent type O FMDV O/ES/2001 strain 4 weeks after the second immunization. Results showed PRV-P1 vaccinated pigs induced high-level neutralizing antibody response to both FMDV and PRV, and strong CTL response against FMD antigen activation. Three of five pigs were completely protected against challenge with FMDV, one pig minimally protected and the other one had increased protection but not complete. However, one pig vaccinated with commercial FMD vaccine developed constant pyrexia. Average levels of antibodies against non-structural 3ABC proteins were significantly lower and efficacy on inhibition of FMDV replication was much increased in swine vaccinated with PRV-P1 than those immunized with commercial FMD vaccine after FMDV challenge. Our results showed that the recombinant PRV-P1 can induce not only humoral and cell-mediated immune responses but also partial protection against FMDV challenge, making it a good candidate for future development of the FMD vaccine.     

艾滋病高危人群中传染性疾病检测情况分析

目的：了解支滋病高危人群中HIV、HBV、HCV和TP四种病原体的感染状况。方法：于泰安市内采集四类高危人群血清，通过酶联免疫吸附实验、明胶颗粒凝集实验和胶体金实验来检测血清中的这四种传染疾病病原体的相应抗体。结果：在艾滋病高危人群2257例标本中，检测出HIV感染者0例；其它三种病原体在高危人群中的感染状况分别是HCY感染者206例（9．13％），乙肝携带者174例（7．71％），梅毒感染者70例（3．10％）。结论：在本市的支滋病高危人群中HCV、HBV和TP都有较高的感染率。     

Efficacy and immunogenicity of recombinant swinepox virus expressing the A epitope of the TGEV S protein

To explore the possibility of developing a vaccine against transmissible gastroenteritis virus (TGEV) infection, a recombinant swinepox virus (rSPV-SA) expressing a TGEV protective antigen has been constructed. Immune responses and protection efficacy of the vaccination vector were assessed in both mice and pig models. An indirect ELISA assay suggested that when mice were vaccinated with rSPV-SA, the level of IgG against TGEV was enhanced dramatically. The cytokine assays were employed and the results indicated that both the Th1-type and Th2-type cytokine levels raised after vaccination with rSPV-SA in mice models. Results from the passive immunity protection test of new born piglets demonstrated that the recombinant live-vector vaccine, rSPV-SA, could 100% protect piglets from the SPV infection, and there was no significant clinical symptom in the rSPV-SA treatment group during this experiment. The data suggest that the novel recombinant swinepox virus is a potential vaccine against TGEV infection.     

Monitoring the uptake of live avian vaccines by their detection in feathers

Protection against diseases caused by the avian viruses, Marek's disease, Infectious laryngotracheitis, chicken anemia and turkey meningoencephalitis is achieved by live vaccines. The application quality is important to assure proper uptake in commercial flocks. We describe a novel evaluation method for the vaccination process by sequential monitoring the vaccine viruses in feathers. Feather collection is easy, non-invasive and non-lethal for the birds, therefore advantageous for monitoring purposes. To demonstrate the vaccine virus presence, an innovative assay of nested real-time amplification was approached because vaccine viruses presence in vivo is less abundant comparing to virulent wild-type isolates.The Marek's disease virus vaccine virus, Rispens/CVI988, in feathers of commercial flock was detected from 4 to 7 days and for at least 3 months post-vaccination, until the survey stopped. As the drinking water route was newly adopted for Infectious laryngotracheitis vaccination, one or two vaccine doses/bird were administered. The virus uptake was detected in feathers between 2 and 20 days-post-vaccination. With a doubled vaccine dose the positivity bird rate was higher. For the first time the chicken anemia vaccine virus presence in chicken feathers was demonstrated between 14 and 35 days-post-vaccination. No previous studies were available, thus in parallel to feathers the vaccine virus was demonstrated in the livers and spleens. The turkey meningoencephalitis vaccine virus uptake in turkey feather-pulps is even more innovative because this is the first turkey virus amplified from feather-pulps. The vaccine virus presence resemble the kinetics of the other 3 viruses, 3–21 days-post-vaccination. Detecting the specific antibodies following vaccination possessed a lower sensitivity than vaccine virus demonstration in feathers. In summary, the presented assay can be adopted for the quality evaluation of the vaccination process in poultry.     

Genetic diversity of Kemerovo virus and phylogenetic relationships within the Great Island virus genetic group

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group, belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae.Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group.For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study.     

