TaqMan实时荧光定量RT-PCR检测甲型肝炎减毒活疫苗病毒含量的研究

目的：建立一种快速定量检测甲型肝炎减毒活疫苗病毒含量的实时荧光定量RT-PCR方法。方法对Gen-Bank中登陆的甲型肝炎减毒活疫苗株（ L-A-1）和其他甲型肝炎病毒基因组全序列比较分析,根据其高度保守的5′端非编码区设计针对甲型肝炎减毒活疫苗株特异性引物与探针,对荧光定量RT-PCR反应条件进行优化,检测该方法的特异性和灵敏性,并对甲型肝炎减毒活疫苗病毒含量进行定量检测。结果该方法对甲型肝炎减毒活疫苗株高度特异,扩增片段为207 bp,不与其他肠道病毒发生非特异性反应。在104 CCID50/管～10-1 CCID50/管之间有良好的扩增曲线,检测的灵敏度可达0．1CCID50～0．01CCID50,比普通RT-PCR高100倍。结论该方法具有快速、灵敏、特异、重复性好等优点,可应用于甲型肝炎减毒活疫苗生产过程中病毒含量滴度测定及指导疫苗成品的配制。     

肠道病毒、乙脑病毒和腮腺炎病毒多重RT-PCR检测方法的建立

为建立针对肠道病毒(enterovirus,EV)、乙脑病毒(Japanese encephalitis virus,JEV)和腮腺炎病毒(mumps virus,MUV)的多重RT-PCR检测方法,分别选择肠道病毒的5′UTR基因、乙脑病毒的E基因和腮腺炎病毒M基因设计3对引物,建立同时检测肠道病毒、乙脑病毒和腮腺炎病毒的多重RT-PCR方法.以中国地区流行的与脑炎相关的麻疹病毒和风疹病毒cDNA为模板验证该检测方法的特异性,同时以梯度稀释的不同滴度的脊髓灰质炎病毒、乙脑病毒、腮腺炎病毒评估该检测方法的检出限.所建立的3种病毒的多重RT-PCR方法可同时或分别特异扩增肠道病毒、乙脑病毒和腮腺炎病毒的152 bp、429 bp和274 bp基因片段,基因序列分别与脊髓灰质炎病毒Sabin 1型5′UTR基因片段、乙型脑炎病毒SA-14-14-2株E基因片段及腮腺炎病毒S79基因片段序列一致;检出限分别达到78.1 CCID50/mL、312.5 PFU/mL、156.2 CCID50/mL;而对麻疹病毒和风疹病毒的扩增均为阴性.所建立的多重RT-PCR特异性和检出限良好,可用于上述3种脑炎病毒的快速检测.     

H7亚型禽流感病毒一步法RT-PCR检测方法的建立

通过分析流感数据库45个H7亚型禽流感病毒的HA序列,在保守区内设计并合成引物,建立了一步法RT-PCR检测方法,扩增片段大小为501bp。通过对H7亚型禽流感病毒尿囊液和棉拭子浸出液不同滴度检测,证实病毒尿囊液最低检出量为105.5EID50/mL;阳性棉拭子最低检出量为103EID50/mL。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原进行检测,仅有H7亚型AIV有特异性目的条带,与其他均无交叉反应。从脏器及咽喉、泄殖腔棉拭子样品的病毒分离和RT-PCR方法比较,表明在10-1的样品浓度下,两者可以达到相同的检出量。表明该一步法RT-PCR方法具有特异性强、敏感性高和准确率高的特点。     

人传染性软疣病毒快速PCR检测方法的建立

为了能够对传染性软疣病毒(MCV)感染做出准确快速的实验室诊断,从14例临床MCV患者皮疹处提取获得病毒DNA,设计引物并进行PCR扩增获得预期的167bp的片段,经测序并与已报道的序列比对,完全一致。而使用痘病毒科其他病毒的特异引物(正痘病毒和副痘病毒属)则没有特异扩增条带。将病毒DNA进行系列稀释后行PCR扩增,结果表明PCR检测方法的敏感性为5×10-4ng/μL。     

JEV-DNA实时荧光定量标准品的构建

利用TaqMan荧光定量PCR技术,建立JEV-DNA定量标准品的制备方法。通过处理JEV减毒活疫苗提取病毒RNA,进行RT-PCR扩增目的片段,与T载体连接,转化感受态细胞,T-A克隆,测序鉴定后定量。获得预期的重组质粒,建立的标准曲线有较大的线性范围。此法制备的重组质粒标准品可用于对病毒载量进行测定。     

