一个引起玉米矮花叶病的甘蔗花叶病毒基因组全序列测定及其结构分析

测定了从浙江省呈现矮花叶病症状的玉米上分离得到的一个马铃薯Y病毒属病毒RNA的核苷酸全序列. 该病毒分离物的RNA基因组由9596个核苷酸组成(不包括polyA尾). 单一的ORF由9192个核苷酸组成, 编码一个分子量为346.1 ku的聚合蛋白. 该蛋白结构特征与高粱花叶病毒(SrMV)中国甘蔗分离物和一个玉米矮花叶病毒(MDMV)保加利亚分离物基因组编码的蛋白非常相似. 序列分析表明, 该病毒分离物与甘蔗花叶病毒(SCMV)各分离物(已报道的仅为基因组3′末端序列)同源性最高, 与SrMV和MDMV同源性次之, 而与约翰逊草花叶病毒(JGMV)同源性最低. 根据马铃薯Y病毒属区分不同病毒和株系的分类标准, 报道的玉米病毒分离物应当鉴定为SCMV的一个株系. 然而, 该分离物在HC-Pro, P3和CI蛋白区域和SrMV中国甘蔗分离物具有极高的氨基酸同源性.     

马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

马尾松毛虫质多角体病毒(湖南株)基因组S7节段(AY180908)cDNA克隆及序列分析结果表明S7由1501个碱基组成,编码由448个氨基酸组成的分子量为49.8 kDa的多肽P50.5′末端和3′末端具有5′-AGTAA-3′和5′-GTTAGCC-3′末端保守序列.该基因组与舞毒蛾质多角体病毒1型和家蚕质多角体病毒1型S7节段有很高的同源性,核苷酸序列同源性分别为97.2%和87.0%,氨基酸序列同源性分别为98.7%和92.8%.P50多肽与人型支原体的 DnaK样蛋白在C-末端有相似性.本文报道了编码P50 C259的cDNA序列的克隆并作了原核表达,当用1.0 mmol/L IPTG 诱导2h,分子量约为37.3 kDa的融合蛋白在大肠杆菌BL21中得到大量表达.     

大豆花叶病毒外壳蛋白基因的克隆和其在大肠杆菌中的表达

通过多聚酶连锁反应(PCR),我们合成了包括病毒外壳蛋白基因在内的病毒基因组3’端区域,并完成了其全部序列分析,比较SMV(北京分离物)和SMv—N株的序列发现;外壳蛋白基因的核苷酸序列同源性达93．D％,其氨氢基酸序列同源性高达98．5％,就基因组3,端非编码序列而言,其同源性达88．8％。Western b1ot分析结果表明所克隆的cDNA片段在大肠杆菌JMl07中能表达正常的病毒外壳蛋白。     

马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

马尾松毛虫质多角体病毒(湖南株)基因组S7节段(AY180908)cDNA克隆及序列分析结果表明：S7由1501个碱基组成,编码由448个氨基酸组成的分子量为49．8kDa的多肽P50。5’末端和3’末端具有5’-AGTAA-3’和5’-GTTAGCC-3’末端保守序列。该基因组与舞毒蛾质多角体病毒1型和家蚕质多角体病毒1型s7节段有很高的同源性,核苷酸序列同源性分别为97．2％和87．0％,氨基酸序列同源性分别为98．7％和92．8％。P50多肽与人型支原体的DnaK样蛋白在C-末端有相似性。本文报道了编码P50 C259的cDNA序列的克隆并作了原核表达,当用1．0mmol／L IPTG诱导2h,分子量约为37.3kDa的融合蛋白在大肠杆菌BL21中得到大量表达。     

