麻疹病毒全长cDNA构建及其感染性的研究

为发展新型疫苗和改造目前使用的麻疹病毒疫苗,以麻疹病毒疫苗株为模板,构建了具有感染性的麻疹病毒cDNA克隆.用RT-PCR分6段扩增出麻疹病毒全长基因,通过酶切、拼接构建麻疹病毒疫苗株CC-47的全长正链cDNA序列,并精确地置于T7启动子控制下与丁型肝炎病毒核酶序列之前.克隆麻疹病毒CC-47株蛋白N、P、L编码区质粒并置于T7启动子控制下,用4个质粒共转染哺乳动物细胞,在表达T7 RNA聚合酶的重组痘苗病毒VTF7-3的作用下进行病毒拯救.经免疫荧光、PCR等方法检测证实,获得了具有感染性的麻疹病毒.所拯救的病毒在哺乳动物细胞连续传3代后,仍能检出病毒抗原和核酸.     

浙江省麻疹病毒的基因特征分析

目的分析浙江省流行的麻疹病毒(MV)的基因特征,为更好地防控麻疹提供科学参考。方法从GenBank检索并下载浙江省所有麻疹病毒株、中国麻疹疫苗株和WHO推荐参考株的血凝素蛋白(H)和核蛋白(N)的基因组序列,利用MEGA 6.0软件进行比对分析,构建种系进化树,确定浙江省麻疹病毒流行的基因型别,并进行同源性和基因变异分析。结果浙江省麻疹病毒以H1基因型为主(27株),其他基因型为辅(2株B3型)。H1a亚型(21/27)占绝对优势,其次为H1b亚型(6/27)。浙江省所有毒株间H蛋白的氨基酸同源性为96.9%~100.0%,与疫苗株Shanghai-191和Changchun-47的同源性均为95.0%~96.0%。浙江省所有毒株间N蛋白羧基末端的氨基酸同源性为82.7%~100.0%,与疫苗株Shanghai-191的同源性为85.8%~89.5%,与疫苗株Changchun-47的同源性为87.3%~91.0%。结论麻疹病毒H1基因型为浙江省麻疹流行的优势株,与现行参考疫苗株(A基因型)差异较大,因此针对麻疹病毒H1基因型疫苗的研制是今后浙江省麻疹病毒防控的关键。     

中国小反刍兽疫病毒分离株China/Tib/07全基因组序列测定与分析

小反刍兽疫病毒属于副黏病毒科麻疹病毒属成员。本研究对我国首次分离的小反刍兽疫病毒株China/Ti-bet/07进行了全基因组序列测定及分子生物学特征分析。根据GenBank公布的小反刍兽疫病毒基因组序列设计引物通过RT-PCR扩增病毒基因组内部序列,通过3′和5′-RACE获得病毒基因组末端序列。序列测定与分析的结果表明,China/Tib/07株全长15948bp,预测编码6种结构蛋白和2种非结构蛋白,与已发表小反刍兽疫病毒基因组的长度和结构相似;在系统进化上与西南亚流行毒株有很高的同源性(91.6%～98.1%);与麻疹病毒属的其它成员相比,与牛瘟病毒的同源性最高(64.3%)。     

我国麻疹病毒流行株的H和N基因变异速率探讨

从GenBank检索并下载了我国近40年的麻疹病毒血凝素蛋白(H)和核蛋白(N)的全基因组序列,用Bioedit7.0软件对参考株和代表株进行序列比对,并进行氨基酸进化树的构建,进一步从氨基酸层面分析麻疹流行株的变化速率。结果显示麻疹病毒H蛋白发生的变异,表现为近十年的变异率(1.33×10-3)大约为前三十年变异率(0.95×10-3)的1.4倍,而N蛋白发生的变异,表现为近些年的变异率(1.90×10-3)大约为前三十年变异率(1.01×10-3)的1.8倍。本研究结果提示麻疹病毒近年变异率有增快的趋势,今后需加强对麻疹病毒基因型变异和演变的监测。     

