中国狂犬病毒设苗株（5aG株）糖基因的克隆与序列分析

用逆转录－聚合酶链反应方法从中国犬疫苗株病毒感染细胞中扩增得到该株病毒糖蛋白基因，并进行序列测定。结果表明该基因开放阅读框架全长１５７５ｂｐ，编码５０５个氨基酸的成熟糖Ｎ－端１９个氨基酸的信号肽，该基因与其他株系相应基因比较，核酸序列同源性为８８％－９１％，氨基酸序列同源性为８７－９０％，其中膜外区同源性高于膜内区及跨膜区同源性。     

中国广西狂犬病毒野毒株（CGX89－1株）糖蛋白基因核酸序列测定和分析

本文报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％。核苷酸序列和巴斯德株（ＰＶ株），国际标准攻击毒株（ＣＶＳ株），中国狂犬病毒疫苗株（３ａＧ株）相比，同源性分别为８４．１％，８３．１％和８４．５％。其推导的氨基酸序列和ＰＶ株、ＣＶＳ株和３ａＧ株相比其同源性分别为９２．４％，８９．７％和８９．５％。ＣＧＸ８９－１株也具有３个Ｎ－糖基化位点，分别位于第３７位、１５７位和３１９位的氨基酸残基上。糖蛋白的膜外区部分重要抗原位点和ＰＶ株、ＣＶＳ株及３ａＧ株具有较高的一致性。     

狂犬病毒5aG株核蛋白基因的克隆及序列分析

用ＲＴ、ＲＡＣＥ、ＰＣＲ等方法，从中国狂犬疫苗株（５ａＧ）病毒感染的鼠脑组织中扩增病毒核蛋白基因，用双链测序法进行了全基因核酸序列分析。结果表明：该基因开放阅读框架全长１３５３ｂｐ，编码４５０个氨基酸。与其它３株狂犬病毒核蛋白相比，核酸序列同源性为９０％～９６％，氨基酸序列同源性为９５％～９６％。表明核蛋白保守性较好。     

5株呼吸道合胞病毒地方株F蛋白基因序列分析

目的呼吸道合胞病毒（ＲＳＶ）Ｆ蛋白是ＲＳＶ感染免疫中最重要的病毒蛋白，为了解我国ＲＳＶ地方株Ｆ蛋白的基因状况和变异特征，随机选取北京、广州、长春和河北四个地区具有不同流行特征的ＲＳＶ地方株（Ａ亚型）５株，进行ＲＳＶＦ蛋白全基因的核苷酸序列分析。方法以提取的病毒ｍＲＮＡ为模板进行ＲＴ－ＰＣＲ扩增、目的基因的克隆及序列测定，对地方株及原型株的序列进行比较分析。结果地方株Ｆ蛋白基因与原型株Ａ２株有很高的同源性，核苷酸全序列的同源性为９５．１％～９６．１％，氨基酸同源性为９６．７％～９７．４％。核苷酸有义突变率为２２．６％～２５．９％。３非编码区的核苷酸序列比蛋白编码区变异显著。河北地方株（Ｅ７３株）在３非编码区有６个核苷酸的插入。Ｆ２亚单位的氨基酸变异高于Ｆ１亚单位。在北京地方株（ＺＨＳ１３株）Ｆ１亚单位内，由具有中和能力单克隆抗体所识别的抗原表位区中存在一个氨基酸的变异。结论我国ＲＳＶ地方株与原型株之间的Ｆ蛋白基因尽管存在一定的变异，但仍有很高的同源性。地方株间Ｆ蛋白的核苷酸、氨基酸变异的位置及形式很相似，提示我国ＲＳＶ的不同流行特征可能并非由于Ｆ蛋白的基因变异所致。     