Relaxation of purifying selection on live attenuated vaccine strains of the family Paramyxoviridae

In wild-type sequences of three paramyxoviruses (measles virus, mumps virus, and Newcastle disease virus), nucleotide diversity at both non-coding sites and at nonsynonymous sites in coding regions was significantly reduced in comparison to that at synonymous sites. Likewise, both the mean and variance of gene diversity at nonsynonymous polymorphic sites were reduced in comparison to non-coding and synonymous sites. Neither of these patterns, which reflect the action of purifying selection against deleterious mutations at nonsynonymous and non-coding sites, were seen in the case of live attenuated vaccine strains, implying that purifying selection has been substantially relaxed on the latter, potentially affecting their biological properties, including antigenicity and vaccine effectiveness. Since the accumulation of mutations increases as a function of the number of generations of replication, these findings highlight the utility of minimizing the number of generations between the original vaccine master seed and the strains used in vaccination, along with periodic monitoring of the extent of sequence evolution.     

对分离自褐家鼠的汉滩型和汉城型病毒的毒力测定

目的 比较分离自褐家鼠的汉滩病毒(HTNV)CGRn5310株和汉城病毒(SEOV)HR54株的毒力差异,探索汉坦病毒(HV)"溢出"到非宿主动物后的毒力变化.方法 选取从贵州省褐家鼠中分离的CGRn5310株HTNV和从河南省褐家鼠分离的HR54株SEOV,用不同稀释度的病毒悬液经脑内接种于昆明乳鼠,观察小鼠发病症状,测定其半数致死量(LD_(50)),并用免疫荧光法检测脑与肺组织内HV特异抗原.结果 接种2株病毒后的小鼠生长缓慢,均出现不同程度的神经症状.CGRn5310株的LD_(50)是10~-6.42,HR54株的LD_(50)是10~-4.51.在死亡小鼠的脑与肺组织中均能检测到HV特异抗原,而对照组脑与肺组织内未检测到病毒抗原.结论 从褐家鼠中分离到的CGRn5310株HTNV的毒力大于从褐家鼠中分离到HR54株SEOV,这可能意味着HV"溢出"到非宿主动物后毒力变化较小.     

静脉注射毒品人群中HIV、HBV和HCV感染的现况研究

目的了解静脉注射毒品人群中人类免疫缺陷病毒（HIV）、乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）的感染情况。方法从四川、湖南、广西和新疆等地静脉注射毒品人群中采集血液样本2025份，应用酶联免疫试剂盒检测抗-HIV、抗-HCV抗体和HBsAg。结果红静脉沣射毒品人群中，抗-HIV、抗-HCV及HBsAg的阳性率分别为14．7％～30．4％、60．7％～85．5％和6．6％～22．4％，其HIV／HBV、HIV/HCV、HCV／HBV和HIV／HCV／HBV合并感染率分别为0％～0．4%、11．6％～27．2％、2．3％～14．3％和1．6％～4．8％。结论静脉注射毒品人群中HIV、HBV和HCV的感染率均高于正常人群，其中HIV与HCV合并感染率最高。     

浙江省局部地区2002～2003年流行的无菌性脑膜脑炎的病原学检测与分析

浙江省局部地区 2 0 0 2～ 2 0 0 3年多次出现无菌性脑膜脑炎的流行。为对疫情进行病原学检测与分析 ,采集了临床标本 ,采用HEp 2、RD、Vero等细胞分离病毒 ,并用肠道病毒通用引物进行特异性逆转录 聚合酶链反应 (RT PCR)扩增。结果显示 :5 1份脑脊液标本中分离出埃可病毒 30型 (ECHO3 0 ) 5株 ,柯萨奇病毒B组 5型 (Coxsack ievirusB5,Cox B5) 1株 ;6 8份粪便标本中分离出ECHO3 0 34株 ,Cox B51株。RT PCR的结果与上述病毒分离结果相一致。此外 ,对采集的 1 5例患者的急性期、恢复期双份血清测定了特异性中和抗体 ,其中 1 3例患者抗体呈≥ 4倍增长。证实引起浙江省局部地区 2 0 0 2～ 2 0 0 3年无菌性脑膜脑炎的主要病原为ECHO3 0 。     

中国大陆首例输入性寨卡病毒病病例调查分析

目的 分析2016年2月9日中国大陆首例确诊寨卡病毒病病例的流行病学特征,为防控寨卡病毒病提供参考依据。方法 对中国大陆首例确诊寨卡病毒病病例进行流行病学调查并临床观察病例同行者与接触者。采集病例血清、尿液等标本,应用荧光定量RT-PCR进行核酸检测。结果 该病例确诊为寨卡病毒病,于第18病日解除隔离、痊愈出院。病例第10病日血清标本和第11~13病日尿液标本检测均为寨卡病毒核酸阳性。该病例系江西省赣县籍外派工作回国人员,发病前在寨卡病毒流行地区有蚊虫叮咬史,同行者与接触者医学观察期内均未出现寨卡病毒病症状。结论 该病例为中国大陆首例寨卡病毒感染确诊病例,系境外输入性病例,感染来源与在寨卡疫源地委内瑞拉蚊虫叮咬有关。