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。     

重庆地区急性呼吸道感染患儿呼吸道合胞病毒流行特征及其G基因序列分析

目的 分析重庆地区2008-2009年度急性呼吸道感染住院患儿呼吸道合胞病毒(respiratory syncytial virus,RSV)的亚型流行情况,并了解优势流行株BA株的G蛋白基因特征.方法 采集2008年4月-2009年3月全年于重庆医科大学附属儿童医院因急性呼吸道感染住院的508例患儿鼻咽深部分泌物,用RT-PCR方法检测RSV并进行亚型鉴定,选取29例B亚型和10例A亚型RSV阳性标本,用RT-PCR的方法扩增全长G蛋白并测序.结果 在508例标本中,RSV阳性126例(24.8%),其中检测出A亚型43例(34.1%),B亚型80例(63.5%),A、B亚型混合感染3例(2.4%).所测的10株A亚型的G基因与标准株A2的核苷酸同源性为91.4%～92.0%,均属GA2基因型;29株B亚型的G基因与标准株CH18537的核苷酸同源性为92.0%～93.0%,其中19株均为具有60个高度重复核苷酸插入的BA株.B亚型流行株与CH18537标准株相比,G基因有多种核苷酸变异如缺失、插入等,尤其在G蛋白近C端1/3处的高变区.结论 2008-2009年RSV仍是重庆地区儿童急性呼吸道感染的主要病原,与既往两年A亚型优势流行不同,2008-2009年度B亚型毒株流行占优;近年新发现的BA株可能已成为本地区优势流行株,BA株G基因变异是否导致G蛋白功能增强,进而促进其优势流行尚有待研究.     

中国呼吸道合胞病毒地方株G蛋白在重组痘苗病毒中的表达

目的　构建表达中国呼吸道合胞病毒 (RSV)地方株G蛋白基因的重组痘苗病毒 ,以用于RSV感染防治的研究。方法　用基因克隆技术将中国RSV地方株G蛋白基因插入到痘苗病毒载体中 ,与痘苗病毒共感染获得重组病毒。用免疫印迹、ELISA、蚀斑减少试验等方法检测表达产物的免疫原性及生物活性。结果　RSV地方株G蛋白在痘苗病毒中获得较好的表达 ,表达产物的糖基化程度较高 ,且该重组病毒免疫家兔可诱发特异性抗体产生。蚀斑减少试验证明 ,用该表达产物制备的抗血清具有中和病毒的活性。结论　重组病毒表达的中国RSV地方株G蛋白具有较好的抗原性、免疫原性等生物活性     

呼吸道合胞病毒分离株N蛋白编码基因特征分析

目的 阐明人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)A和B亚型病毒分离株的核蛋白(nucleoprotein,N)编码基因特征.方法 采用逆转录-聚合酶链反应(revelrse transciptionpolymerase chain reaction,RT-PCR)对北京2004年分离的HRSV代表株(2株A亚型和2株B亚型)进行N基因全长序列扩增、测序,并和GenBank下载的所有HRSV病毒的N基因全长序列进行对比和分析.结果 2株HRSV A亚型分离株与A亚型Long株(标准株)的N蛋白的核苷酸和氨基酸差异分别为36～40个(3.1%～3.4%)和4个(1.0%);2株HRSV B亚型分离株与B亚型CH18537株(标准株)的N蛋白之间的核苷酸和氨基酸差异分别为17(1.4%)和1(0.3%)个.4株HRSV代表株和从GenBank下载的HRSV的N蛋白之间的核苷酸和氨基酸差异分别为3～172个(0.25%～14.63%)和0-18个(0～4.6%).结论 N基因在HRSV基因组中是较为保守的基因,A或B亚型的型内差异相对较小;但A和B亚型之间N基因序列有较大变异,变异平均分配于整个N基因中;本研究为HRSV基因快速诊断试剂的研制提供了基因信息学数据.     