逆转录-聚合酶链反应在乙型脑炎减毒活疫苗生物反应罐清洁验证中的应用

为了对乙型脑炎减毒活疫苗生物反应罐清洁后乙型脑炎病毒(JEV)检测方法进行探讨,从GenBank中收录的乙型脑炎病毒的E蛋白基因序列设计一对引物,以乙型脑炎减毒株SA14-14-2培养物提取RNA作为模板,进行逆转录和PCR扩增。结果表明乙型脑炎减毒株SA14-14-2扩增出预期的特异性条带,阴性对照没有扩增出任何条带。聚合酶链反应与血吸附试验比较,有灵敏、快速、稳定性的特点,可用于生物反应罐清洁后乙型脑炎残留病毒的检测。     

RT-PCR方法扩增白蛉热病毒M片段的研究

为建立扩增未知序列白蛉热病毒M片段的RT-PCR方法,本研究选取7个血清组成员和8个未分组血清型,共42株白蛉热病毒为RT-PCR检测对象.通过排列GenBank中已知的4型白蛉热病毒M片段氨基酸序列,选择保守区设计引物.根据保守区各已知病毒的cDNA特异序列合成寡核苷酸,将相同区的寡核苷酸等量混合成为"鸡尾酒"引物,用于RT-PCR.扩增产物经电泳检测,纯化后直接测序.引物对Ph-M-2FM和Ph-M-3RM扩增产物长度约为600bp,从42株病毒中扩增出34株,阳性率为81.0%.另一引物对Ph-M-2FM和Ph-M-4R2M扩增产物长度约为1400bp,扩增出22株病毒,阳性率为52.3%.测出序列经BLAST检索,与GenBank中已知白蛉热病毒同源.本研究首次成功地应用RT-PCR扩增不同血清型未知序列白蛉热病毒的部分M片段,并测出扩增产物序列,为白蛉热病毒属成员的基因鉴定和种系发生关系分析提供了实验手段,并将有助于白蛉热病毒感染的基因诊断.     

广东地区两种兰花病毒病害的分子鉴定及检测

根据已报道的建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)基因组核苷酸序列,在其cp基因上下游设计PCR引物.CyMV预计扩增产物784bp,ORSV预计扩增产物604bp.以采集自广东省顺德的墨兰和文心兰表现病毒病症状的病株叶组织总RNA为模板,进行RT-PCR扩增.对预期大小的5个扩增产物进行克隆和测序,结果表明,来源于不同兰种或同一兰种不同兰场的病样CyMV引物扩增产物核苷酸序列存在少量差异,但均与世界各地的CyMV分离物cp基因高度同源;而来源于不同兰种的病样ORSV引物扩增产物核苷酸序列完全相同,与世界各地的ORSV分离物cp基因高度同源.因此可将侵染广东兰花的两种病毒鉴定为CyMV和ORSV.混合上述两种病毒的 PCR引物,采用双重RT-PCR扩增,对采自广东顺德23个兰场共153份样品进行病毒检测,76份(49.7%)检出CyMV,52份(34.0%)检出ORSV,2份(1.3%)同时检出CyMV和ORSV.     

乙型脑炎病毒减毒中间株SA14—12—1—7基因组全序列的测定

本研究通过对乙型脑炎活疫苗减毒过程中间株SA14 12 1 7株进行全序列测定和分析 ,进一步了解乙脑活疫苗减毒及其稳定性的分子机制。根据已发表的SA14 14 2株及SA14 株的序列 ,设计 6对重叠引物 ,涵括整个乙脑病毒的基因组 ,通过RT PCR扩增出SA14 12 1 7株的各cDNA片段 ,分别克隆到pGEM T载体 ,转化至TG1受体菌中 ,挑取阳性克隆进行鉴定后测序。结果表明SA14 12 1 7株基因组全序列长 10 976个核苷酸 ,从 96到 10 394为一个长开放读码框 ,编码 3432个氨基酸。与野毒株SA14 和疫苗株SA14 14 2的核苷酸序列和氨基酸序列相比 ,同源性均在 99%以上 ,突变位点分散于各个区域 ,E区有 5个位点与疫苗株一致而与野毒株不同 ,3个位点与野毒株一致而与疫苗株不同 ,推测与其容易产生回复突变、恢复毒力有关。此外 ,NS3、NS5和 3′NTR的几个位点可能与病毒毒力稳定性相关。综上所述 ,乙脑病毒减毒中间株的基因组全序列基本类似于已发表的序列 ,若干突变位点影响病毒的弱毒性及毒力的稳定性。全序列的测定对于研究疫苗株的减毒机理具有重要意义     

