两株中国腮腺炎野毒SH基因cDNA直接测序和克隆测序

对来源于兰州和上海两市的两株腮腺炎病毒野毒的小疏水蛋白（ＳＨ）基因及其旁侧区ｃＤＮＡ的ＰＣＲ扩增产物,分别进行直接测序和克隆测序。用直接测序法测定２株腮腺炎野毒３７８个核苷酸序列,在此范围内存在１６个核苷酸（４．２％）差异,其中１１个（２．９％）位于编码区序列中,所推导的ＳＨ蛋白序列有６个氨基酸（１０．５％）差异。克隆测序法测定的这两株腮腺炎野毒的核苷酸序列相当于直接法的７８－３７８位核苷酸。两处     

猴泡沫病毒SFV-1前病毒pol基因克隆及序列分析

采用nested-PCR从猕猴肾组织细胞中获得同源性较高、具有高度保守序列的、长度为465 bp 大小的猴泡沫病毒SFV-1的 pol 基因的cDNA,将其克隆到pMD18-T载体中,并对其进行序列分析,分析结果表明所克隆的基因片断为猴泡沫病毒SFV-1 pol 基因片段,与文献中已发表的来源于灵长类动物SFV-1、SFV-1A、SFV-1B、SFV-2、 SFVmac、SFVkm的 pol 基因核苷酸序列进行比对,显示SFVkm1与上述病毒 pol 基因核苷酸同源性分别为91.06%、90.59%、90.82%、90.21%、89.41%、91.53%.对 pol 基因克隆和序列分析是了解和掌握SFV病毒分子流行病学及其变异趋势的重要手段,7 种不同基因型的泡沫病毒的系统进化树分析显示了SFVkm1与它们之间的相互联系.     

猴泡沫病毒间接免疫荧光检测方法的建立及初步应用

目的建立检测血清中猴泡沫病毒（SFV）抗体的间接免疫荧光方法,为检测实验用猴群中SFV的感染情况提供参考依据。方法用SFV-1病毒感染BHK-21细胞,待50%细胞出现病变时,用胰蛋白酶消化细胞后以2×107/mL浓度40μL的细胞滴到10孔镀膜的玻片上,丙酮固定。利用制备的抗原片通过间接免疫荧光法对34份猴血清标本进行检测。结果建立了检测SFV抗体的间接免疫荧光染色方法,SFV抗体阳性19例,15例血清检测为阴性。结论本方法具有良好的特异性,可作为SFV检测的可靠方法。     

一株腮腺炎野毒分离传代前后其SH基因cDNA的测序比较

为证实鸡胚分离传代是否引起腮腺炎病毒基因组高变区小疏水蛋白（ＳＨ）基因的变异,用来源于上海的腮腺炎野毒Ｗｓｈ３株在鸡胚尿囊腔分离前和传代５次后测定ＳＨ基因及其旁侧区３８０个核苷酸的ｃＤＮＡ序列,两者结果相同,未见该基因的突变。该基因核苷酸测序可用于腮腺炎病毒的分子流行病学和毒株基因鉴别研究。     

Nested-PCR检测恒河猴泡沫病毒

目的用nested-PCR检测恒河猴泡沫病毒SFV的前病毒形式。方法从SFV-1的pol区域选择两对引物分别对原代猴肾细胞（rhesus monkey kidney,RMK）及猴外周血淋巴细胞（peripheral blood lymphocytes,PBLs）进行体外扩增,扩增产物经1.0%的琼脂糖凝胶电泳,证实其特异性。阳性对照使用具有典型泡沫样病变的RMK377细胞株的前病毒DNA,阳性对照的PCR扩增产物经测序证实,阴性对照为恒河猴的SRV-1cDNA。结果nested-PCR能快速灵敏的直接从猴外周血淋巴细胞检出SFV的前病毒形式,与RMK细胞培养结果基本相一致。结论本实验所建立的检测SFV的nested-PCR法能快速准确的检测猴群中的SFV的带毒情况,对于提高实验猴的质量具有重要意义。     

一株产生EB病毒的绒猴淋巴细胞株的建立

用Ｂ９５－８细胞株产生的ＥＢ病毒，转化棉顶绒猴外周血淋巴细胞，获得ＫＭＴ３细胞株。该细胞株能产生高滴度的ＥＢ病毒。含ＥＢ病毒衣壳抗原的自然阳性细胞率为３￣５％，激活后达５０￣６０％，其培养上清液可直接转化人淋巴细胞得到传代细胞系。ＫＭＴ３细胞染色体数２ｎ＝４６条。     

猴泡沫病毒（SFV）RT-nestedPCR检测方法的建立及初步应用

目的建立实验猴群及相关生物制品猴泡沫病毒（SFV）的PCR检测方法。方法选择SFV-1、SFV-3、SFVCPZ前病毒序列的pol基因同源性较高的区域设计嵌套引物对SFV-1毒种进行RT-nestedPCR扩增并克隆测序,以确定其准确性,通过验证方法的特异性和敏感性,初步应用该方法对恒河猴外周血淋巴细胞（PBLs）,常用猴肾传代细胞及猴源性生物制品进行检测。结果经RT-nestedPCR扩增出的片断与SFV-1 cDNA序列同源性达到99%,对10只恒河猴的检测结果为5只阳性,5只阴性,对常用猴肾传代细胞及脊髓灰质炎疫苗的检测结果均为阴性。结论所建立的SFV RT-nestedPCR检测方法能准确的检测出恒河猴SFV的感染情况,对控制实验猴群的质量具有重要意义。该方法可用于检测猴源性生物制品中SFV的污染情况,为保证生物制品应用的安全性提供一定依据。     