北京地区2000-2004年分离的呼吸道合胞病毒B亚型株G蛋白基因的序列分析

目的 了解近年来北京地区流行的B亚型呼吸道合胞病毒（BSV）G蛋白的基因特征。方法 2000年冬季至2004年冬季自首都儿科研究所附属儿童医院收集的急性呼吸道感染患儿呼吸道标本，从中分离到B亚型RSV毒株，每年随机选取1至2株毒株，用RT-PCR扩增其G蛋白全基因后克隆至pBS-T载体中，筛选出阳性克隆后测序，并与B亚型标准株（CH18537）和文献报道的毒株序列进行比较分析。结果 所测毒株G蛋白全基因核苷酸长度分别为915、921和981bp，推导的氨基酸长度分别为292、293、312和315从。分离株与B亚型标准株CH18537间存在着明显的差异，CH18537株同分离株间的核苷酸的同源性为91．9％～93．7％，氨基酸的同源性只有85．0％～89．0％。分离株间核苷酸的同源性为93．4％～98．8％，氨基酸的同源性为88．2％～98．6％。氨基酸的变异主要集中在胞外区一个高度保守区的两端，而胞内区和跨膜区相对保守。此外，在进行G蛋白基因分析的8株B亚型分离毒株中，我们发现有3株BSVG蛋白在490～495位核苷酸缺失，3株RSVG蛋白在c末端791位后出现了60个核苷酸的插入，导致C末端259位后出现20个氨基酸的插入。这60个核苷酸与相邻的前60个核苷酸高度重复，只出现3至4个核苷酸的差异。该3株分离株与文献报道的有60个核苷酸插入的两株（S00-4和BA4128／99B）核苷酸和氨基酸同源性分别为97．5％～98．6％和95．5～98．1％，在插入的20个氨基酸中，有高达50％（9～10个氨基酸）左右的丝氨酸和苏氨酸氧连接的糖基化位点。结论 RSVB亚型G蛋白基因变异存在着多样性，分离株既有核苷酸替代，又有基因缺失和插入，还有糖基化位点的改变和氨基酸长度的改变。这种变异使G蛋白高度糖基化，提示最终可能导致病毒抗原性的改变。北京分离的有60个核苷酸插入的变异株与日本及西班牙报道的变异株有非常近的亲缘关系，提示这种G蛋白基因中有60个核苷酸插入的B亚型变异株已经在子代病毒中形成稳定遗传并在世界范围内传播。     

呼吸道合胞病毒G蛋白胞外区基因克隆及序列分析

目的 对呼吸道合胞病毒Ｇ蛋白胞外区基因进行克隆及序列分析。方法 从ＲＳＶ感染的喘息型支气管炎患儿咽试子分离的病毒培养物中提取病毒ＲＮＡ,进行逆转录及ＰＣＲ扩增,克隆到Ｇ蛋白胞外区基因ＤＮＡ序列分析。结果 克隆片段全长６９０ｂｐ,编码一个含有２２９个氨基酸的多肽。与５个ＧｅｎＢａｎｋ中不同的ＲＳＶ分离株（包括Ａ,Ｂ亚型）的Ｇ蛋白胞外区的氨基酸序列相比,Ａ亚型的ＲＳＶ毒株氨基酸水平的同源率为９５．２％     

呼吸道合胞病毒F蛋白研究进展

人呼吸道合胞病毒（RSV）融合蛋白（F蛋白）为病毒表面结构蛋白,可以刺激机体产生保护性抗体和细胞免疫反应。因此对F蛋白的深入研究将有助于了解病毒感染的机理,从而为研究RSV的免疫机理、研制特定部位的亚单位或多肽疫苗提供帮助。     

呼吸道合胞病毒NS1基因原核表达及其免疫原性分析

目的：原核表达呼吸道合胞病毒（RSV）非结构NSl蛋白,并对其免疫特性进行研究。方法采用PCR技术获得NS1基因,将其插入pET41a（＋）载体上,导人大肠杆菌后诱导表达;利用GST亲和层析柱对诱导表达的NS1蛋白进行纯化,免疫动物制备抗体,Westernblot法鉴定抗体的特异性。结果重组质粒经酶切鉴定和DNA序列测序完全正确．经IPTG诱导后融合蛋白在大肠杆菌BL21-DE3中得到高效表达．表达产物经超声破碎和GST亲和层析纯化获得重组蛋白,分子量约42000Mr,以抗NS1蛋白的多克隆抗体为一抗进行Westernblot检测,结果只有RSV感染细胞的样品在15000Mr处有特异性带显示,其他病毒感染的细胞和阴性对照没有相应条带。结论NSl基因得到高效表达,免疫制备得到的抗体与RSV感染细胞有特异性反应,与其他常见呼吸道病毒感染细胞无交叉免疫反应。     

人类呼吸道合胞病毒粘附（G）蛋白的研究进展

HRSVG蛋白是性质独特的、高度变异性粘附蛋白，在诱导保护性免疫和致病机制方面起重要作用。HRSV感染严重程度可能与G蛋白有关。探讨G蛋白的免疫致病机理将有助于掌握HRSV的致病机制，了解清除HRSV和其它病毒的可能机制。     