乙型脑炎减毒活疫苗生产株SA14-14-2全基因组序列测定及基因遗传稳定性

目的对乙型脑炎减毒活疫苗生产株SA14-14-2株进行全基因组序列测定和分析,并观察该生产株在疫苗制备过程中的基因遗传稳定性。方法根据DNA序列数据库(Gen Bank)公布的SA14-14-2株的序列,设计合成7对引物,提取疫苗生产株SA14-14-2及其工作种子批生产的3批原液、3批成品疫苗的病毒RNA,通过RT-PCR方法扩增SA14-14-2株的cDNA片段,分别克隆到pGEM-T载体,转化至大肠埃希菌DH5α中,挑取阳性菌落克隆、鉴定后测定全序列并对序列进行比较分析,观察毒株在传代的过程中病毒滴度的稳定性。结果乙型脑炎减毒活疫苗生产株SA14-14-2株基因组全长10 976 bp,编码3 433个氨基酸。3批原液和3批成品疫苗的基因组全长为10 977 bp,比较分析发现,在3'端非编码区10 701处多一个G核苷酸的插入。与DNA序列数据库(Genbank)登录号为D90195的全长序列同源性分别是99.9%、99.9%、99.9%、99.9%、99.8%、99.9%、99.8%,其中E蛋白的同源性均为100%。SA14-14-2生产株的病毒滴度为7.22 lg PFU/mL,原液和成品疫苗的滴度分别为7.32、7.23、7.32、6.86、6.92、6.70 lg PFU/mL。结论乙型脑炎减毒活疫苗生产株SA14-14-2基因稳定,具有良好的一致性,为乙型脑炎减毒活疫苗的质量的稳定性提供了可靠依据。     

No evidence of murine leukemia virus-related viruses in live attenuated human vaccines

Background

The association of xenotropic murine leukemia virus (MLV)-related virus (XMRV) in prostate cancer and chronic fatigue syndrome reported in previous studies remains controversial as these results have been questioned by recent data. Nonetheless, concerns have been raised regarding contamination of human vaccines as a possible source of introduction of XMRV and MLV into human populations. To address this possibility, we tested eight live attenuated human vaccines using generic PCR for XMRV and MLV sequences. Viral metagenomics using deep sequencing was also done to identify the possibility of other adventitious agents.

Results

All eight live attenuated vaccines, including Japanese encephalitis virus (JEV) (SA-14-14-2), varicella (Varivax), measles, mumps, and rubella (MMR-II), measles (Attenuvax), rubella (Meruvax-II), rotavirus (Rotateq and Rotarix), and yellow fever virus were negative for XMRV and highly related MLV sequences. However, residual hamster DNA, but not RNA, containing novel endogenous gammaretrovirus sequences was detected in the JEV vaccine using PCR. Metagenomics analysis did not detect any adventitious viral sequences of public health concern. Intracisternal A particle sequences closest to those present in Syrian hamsters and not mice were also detected in the JEV SA-14-14-2 vaccine. Combined, these results are consistent with the production of the JEV vaccine in Syrian hamster cells.

Conclusions

We found no evidence of XMRV and MLV in eight live attenuated human vaccines further supporting the safety of these vaccines. Our findings suggest that vaccines are an unlikely source of XMRV and MLV exposure in humans and are consistent with the mounting evidence on the absence of these viruses in humans.     

马传染性贫血病毒美洲流行株与我国弱毒疫苗株鉴别诊断方法的研究

马传染性贫血病毒(Equine infectious anemia virus,EIAV)是反转录病毒科慢病毒属的成员,是马传染性贫血病的病原。二十世纪七十年代我国就研制出马传染性贫血驴白细胞弱毒疫苗,成为世界第一个成功地应用该疫苗控制了我国的马传贫的发生[1]。而且我国的马传贫弱毒疫苗对异源的美国、古巴和阿根廷等毒株也有很高的保护率[2]。因此将我国的马传贫驴细胞弱毒疫苗推向国际市场成为可能。然而目前制约该苗出口的技术问题是现行的OIE推荐的琼脂扩散实验和ELISA等血清学方法不能鉴别自然感染马与我国弱毒疫苗免疫马,针对这个关键问题,本试验…     

提高麻疹减毒活疫苗病毒滴度的研究

病毒滴度是麻疹减毒活疫苗的主要质量指标之一。本文结合生产实际,就有关提高麻疹疫苗病毒滴度的影响因素进行了探讨。试验说明,生产用毒种的质量十分重要,要保证麻疹疫苗的高滴度和生产的稳定,需要建立适合麻疹毒种的培养条件并保持培养条件一致,从而必须加强生产过程的全面质量管理。     