汉滩病毒Z10株G1糖蛋白编码区克隆及序列分析

目的 汉滩病毒浙１０（Ｚ１０）株Ｇ１糖蛋白编码区的克隆、核苷酸序列分析，提供我国应用最为广泛的肾综合征出血热（ＨＦＲＳ）疫苗株序列资料。方法 反转录ＰＣＲ法扩增Ｇ１基因片段，克隆入ｐＧＥＭ－Ｔ载体，双脱氧链终止法测定核苷酸序列。结果 测定１４４９个核苷酸，可编码４８３个氨基酸。与Ｉ型７６／１１８、Ｌｅｅ、Ｈｏｊｏ株比较核苷酸同源性分别为８７％、８６％、８６％，与Ⅱ型Ｒ２２株同源性为６７％。比较氨基     

中国分离乙脑病毒与灭活疫苗株(P3株)E基因差异分析

目的 分析我国近年来从蚊虫及患者标本中分离的乙脑病毒与灭活疫苗株（P3株）之间在E基因区段核苷酸及氨基酸差异。方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列，通过Clustal X（1．8）、DNASTAR、GENEDOC（3．2）等生物学软件进行分析。结果 P3株与福建分离株之间核苷酸同源性在98．3％-98．5％之间、氨基酸同源性在98．2％-98．6％之间；P3株与上海分离株之间核苷酸同源性在88．0％-88．5％之间、氨基酸同源性在98．0％-98．4％之间。E基因区段500个氨基酸中P3株与所有新分离乙脑病毒株之间共存在19个位点的差异，其中在E-76、E-306、E-408处所有新分离毒株与P3株存在共同的差异；在E-160、E-487处福建分离株与P3株存在共同差异；上海分离株与P3株在E-129、E-222、E-227、E-366存在共同差异。结论 上海蚊虫中分离的Ⅰ型乙脑病毒和福建省脑炎患者中分离的Ⅲ型乙脑病毒与P3株在E基因的部分氨基酸位点存在差异，但均不处在影响病毒生物学特性的关键位点。     

中国登革2型病毒分离株乳鼠致病性差异与基因变化关系的研究

本文报道１９８７年从广西病人血清中分离的登革２型（Ｄ２）病毒Ｄ２－４３株和１９８５年从海南病人血清中分离的Ｄ２－０４株乳鼠致病性的差异与基因变化的关系。结果表明Ｄ２－４３株对乳鼠致病，Ｄ２－０４株不致病。Ｄ２－４３株和Ｄ２－０４株Ｃ到ＮＳ１基因的读码框架基本相同，均由３３８１核苷酸组成，编码氨基酸总数１１２７。包含三个结构蛋白Ｃ．ＰｒＭ（Ｍ）、Ｅ和一个非结构蛋白ＮＳ１。该两株病毒核苷酸序列同源性为９３．８％，氨基酸序列的类似性为９１．３％。Ｃ和Ｅ基因同源性为９５．０％～９５．８％，ＮＳ１基因为９２．２％，Ｍ基因为８６．７％，两株之间核苷酸序列的主要差异是在Ｍ基因。两株病毒的Ｃ－ＮＳ１基因与国际参考毒株比较，４３株与ＪＡＭ株类侧性最高，其次是ＮＧＣ株，与Ｓ１株类似性最小。而０４株只有Ｃ、Ｅ和ＮＳ１与ＪＡＭ株最高，ＰｒＭ（Ｍ）都与ＮＧＣ株最高。４３株与０４株的Ｍ基因相比较，０４株的ＰｒＭ（Ｍ）基因的同源性低于４３株，说明两株与国际参考毒株比较，主要差异也在Ｍ基因，乳鼠致病性差异可能与Ｍ基因变化有关。     