中国分离巴泰病毒基因组全编码区序列测定和分析

采用RT-PCR和TAIL-PCR方法,首次对我国分离的巴泰病毒(YN92-4株)基因组的全编码区进行序列测定和分析。结果显示,YN92-4株病毒基因组由S、M、L三个片段组成,长度分别为947、4 371、6 860个核苷酸。其中,S片段基因编码由234个氨基酸残基组成的核衣壳蛋白和由102个氨基酸残基组成的非结构蛋白,M片段基因编码由1 435个氨基酸残基组成的前体蛋白,L片段基因编码由2 239个氨基酸残基组成的RNA聚合酶。与国外其它地区的巴泰病毒分离株进行基因组全编码区序列比较后发现,YN92-4株与日本牛血清分离株(ON-7/B/01株)在S、M片段核苷酸(氨基酸)的同源性最高,分别为97.7%(100%)和95.7%(98%);由于本研究首次开展对巴泰病毒L基因片段核苷酸序列的研究,因此国际基因库尚无可参考的序列信息,本研究比较了我国分离的巴泰病毒与同一血清组的代表病毒Bunyamwera病毒L片段的核苷酸和氨基酸序列同源性,分别为73.5%和81.6%。系统进化分析显示,YN92-4株基因组与其它巴泰病毒分离株在各自分支下形成独立分支。本研究提示我国分离的巴泰病毒YN92-4株未发生基因重配(...     

我国麻疹病毒流行株的H和N基因变异速率探讨

从GenBank检索并下载了我国近40年的麻疹病毒血凝素蛋白(H)和核蛋白(N)的全基因组序列, 用Bioedit7.0软件对参考株和代表株进行序列比对,并进行氨基酸进化树的构建,进一步从氨基酸层面分析麻疹流行株的变化速率.结果显示麻疹病毒H蛋白发生的变异, 表现为近十年的变异率(1.33×10-3)大约为前三十年变异率(0.95×10-3)的1.4倍,而N蛋白发生的变异,表现为近些年的变异率(1.90×10-3)大约为前三十年变异率(1.01×10-3)的1.8倍.本研究结果提示麻疹病毒近年变异率有增快的趋势,今后需加强对麻疹病毒基因型变异和演变的监测.     

我国登革 4型病毒 B5株基因组全序列的测定及分析(英文)

 对我国登革 4型病毒 B5株 (D4- B5)基因组进行全序列测定及分析 ,为研究病毒基因组结构与功能的关系及研制新型登革疫苗奠定基础 .根据登革 4型病毒 81 4669株的序列设计特异引物 ,通过 RT- PCR扩增出 D4- B5株不同长度的片段 ,分别克隆到 p GEM- T载体 ,将挑取的阳性克隆进行 PCR、酶切鉴定及序列测定 .结果显示 ,D4- B5株的基因组全长 1 0 665nt,5′和 3′非编码区分别为 1 0 1 nt和 40 3nt,中间一个长 1 0 1 61 nt的开放读码框架 ,编码 3387个氨基酸 .与 D4- 81 4669株比较 ,两者核苷酸序列同源性为 93.0 8% ,氨基酸序列同源性为 96.58% .D4- B5株的基因组全序列与 D4- 81 4669株类似 ,但也有较大差异 .同源进化分析表明 ,D4- B5株的基因型为 型 ,与登革 4型病毒菲律宾分离株亲缘关系较近 .这是首次报道的我国登革 4型病毒分离株基因组全序列 ,对研究病毒基因组结构与功能的关系 ,探讨我国毒株的地理来源及研制适合我国人群的新型登革疫苗具有一定的意义 .     

狂犬病病毒aG株全基因组序列测定及遗传稳定性分析

目的探究狂犬病病毒(Rabies virus,RV)aG株全基因组序列特征及遗传稳定性。方法严格按疫苗生产工艺进行传代,提取主种子批、工作种子批及疫苗原液病毒RNA,通过RT-PCR技术扩增全基因组各片段基因,然后分别将其克隆到p GEM-T载体中,并进行序列测定;采用DNAStar软件包对aG株与Gen Bank中4aGV参考株(JN234411)以及18株基因1型RV参考株进行同源性分析。结果 aG株全基因组由11 925个核苷酸组成,共编码3 600个氨基酸。疫苗原液与主种子批全基因组核苷酸和氨基酸同源性均为100%,而工作种子批与主种子批的核苷酸与氨基酸同源性分别为99.97%和99.92%;aG株与4aGV参考株全基因组核苷酸与推导的氨基酸同源性均为99.9%,其与18株基因1型参考株核苷酸与氨基酸序列同源性分别为84.2%~97.6%和93.7%~98.3%;aG株传代病毒与4aGV参考株全基因组氨基酸序列高度保守,且各主要功能区未发生变异。结论狂犬病病毒aG株在实验室长期生产传代过程中,全基因组遗传特性稳定。     