一株辛德毕斯病毒的基因组序列测定及分析

目的：对引进的一株辛德毕斯病毒的基因组序列进行测定,阐明其与已报道毒株序列的关系。方法：对辛德毕斯病毒基因组编码区进行分段RT-PCR扩增,对非编码区采用RACE法进行扩增,将扩增产物直接进行测序,应用DNAStar软件将测序结果拼接得到基因组序列,采用MEGA3.1软件对9株辛德毕斯病毒基因组序列进行系统进化发生树的构建。结果与结论：此株辛德毕斯病毒基因组共11663nt,编码3745个氨基酸残基,其中5＇端的2/3基因组编码4种非结构蛋白NSp1、NSp2、NSp3和NSp4,3＇端的1/3基因组编码5种结构蛋白E1、E2、E3、6K和C;结构基因和非结构基因之间有48nt的连接区为非翻译区;病毒基因组5＇末端和3＇末端分别有59、318nt的非编码区;序列同源性分析结果表明,此株病毒与S.A.AR86株的同源性最高,两者核苷酸序列的同源性为99.7%,氨基酸序列的同源性为99.6%,而与本室保存的另一辛德毕斯病毒MEI株的遗传进化关系稍远,系统进化发生树处于不同分支上。     

我国禽脑脊髓炎病毒分离株全基因组的测定

测定了我国禽脑脊髓炎病毒(avian encephalomyelitis virus,AEV)分离株L2Z株的全基因组核苷酸序列.该病毒株的3′和5′非编码区核苷酸序列用3′和5′RACE(cDNA末端快速扩增)法获得.基因组全长为7 059个核苷酸残基,包括494个核苷酸残基的5′非编码区、6 402个核苷酸残基的开放阅读框和136个核苷酸残基的3′非编码区及poly(A)尾巴.与已发表的AEV疫苗株1 143的基因组序列比较发现,它们之间核苷酸和氨基酸的同源性分别为98%和97.6%.结构蛋白(VP1～VP4)中,主要宿主保护性免疫原蛋白VP1氨基酸之间差异较小.与小RNA病毒科其它病毒属相比,在非结构蛋白3D中,预测的8个RNA依赖性RNA聚合酶主要结构域中的4个高度保守.从而进一步确认了AEV的分子特性.     

小麦黄花叶病毒基因组核苷酸序列分析

以小麦黄花叶病毒 (WYMV)HC分离物为材料 ,合成病毒基因组cDNA并进行序列分析 .结果表明 ,病毒RNA1共有 76 2 9个核苷酸 ,编码 1种由 2 40 7个氨基酸组成的多聚蛋白 ,切割产生病毒外壳和其他 7种可能的蛋白质 .该病毒的RNA2共有 36 39个核苷酸 ,编码 1种由 90 3个氨基酸组成的蛋白 ,可能切割产生 2种非结构蛋白 .WYMV虽然与小麦梭条花叶病毒 (WSSMV)在基因组结构上具有相似性 ,但是两者间在核苷酸及氨基酸水平上的同源性却分别低于 70 %和 75 % .由此结果可以确认 ,WYMV与WSSMV是大麦黄花叶病毒属 (Bymovirus)的 2种不同病毒 .     

黄瓜花叶病毒山东株(CMV\|SD)RNA2全长cDNA克隆的构建及全序列分析

分别利用5’RACE和3’RACE确定了CMV\|SD RNA2的5’和 3’末端序列,在此基础上,利用RTPCR得到了RNA2的5’端一半的cDNA克隆pC25和3’端一 半的cDNA克隆pC23,并通过拼接构建了RNA2全长cDNA克隆pC2F。通过对pC25和pC23进行序列 测定,得到了RNA2的全序列。序列分析结果表明CMVSD RNA2由3048nt组成,其中存在2个 部分重叠的阅读框ORF1(79～2652nt)和ORF2(2414～2746nt),分别编码858aa的2a蛋白和111 aa的2b蛋白,并在2a蛋白的序列中发现了动植物病毒复制酶所特有的两个保守序列。该株系 RNA2核苷酸序列与分属CMV I亚组的Fny株系和II亚组的Q株系RNA2的核苷酸序列同源性分别 为917%和756%;2a蛋白的氨基酸序列同源性分别为938%和677%,2b蛋白的氨基酸序 列同源性分别为830%和513%。同源性比较的结果表明SD株系属于CMV I亚组。     

小麦黄花叶病毒基因组核苷酸序列分析*1)

以小麦黄花叶病毒(WYMV)HC分离物为材料,合成病毒基因组cDNA并进行序列分析.结果表明,病毒RNA1共有7 629个核苷酸,编码1种由2 407个氨基酸组成的多聚蛋白,切割产生病毒外壳和其他7种可能的蛋白质.该病毒的RNA2共有3 639个核苷酸,编码1种由903个氨基酸组成的蛋白,可能切割产生2种非结构蛋白.WYMV虽然与小麦梭条花叶病毒(WSSMV)在基因组结构上具有相似性,但是两者间在核苷酸及氨基酸水平上的同源性却分别低于70%和75%.由此结果可以确认,WYMV与WSSMV是大麦黄花叶病毒属(Bymovirus)的2种不同病毒.     