痘苗病毒天坛株基因组病毒生长因子基因结构的研究

克隆了位于我国痘苗病毒天坛株基因组左端的病毒生长因子（ＶＧＦ）基因并对其编码多肽的结构特点进行了分析。结果表明，由天坛株ＶＧＦ基因所推导的氨基酸序列具有典型的上皮生长因子（ＥＧＦ）超家族成员的特征序列和结构特点，在核苷酸及氨基酸水平上天坛株ＶＧＦ与其他正痘病毒ＶＧＦ的同源性在８５％～９５％之间，氨基酸残基的缺失和插入等变异主要集中出现在多肽的信号肽和穿膜区，推测对功能的影响不大。此外，我们发现由天坛株基因组右末端的开放读码框架（ＯＲＦ）ＴＢ２２Ｌ推导的氨基酸序列与天坛株ＶＧＦ多肽第６７位至１４０位完全相同，提示ＯＲＦＴＢ２２Ｌ可能是天坛株基因组第二个ＶＧＦ基因的变异产物。     

中国广西狂犬病毒野毒株（CGX89—1株）糖蛋白基因核…

本报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％     

登革4型病毒包膜蛋白基因的克隆和部分核苷酸序列测定

利用反转录多聚酶链反应（ＲＴ－ＰＣＲ）技术分离并克隆了登革４型０１株病毒的包膜蛋白（Ｅ）基因，并测定了部分核苷酸序列，发现该株与国外发表的一株相应区域核苷酸序列的同源性为９３．４％，由此推测出氨基酸序列的同源性为９２．１％。     

A DNA vaccine that encodes rabies virus glycoprotein lacking transmembrane domain enhances antibody response but not protection

Rabies virus (RV) glycoprotein (gp) consists of three domains: cytoplasmic, transmembrane and ectodomain. It occurs in a complete, membrane-bound form within the infected cell, but it is released from them in a deleted, secreted form lacking the transmembrane domain. This study was performed to test the importance of the transmembrane domain for the capability of the RV gp gene, introduced into mice via a recombinant plasmid (DNA vaccine), to induce immune response and protection against challenge. Although the antibody response to the secreted form of gp was higher than that to complete gp, the protective efficacy of the respective DNA vaccine against challenge was not better than that of the DNA vaccine inducing complete gp. This indicates that the transmembrane domain of RV gp is important for generating protection against rabies and should be present in RV DNA vaccines.     

Nucleotide and deduced amino acid sequences of the glycoprotein gene of rabies virus vaccine strain Vnukovo-32

Summary The glycoprotein gene of the rabies virus vaccine strain Vnukovo-32 was sequenced and the deduced protein sequence was analyzed and compared with that of various laboratory and street strains. The amino acid sequence homologies of strain Vnukovo-32 were compared with fixed strains ERA, SAD B19, PV, HEP-Flury, CVS and two street strains, canine and CXX89-1, were 98.9 %(6 replacements), 98.3% (9), 96.2% (20), 91.4% (45), 87.0% (68), 93.5% (34) and 91.4% (45), respectively. Sequence alignments of the proteins revealed that the most conserved region is the ectodomain, whereas the transmembrane and cytoplasmic domains showed significant divergence.     

Spectroscopic characterization of two peptides derived from the stem of rabies virus glycoprotein

Rabies virus glycoprotein (G) is a trimeric type I transmembrane glycoprotein that mediates both receptor recognition and low pH-induced membrane fusion. Electron microscopy has indicated that the ectodomain of protein G is made of a globular head and a stem. In order to characterize the putative stem region at the molecular level, we designed two peptides, P(S) and P(L), which were produced as GST fusion proteins in bacteria. Peptide P(S) extends from amino acid (aa) 374 to aa 428 whereas peptide P(L) extends from aa 368 down to the end of the ectodomain of G (aa 439). Their secondary and quaternary structures have been studied with spectroscopic and biophysical methods. We show that these isolated peptides are monomeric and poorly structured in aqueous solution. However, circular dichroism (CD) in presence of 2,2,2-trifluoroethanol and NMR data indicate that this region may adopt a alpha-helical conformation in the complete glycoprotein.     