日本脑炎病毒SA14-14-2 E蛋白结构域Ⅲ的抗原性和免疫原性分析

为了表达日本脑炎病毒囊膜蛋白(E蛋白)结构域DⅢ区,了解其作为亚单位疫苗的可能性,本研究根据SA14-14-2病毒株序列(GenBank Accession No.D90195)设计两条引物,以全长JEV感染性克隆pBR-JTF为模板,通过PCR扩增出JEVE蛋白DⅢ的cDNA片段,构建了原核表达载体pET-JEDⅢ,转化大肠杆菌Rosetta(DE3)进行融合表达。融合蛋白为可溶性表达,表达量约占菌体蛋白的75%。用纯化后蛋白免疫新西兰兔和BALB/C鼠,通过ELISA,Western blotting,噬斑减少实验,及乳鼠攻毒实验验证JEDⅢ的抗原性和免疫原性。Western blotting及ELISA结果表明纯化后的表达产物具有良好的抗原性,纯化的JEDⅢ蛋白免疫新西兰兔,可以获得高达1:7×105滴度的抗JEV特异性抗体;JEDⅢ蛋白免疫BALB/C鼠,可以获得1:8.2×104滴度的抗JEV特异性抗体。并且获得1:256滴度的中和抗体,乳鼠攻毒实验能达到75%的保护效果。以上结果说明本研究表达、纯化的重组JEDⅢ蛋白,免疫小鼠以及兔后,能产生抗JEV的特异性抗体,中和性抗体,能够保护部分乳鼠接受毒...     

我国肾综合征出血热疫苗生产株LR1株的全基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列 ,了解该株分子基础 ,从提取的细胞总RNA逆转录PCR扩增 ,产物纯化后克隆T载体纯化后测序 ,结果证明 ,LR1株全基因组序列由L6 5 33、M36 16、S片段的16 92个核苷酸组成 ,依各自读码框架分别编码 2 15 1、1135、42 9个氨基酸。序列同源比较分析表明 ,LR1毒株与国外HTN型毒株高度同源 ,属同一亚型 ,尤其与HTN代表株 76 - 1183个片段同源率高达 99 3%～ 99 8% ,而与国内的HTN型病毒差异较大 ,同源率仅为 79 4%～ 84 6 %。氨基酸比较也显示了同样的结果。     

Effect of urea and uric acid on the hemolytic activity of Sendai virus

It was found that haemolytic activity of Fushimi strain of Sendai virus multiplied in allantoic cavity of chicken embryos is independent on its haemagglutinating titer and also on allantoic fluid urea and uric acid content. It was shown in experiments with embryonated eggs that these two compounds have no also influence on haemolytic activity induction in Sendai virus. Moreover, the results of an experiment in which allantoic fluid was replaced by Eagle's liquid suggest that most probably the other components present in allantoic fluid do not also influence the appearance of haemolytic activity of this virus.     

A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.     

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice

The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live‐attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live‐attenuated vaccine, whereas the titers of IgG2a induced by the live‐attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus‐specific IFN‐γ⁺ CD4⁺ and CD8⁺ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus‐specific IL‐17⁺ CD4⁺ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL‐17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live‐attenuated and recombinant MVA vaccines.     

表达乙型脑炎病毒E蛋白重组减毒沙门菌活载体疫苗株的构建

目的:构建表达乙型脑炎病毒(JEV)E蛋白的口服重组减毒鼠伤寒沙门菌活载体疫苗株。方法:克隆JEV E基因,将其插入表达载体pYA3341中,构建重组质粒pYA3341-E,将重组质粒电转入鼠伤寒沙门菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E);鉴定重组菌E蛋白的表达,测定重组菌的稳定性、生长曲线、安全性,以及小鼠的免疫试验和血清中和试验。结果:酶切鉴定和序列测定证实重组质粒构建成功;SDS-PAGE检测有目的蛋白条带;Western印迹证实表达的E蛋白能与猪抗JEV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可较稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用,安全可靠;小鼠口服重组菌免疫,ELISA检测产生了抗JEV抗体;中和试验表明产生的抗体具有中和活性。结论:构建了能稳定表达JEV E蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550(pYA3341-E),为研究乙型脑炎口服基因工程疫苗奠定基础。     

乙型脑炎疫苗研究进展

乙型脑炎（乙脑）的流行是世界性的公共卫生问题之一,其流行范围正逐步扩大。目前对于乙脑尚无特效治疗手段,因此预防乙脑的发生尤为重要。随着对日本脑炎病毒研究的深入,利用基因工程技术研制新型候选疫苗已成为预防乙脑新的发展方向。我们简要综述了乙型脑炎病毒的基因组结构及其蛋白功能,以及国内外乙型脑炎疫苗研发的新进展。