西瓜花叶病毒中国分离株全基因组核苷酸序列测定

西瓜花叶病毒(Watermelon mosaic virus,WMV)是马铃薯Y病毒属(Potyvirus)成员,主要危害西瓜和甜瓜,引起花叶病。在田间,该病害主要由蚜虫以非持久性方式传播。西瓜和甜瓜花叶病在国内陕西、山东、云南、辽宁、山西、新疆、河南和黑龙江等地广泛发生[1-6]。从20世纪80年代中期开始发生,逐渐上升为普遍发生的主要病害。我国大部分地区因西瓜和甜瓜病毒病造成的损失为30%~50%,甚至会绝产,西瓜花叶病毒已经成为制约西瓜和甜瓜高产稳产最主要的因素之一[7]。到目前为止,多数工作集中在对西瓜和甜瓜病毒病的鉴定,在分子生物学上仅限于对CP基因…     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.     

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.     

Complete nucleotide sequence of the Eastern equine encephalomyelitis virus genome

The complete nucleotide sequence of the genomic 42S RNA of Eastern equine encephalitis virus has been defined for the first time. The strategy of this viral genome occurred analogous to the ones of the other alfa viruses. The comparison of amino acid sequences of E1 and E2 proteins from the two strains of the virus has revealed a number of differences. Partially, they are localized in the hydrophilic regions of the protein molecules and evidently participate in organization of the specific antigenic structures. The amino acid sequences of all viral proteins have been comparatively analysed with the sequences of the analogous proteins of other known alfa viruses.     

甲型肝炎减毒活疫苗(H2株)快速繁殖株不同代次全基因序列比较

为探索甲型肝炎减毒活疫苗 (H2株 )细胞适应的分子机制 ,将甲型肝炎减毒活疫苗毒株(HAVH2K7)在人胚肺二倍体细胞KMB17上快速连续系列传代增强适应 ,繁殖周期由原来的 2 8d缩短为 14d ,连续传代后抗原滴度和感染性滴度不断增加 ,传至 2 2代抗原滴度达 1∶10 2 4 ,感染性滴度lgCCID50 (每ml)为 7 83 .分别将第 6代和第 2 2代病毒用AC PCR法和PCR法扩增 .扩增片段分别与pGEM T载体连接得到重组质粒 ,测定cDNA插入片段的序列 .对 2个不同代次全基因序列及氨基酸序列比较分析表明 ,HAVH2K7适应至第 6代时 ,整个基因组有 6个核苷酸变异 ,全部位于编码区内 ,变异率为 0 0 7% ,导致 3个氨基酸变化 ,分别位于VP2 (A S) ;2C(N H) ;3A(R C) .适应至第 2 2代时 ,整个基因组出现 18个核苷酸突变 ,变异率为 0 2 4 % ,13个是该代次产生的 ,变异最大区域 5′端非编码区 (5′UTR)有 5个核苷酸变异 .编码区突变导致 7个氨基酸变化 ,其中 4个氨基酸变化是该代次在 6代基础上特有的变异 (2C ,Q P ;3A ,A S ;3C ,T A ;3D ,V G) .2C区是编码区变异最多区域 ,共有 4个核苷酸突变 ,在 6代变异基础上出现 3个新突变 ,导致 1个氨基酸变异 .说明 5′UTR的2C区变异对病毒的翻译效率、感染性滴度提高具有重要作用 .     

Sequence variation within a nonstructural region of the hepatitis G virus genome.

Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nucleotide positions 5829 to 8421 were designed and used to analyze serum specimens obtained from patients with community-acquired non-A, non-B hepatitis who were HGV RNA positive. One set of primers was found to be most efficient in detecting HGV and was subsequently used to test 162 HCV-positive and 11 HCV-negative plasma units obtained from individual paid donors. HGV RNA was detected in 30 (17.3%) plasma units, 2 of which were found among the 11 HCV-negative specimens. A complete set of nine PCR fragments was obtained from two patients with community-acquired acute non-A, non-B hepatitis and from four paid donors. All PCR fragments were sequenced and were shown to have a nucleotide similarity of 85.9 to 92.3% and a derived amino acid similarity of 96.0 to 99.0%. The majority of nucleotide changes occurred in the third position of codons. The HGV nucleotide and protein sequences obtained in this study were compared with HCV sequences. Based on this analysis the 2.6-kb fragment was predicted to encode the C-terminal part of the putative NS4b, the entire NS5a, and almost the complete NS5b proteins. Putative protease cleavage sites separating these proteins were also predicted. In serial specimens obtained from the two HGV-infected patients, no significant variations were found in the HGV nucleotide and derived amino acid sequences over time. The HGV sequences obtained from one patient showed no changes over 6 months, whereas more than 99.0% homology was observed for sequences from the second patient over 2.5 years. Heterogeneity analysis performed on 10 sequences obtained in this study and corresponding regions from 6 known full-size sequences of the HGV genomes demonstrated notable discrete heterogeneity consistent with the existence of HGV genetic groups or types.     

Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.     

我国登革3型病毒广西80-2株基因组全序列分析

对我国登革 3型病毒 80 2株基因组进行全序列测定 ,为了解其基因组结构与功能的关系提供依据 .根据登革 3型病毒H87株的序列设计并合成引物 ,应用RT PCR和RACE法 ,对 80 2株基因组RNA进行扩增、克隆测序后获得我国登革 3型病毒广西株基因组序列 .该株病毒基因组全长10 696nt ,不含poly(A)尾 ,4种碱基数分别为A :3 4 3 7,C :2 2 15,G :2 773 ,U :2 2 71.包含一个读码框架 ,自 95至 10 2 67位 ,共 10 170个碱基 ,编码 3 3 90个氨基酸 ,5′和 3′非编码区长度分别为 94nt和4 3 2nt.与H 87株比较 ,核苷酸和氨基酸序列同源性均在 99%以上 ,有 2 8个碱基发生改变 ,其中 2 6个碱基突变发生在读码框架内 ,碱基转换 18个 ,颠换 10个 ;碱基突变引起 14个氨基酸的改变 .80 2株与H87株病毒的基因组全序列同源性高 ,变异度小 .     

Molecular cloning and characterization of the circumsporozoite protein gene of Plasmodium inui isolated from Formosan macaques (Macaca cyclopis) in Taiwan

We characterized the complete nucleic and amino acid sequences of the Plasmodium inui circumsporozoite protein (Pincsp) gene and analyzed nucleotide diversity across the entire Pincsp gene by using 7 field isolates and strains Taiwan I and II obtained from Formosan macaques (Macaca cyclopis) in Taiwan. The length of the circumsporozoite protein ( CSP ) gene ranged from 1077 to 1125 bp. Size polymorphisms were due to variations in the number of tandem repeat units. The non-repetitive (NR) region exhibited high homology (99.1 ～ 100 and 98.7 ～ 100% at the nucleotide and amino acid levels, respectively) and was conserved among the variants (nucleotide diversities, π, of the 5'NR and 3'NR regions were 0.00364 and 0.00392, respectively). In the central repetitive (CR) region, we decomposed the sequences into 2 kinds of repeating amino acid motifs, i.e., a repeat unit R1, PA(P/A)(P/A)A(E)GG (n = 11-13), and a following repeat unit R2: P(A/G)(A/P/G)(P/Q)AQ(N/K) (n = 9-10). Analyzing these repeat sequences showed evidence of 3 genetic mechanisms for generating variations in the repeats of the Pincsp gene, i.e., point mutation, insertion, and recombination. These findings suggest that polymorphisms in the Pincsp gene are essentially limited to the CR region, which showed much greater variability in terms of length, number of repeats, and sequence.