Molecular characterization of the full-length genome of a rabies virus isolate from India

Rabies is an important public health problem in South East Asia, with cases in this part of the world contributing to about 70% of the global burden. A large number of rabies cases occur in India, however, there is no organized system of surveillance and hence there is a lack of reliable data. Moreover, comprehensive molecular epidemiological studies have not been performed on Indian virus isolates. In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus obtained from the brain of an infected patient. Comparison of the genomic sequence with those of the ten fully sequenced rabies strains available in GenBank showed nucleotide homology ranging from 97% with AY956319 to 81% with AY705373. Amino acid homology of nucleoprotein ranged from 99.7% with AY352493 to 92% with DQ875051. In case of the glycoprotein gene, the homology ranged from 98.8% with AY956319 to 87.2 % with AY705373. An extensive nucleoprotein, glycoprotein, and full-length genome-based phylogenetic analysis was performed along with sequences available from the GenBank. Phylogenetic analysis of the complete genome sequence indicated that this isolate exhibited close homology with the ex Indian strain AY956319. Primer sequences and the scheme of amplification can be availed from the authors on request.     

中国狂犬病毒疫苗株（5aG）糖蛋白基因在痘苗病毒中的表达及免疫效果观察

将克隆到的中国狂犬病毒疫苗株（５ａＧ）的糖蛋白基因重组到痘苗病毒ＴＫ区，并在痘苗病毒Ｐ１１启动子的控制下，构建了狂犬－痘苗重组病毒（ＶＶａＧ）。经间接免疫荧光和Ｗｅｓｔｅｒｎ免疫印染证明，重组病毒ＶＶａＧ能良好地表达狂犬病毒糖蛋白，其分子量约为６６００。用ＶＶａＧ免疫小鼠，７ｄ便可诱生较高的狂犬病毒中和抗体，２１ｄ达４１６９，并能１００％保护狂犬病毒本毒株和国际标准攻击毒（ＣＶＳ）的致死量攻击。     

Characterization of nucleoprotein gene sequence of an Indian isolate of rabies virus

Rabies occurs in all parts of Indian sub-continent except Andaman and Nicobar and Lakshadweep group of islands. The full-length nucleoprotein (N) gene sequence of a rabies virus isolate from India is reported for the first time and the same has been compared with available N gene sequences from the database. A central domain of 230 amino acids (aa) from aa 141 to aa 370 exhibited more than 95% similarity. There were 8 amino acid positions (aa 29, 32, 38, 84, 119, 379, 438, and 439) at which substitution was unique for Indian isolates but common for laboratory strains. In antigenic epitopes, except for a single amino acid difference at the antigenic site IV, the amino acids were conserved. The Indian isolate also possessed two Bam HI sites (aa 247 and 278), while the other Asian isolates had only one site at aa 278 or were not digested with Bam HI at all. Phylogenetic analysis also demonstrated that the Indian isolate was closely related to the Sri Lankan isolate and grouped in the cluster that comprised of the isolates from other Asian countries namely China and Pakistan.     

European and American Subgroup C Isolates of Avian Metapneumovirus belong to Different Genetic Lineages

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.     

Sequence of a bovine herpesvirus type-1 glycoprotein gene that is homologous to the herpes simplex gene for the glycoprotein gB

The 130-kDa bovine herpesvirus-1 (BHV-1) glycoprotein GVP 6 was found to cross-react immunologically with the herpes simplex glycoprotein gB. Antibodies in polyclonal serum against gB immunoprecipitated GVP 6 and its cleavage products from a lysate of BHV-1-infected cells. Conversely, polyclonal serum against GVP 6 precipitated gB from HSV-1-infected cell lysates. Sera against the other glycoproteins did not demonstrate cross-reactivity. A 3.6-kb Kpnl-Hpal fragment of BHV-1 DNA that hybridized to the gene for gB was cloned and the nucleotide sequence of both strands was determined. The longest codon reading frame in the fragment coded for a protein that showed extensive homology with gB1 and related sequences from pseudorabies virus, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus. The strongest homologies were observed in two segments of the ectodomain, the transmembrane domain, and sequences adjacent to the transmembrane domain.