一株产生EB病毒的狨猴淋巴细胞株的建立

用Ｂ９５８细胞株产生的ＥＢ病毒,转化棉顶狨猴外周血淋巴细胞,获得ＫＭＴ３细胞株。该细胞株能产生高滴度的ＥＢ病毒。含ＥＢ病毒衣壳抗原的自然阳性细胞率为３～５％,激活后达５０～６０％,其培养上清液可直接转化人淋巴细胞得到传代细胞系。ＫＭＴ３细胞染色体数２ｎ＝４６条     

泡沫病毒的基因内部启动子

泡沫病毒的基因内部启动子刘佳建耿运琪（南开大学生命科学学院,天津３０００７１）关键词泡沫病毒内部启动子反式激活因子泡沫病毒在分类上属反转录病毒科（Ｒｅｔｒｏｖｉｒｉｄａｅ）的泡沫病毒属（Ｓｐｕｍａｖｉｒｕｓ）,因其感染细胞后可诱导产生类似泡沫的大量空...     

猴血中SFV病毒基因PCR检测方法的建立及应用

针对猴泡沫病毒SFV（Simian Foany Virus）多聚酶区（pol区）设计两对引物,以猴血DNA为模板者嵌套式PCR扩增,得到500bp左右基因片段,克隆进入pUC-19载体,经测序鉴定为SFV465bp的pol区基因片段,将此段序列与SFV各型425bp的pol区基因片段进行同源性比较,它与SFV－1型的同源性最高,为92.00%。在此基础上,用这两对引物对158例猴血DNA进行检测,阳性54例,阳性率为34.2%,发现猴群中有较高的SFV病毒的感染。     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

Replication of Simian Foamy Virus in Monkey Kidney Cells

The structure of foamy virus, its mode of maturation, and the origin of vacuoles in monkey kidney cells are described.     

A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions

Simian immunodeficiency virus (SIV) is an important lentivirus used as a non-human primate model to study HIV replication, and pathogenesis of human AIDS, as well as a potential vector for human gene therapy. This study investigated the role of single-stranded purines (ssPurines) as potential genomic RNA (gRNA) packaging determinants in SIV replication. Similar ssPurines have been implicated as important motifs for gRNA packaging in many retroviruses like, HIV-1, MPMV, and MMTV by serving as Gag binding sites during virion assembly. In examining the secondary structure of the SIV 5′ leader region, as recently deduced using SHAPE methodology, we identified four specific stretches of ssPurines (I-IV) in the region that harbors major packaging determinants of SIV. The significance of these ssPurine motifs were investigated by mutational analysis coupled with a biologically relevant single round of replication assay. These analyses revealed that while ssPurine II was essential, the others (ssPurines I, III, & IV) did not significantly contribute to SIV gRNA packaging. Any mutation in the ssPurine II, such as its deletion or substitution, or other mutations that caused base pairing of ssPurine II loop resulted in near abrogation of RNA packaging, further substantiating the crucial role of ssPurine II and its looped conformation in SIV gRNA packaging. Structure prediction analysis of these mutants further corroborated the biological results and further revealed that the unpaired nature of ssPurine II is critical for its function during SIV RNA packaging perhaps by enabling it to function as a specific binding site for SIV Gag.     

猴副流感病毒SV5 PCR检测方法的建立与初步应用

目的建立检测SV5的PCR方法并加以初步应用。方法根据GenBank中报道的SV5序列,针对其中的SH基因设计引物进行PCR反应,扩增产物进行测序并用BLAST软件进行同源性比对,同时利用限制性内切酶的酶切反应以证实此PCR反应的特异性。在此基础上设计巢式PCR提高此方法的灵敏度。利用此方法对20份猴肾源细胞培养物和40份血清标本进行检测。结果利用设计的引物扩增出的序列测序结果证实与报道的SV5SH基因相对位置的序列一致。AccⅢ限制性内切酶可对PCR产物进行特异性酶切。巢式PCR比一次PCR的敏感度有所提高。用此方法检测的20份猴肾源细胞培养物和40份血清标本结果为阴性。结论初步建立了检测SV5病毒的PCR方法,排除实验室用20份猴肾源细胞培养物和40份血清标本SV5的污染。     

Characterization of a cis-Acting Sequence in the pol Region Required To Transfer Human Foamy Virus Vectors

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5′ gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3′ pol region. Although small 5′ and 3′ deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.     

猴艾滋病病毒（SRV）的装配及释放与细胞内骨架系统关系的电镜观察

将猴艾滋病 Ｄ 型逆转录病毒（ Ｓｉｍｉａｎ Ａ Ｉ Ｄ Ｓｔｙｐｅ Ｄｒｅｔｒｏｖｉｒｕｓ , Ｓ Ｒ Ｖ１） 感染的 Ｒａｊｉ 细胞进行选择性抽提制备成“核骨架中间纤维”这一细胞内骨架系统,再结合常规电镜技术和 Ｄ Ｇ Ｄ 包埋去包埋剂电镜技术,对此病毒在宿主细胞内的装配和释放与这一骨架系统的关系进行了研究。结果表明 Ｓ Ｒ Ｖ１ 病毒核壳体的装配是在细胞核附近的胞质中进行的,其“装配中心”是结合在胞质骨架的中间纤维上,正在装配的和已装配好的病毒核壳体都是紧密结合在中间纤维上的。而在核内骨架系统中未观察到病毒粒子。这表明 Ｓ Ｒ Ｖ 的装配可能是依赖于胞质中的中间纤维作支架,且装配好的核壳体可能沿着中间纤维被运送至质膜内侧或胞质内病毒感染后形成的空泡膜上,然后出芽释放     

Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3)

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.