呼吸道合胞病毒的蛋白特征及抗原变异

呼吸道合胞病毒是非节段性的单链负股RNA病毒，是引起婴幼儿下呼吸道感染的重要原因。本文就近年来对呼吸道合胞病毒的蛋白特征和抗原变异的研究作一综述。     

Antigenic and biological criteria of differentiation of the newly isolated strains of respiratory syncytial virus

Respiratory syncytial (RS) virus strains isolated in different years varied by their antigenic and biological properties. The lowest degree of relatedness was found between the "street" virus and the prototype Long strain; the highest occured among the isolates from a given isolation period. Based on the mean indices of efficiency of the virus reproduction in human embryo lung (HEL) cells at 37 degrees C and 39 degrees C as well as on the degree of virus sensitivity to reference antibodies, the isolates from various years could be divided into three groups, namely high, mild and low virulent strains. The incidence of RS virus infections in children depended on the strain characteristic of virus population circulating in a community of children during the long-term observation period of 1976-1979. Cyclic variation was found in isolation rates of RS viruses; the duration of each cycle in different years ranged from 21 to 41 days. The variability of isolation cycles and the frequency of RS virus reinfections were closely related to the biological characteristics of circulating virus strains.     

Genetic variability in envelope-associated protein genes of closely related group A strains of respiratory syncytial virus

The genetic and antigenic diversity present in respiratory syncytial virus (RSV) strains may in part be explained by genetic drift similar to that which occurs with influenza virus B. To study drift in RSV strains, we sequenced the five membrane-associated genes, M, SH, G, F, and M2, from three sets of RSV isolates: one set of seven closely related isolates obtained over 5 years in St. Louis, MO, and two sets of four closely related RSV isolates from other communities. We found nucleotide-variable and conserved regions in all five genes, and the greatest diversity in the SH and G genes. We did not find clear evidence of genetic drift in the seven isolates from St. Louis for any of the five genes. Although the relationships between strains were usually maintained independent of the genes studied, for several isolates there was a dramatic shift in genetic relationships for one of the five genes. Our inability to demonstrate genetic drift and the dramatic shift in genetic relationships between some strains for some genes suggest that we need to better define the mechanisms and rate of change in this virus to accurately define phylogenetic relationships between strains.     

Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration with high mortality rates in swine. It has become increasingly problematic in China. Since the nucleocapsid (N) protein is highly conserved, it is a candidate protein for early diagnosis and vaccine development. In this study, the N genes of 15 PEDV strains were amplified by RT-PCR and cloned into the pMT-19T vector, sequenced, and compared to each other as well as to PEDV reference strains. The nucleotide sequences of the N gene of the Chinese PEDV strains consist of 1326 nucleotides and encode a 441-aa-long peptide. The nucleotide sequences of the fifteen PEDV strains in our study were 96.1-100 % identical to each other, and the deduced amino acid sequences were 94.8-100 % identical. Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 94.2-99.7 % identical to those of the Chinese PEDV strains, and their deduced amino acid sequences were 94.1-99.5 % identical. In addition, the fifteen strains showed a high degree of nucleotide sequence identity to the early domestic strains (98.4-99.7 %) except the LZC strain, but less sequence identity to the vaccine strain (CV777) used in China (94.7-97.7 %). Phylogenetic analysis showed that the Chinese PEDV strains are composed of a separate cluster including three early domestic strains (JS-2004-02, LJB/03 and DX) but differ genetically from the vaccine strain (CV777) and the early Korean strains (Chinju99 and SM98).     

Sequence analysis of a Torque teno canis virus isolated in China

In the present study, a total of 158 fecal samples were collected from diarrheal dogs younger than 1 year old in pet clinic in China. 20 specimens (20/158, 13%) were positive for Torque teno canis virus DNA using detection PCR. One representative positive isolate designated LDL was randomly selected, cloned and sequenced. The complete genome of the LDL Chinese strain was 2799 nucleotides in length and contains three open reading frames (ORFs), which encode 576 (ORF1), 101 (ORF2), and 243 (ORF3) aa. Compared with the human and other animal TTV genomes, the genome of the LDL strain is clearly smaller and shares 95% identity with Japanese cf-TTV10 strain (AB076002). Phylogenetic analysis showed that the present Chinese Torque teno canis virus LDL strain was also closely clustered with the previous Japanese cf-TTV10 strain, and formed a different branch together with Torque teno sus viruses 1 and 2 compared with other Torque teno viruses, Torque teno mini virus, and Torque teno midi virus. Our study demonstrated that Torque teno canis virus is present in China.     