抑制麻疹病毒体外复制的效应性小干扰RNA的鉴定

为了鉴定抑制麻疹病毒体外复制的靶向Rab9GTPase基因效应性小干扰RNA（siRNA）,根据。iRNA设计原则和Rab9GTPase基因的mRNA序列,设计并化学合成8对靶向Rab9GTPase基因的siRNAs和1对阴性对照siRNA,经脂质体法转染Vero—E6细胞株,转染10h后感染麻疹病毒Edmonston株。通过逆转录聚合酶链反应（RT—PCR）检测转染后细胞内Rab9GTPasemRNA水平;通过标准蚀斑试验检测麻疹病毒滴度。同对照组相比,8对靶向Rab9GTPase基因siRNAs中的2对（Rab9-4和Rab9—7）,以时间和剂量依赖性的方式显著地抑制细胞内Rab9GTPasemRNA表达和麻疹病毒的复制（抑制率高达90％以上）,其他的siRNAs对细胞内Rab9GTPasemRNA表达和麻疹病毒的复制的抑制性效应则低于50％。结果表明,Rab9-4和Rab9—7是体外抑制麻疹病毒复制的最有效的siRNAs,这些siRNAs靶序列能被用来深入研究RNA干扰治疗麻疹病毒感染的可能性。     

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%–5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.     

RNA干扰抑制麻疹病毒体外复制的实验研究

为了研究短发夹RNA(shRNA)介导的RNA干扰对麻疹病毒体外复制的抑制作用,构建靶向与麻疹病毒复制密切相关的宿主细胞基因Rab9 GTPase基因特异性shRNA表达载体,分别转染Vero-E6和B95a细胞后感染麻疹病毒Edmonston株和野生株。逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western-blot)检测转染细胞内Rab9 GTPase基因表达水平;标准蚀斑试验测定麻疹病毒滴度。结果显示转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平同对照组相比明显降低,标准蚀斑试验显示麻疹病毒的复制受到显著抑制,抑制率达到90%以上。结果表明载体介导的shRNAs能通过特异性下调Rab9 GTPase基因表达抑制麻疹病毒体外复制,Rab9 GTPase可能成为治疗麻疹病毒感染的RNA干扰靶。     

Unique nature of an attenuated strain of tobacco mosaic virus: autoregulation

An attenuated strain L11A of tobacco mosaic virus (TMV) multiplied like wild type strain L at an early stage of infection in tomato leaves. Four days after inoculation, however, multiplication of L11A was drastically reduced (autoregulation) compared with the constant multiplication of L. In mixed infections, L11A strongly inhibited the multiplication of homologous strain L. Experiments with cucumber mosaic virus (CMV) or tobacco plants revealed that the inhibitory mechanism of L11A is not host-specific but virus-specific, and the autoregulatory mechanism is effective only for TMV. RNA synthesis in L11A infected leaves 4 days after inoculation was studied by polyacrylamide gel electrophoresis. Synthesis of TMV-RNA and its replicative intermediate were strongly inhibited, whereas the replicative form of TMV-RNA and ribosomal RNA were synthesized as in the case of L infection. Synthesis of non-coat-protein was studied by the incorporation of radioactive histidine into subcellular fractions derived from leaves infected with L or L11A for 4 days. Different patterns of the two strains in protein synthesis were noted. At least three proteins were predominantly synthesized in L11A infection. One of them was observed in the mitochondria fraction. From its position in polyacrylamide gel, it could be viral coded 165K protein which is considered to be involved in viral RNA replication. These results suggest that the unique nature of attenuated virus L11A, i.e. autoregulation, resulted from the inhibitory mechanism of viral RNA synthesis due to overproduction of 165K protein and is quite distinct from interferon, intrinsic interference or interference by defective virus.     

Characterization of an RNA-dependent RNA polymerase activity associated with measles virus

An RNA-dependent RNA polymerase activity has been found copurifying with measles virus infectivity and complement-fixing antigen in three Vero cell-grown variants of measles virus: the attenuated Edmonston B strain, the natural non-attenuated Edmonston strain, and a subacute sclerosing panencephalitis isolate, IP-3. Incubation of purified measles virions with immunoglobulin G derived from sera of monkeys hyperimmunized against measles specifically removes activity sedimenting in the density region of measles virions. The requirements of the reaction, which is RNase sensitive, are similar to those reported for other paramyxovirus-associated activities, including detergent, divalent cation, ribonucleoside triphosphates, and a reducing agent. The size classes of RNA synthesized correspond to those found in measles-infected cells, including 50, 35, and 16 to 20S. The product RNA of the Edmonston B virus-stimulated reaction was rendered RNase resistant by annealing with RNA extracted from purified Edmonston B virions. RNA from uninfected Vero cells was ineffective in the annealing reaction.     

Persistent Infection of Cells in Culture by Measles Virus III. Comparison of Virus-Specific RNA Synthesized in Primary and Persistent Infection in HeLa Cells

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.