Group B strains of human respiratory syncytial virus in Saudi Arabia: molecular and phylogenetic analysis

The genetic variability and circulation pattern of human respiratory syncytial virus group B (HRSV-B) strains, identified in Riyadh during the winters of 2008 and 2009, were evaluated by partial sequencing of the attachment (G) protein gene. The second hypervariable region (HVR-2) of G gene was amplified by RT-PCR, sequenced and compared to representatives of different HRSV-B genotypes. Sequence and phylogenetic analysis revealed that all Saudi strains belonged to the genotype BA, which is characterized by 60-nucleotide duplication at HVR-2. Only strains of 2008 were clustered with subgroup BA-IV, while those isolated at 2009 were clustered among the most recent subgroups (particularly BA-X and CB-B). Amino acid sequence analysis demonstrated 18 amino acid substitutions in Saudi HRSV-B strains; among which five are specific for individual strains. Furthermore, two potential N-glycosylation sites at residues 230 and 296 were identified for all Saudi strains, and an additional site at amino acid 273 was found only in Riyadh 28/2008 strain. O-glycosylation was predicted in 42–43 sites, where the majority (no = 38) are highly conserved among Saudi strains. The average ratio between non-synonymous and synonymous mutations (ω) implied stabilizing selection pressure on G protein, with evidences of positive selection on certain Saudi strains. This report provides preliminary data on the circulation pattern and molecular characteristics of HRSV-B strains circulating in Saudi Arabia.     

Antigenic and genomic diversity of central European respiratory syncytial virus strains

Summary. Thirty-two RSV strains recovered during the winter months of 1987/88 to 1993/94 from hospitalized children in Vienna, Austria and Zagreb, Croatia were analysed for antigenic and genetic variations. Twenty-nine of the 32 isolates investigated belonged to group A and 3 to group B, with the majority of infections caused by subgroup A1 (21 of 29). Restriction endonuclease mapping of PCR products derived from parts of the N and G gene of 18 group A strains identified 3 distinct lineages, very similar to those defined by analysis of recurrent epidemics in Birmingham, United Kingdom during the same period. Results of this study povide further information on the global pattern of RSV and show that very similar viruses are present simultaneously in widely separated areas. Received August 4, 1997 Accepted January 30, 1998     

Occurrence of respiratory syncytial virus subtypes A and B strains in Sweden

The subtype characteristics of 22 strains of respiratory syncytial (RS) virus isolated in Sweden were determined by the use of monoclonal antibodies. Eleven antibodies specific for distinct epitopes on five different structural proteins were used in immunofluorescence and radioimmune precipitation assays. One group of 12 isolates were derived from a three-month epidemic during 1984, whereas the other ten virus isolates were recovered during a time period of 13 years (1971-1983). All isolates could be allocated to the previously defined groups of subtype A and B strains of RS virus. During the single epidemic season, five subtype A and seven subtype B strains were found. During the 13-year period a randomly alternating appearance of six subtype A and four subtype B strains was observed. Thus RS virus strains of different subtype characteristics may occur alternately or concomitantly. The possible significance of consecutive infections with RS virus subtypes for immunopathological events deserves further studies.     

Genomic analysis of two porcine encephalomyocarditis virus strains isolated in China

Summary. Two strains of encephalomyocarditis virus (EMCV), designated BJC3 and HB1, were isolated from an aborted fetus and the heart tissue of a dead piglet that had pericardial fluid, respectively. The complete genomic sequences of the two viruses were determined and analyzed. The size of the genomes of BJC3 and HB1 were 7746 and 7735 nucleotides, respectively, including poly(A) tails. Comparative analysis with the genomic sequences of other EMCV strains showed that BJC3 and HB1 shared higher identity (92.5–99.6%) with BEL-2887A/91, EMCV-R and PV21, but lower identity (83.3–84.6%) with EMC-B, EMC-D and D variants, and only 81.0% with Mengo virus. Two amino acid mutations in the leader protein of the two viruses and one amino acid substitution in VP1 of BJC3 were found in comparison to other EMCV strains Phylogenetic analysis based on the amino acid sequences of the entire ORF revealed that the two Chinese isolates BJC3 and HB1 clustered together with the strains BEL-2887/91, EMCV-R and PV21, which belong to the same genetic subgroup as EMCV-30. Our results provide genomic information for EMCV isolated in China.