海南岛两株甲病毒基因组3＇末端核苷酸序列的克隆与分析

目的 从分子水平鉴定海南省分离的两株甲病毒（ＨＢｂ１７和Ｍ１病毒）。方法 采用ＲＴ－ＰＣＲ方法,分别扩增两株病毒基因组的３’末端核苷酸序列,扩增产物经亚克隆,筛选重组子并测定核苷酸序列对序列进行分析。结果 ＨＢｂ１７和Ｍ１病毒分别扩增出约１．６ｋｂ和１．３ｋｂ的特异片段。序列分析表明,在３’末端非翻译区ＨＢｂ１７病毒和Ｍ１病毒与罗斯病毒（国际标准株Ｔ４８病毒）核苷酸同源性为９９％,Ｍ１病毒与鹭山病     

中国分离的甲属病毒YN87448毒株非结构区基因的核苷酸序列测定及分析

目的　研究我国首例从发热病人血清中分离的YN87448病毒的分子致病基础。方法　甲属病毒基因序列保守区设计引物,用逆转录聚合酶链反应(RTPCR)方法扩增,再将扩增片段克隆到pGEMT载体。测定YN87448毒株非结构基因序列。结果　YN87448病毒非结构区基因序列为7613个核苷酸(不包括5′端帽子结构),编码非结构蛋白1(nsP1),非结构蛋白2(nsP2),非结构蛋白3(nsP3),非结构蛋白4(nsP4);有一个起始密码子(ATG)位于第60位碱基;含有2个终止密码子(TGA),分别位于基因组nsP3基因和nsP4基因的末端。YN87448病毒非结构区基因与辛德毕斯病毒家族中唯一能致成年小鼠死亡的S.A.AR86株相应基因的核苷酸序列同源性为988%,但YN87448毒株不致成年小鼠死亡。与S.A.AR86株相比,基因结构上有三点明显差异:YN87448毒株在nsP3区位于基因组5256bp5309bp含54个核苷酸的插入;位于基因组5603bp处3个核苷酸(AGT)的缺失;在nsP3和nsP4之间有乳白突变终止密码子(TGA)。此序列已输入GeneBank,序列号为AF103734。结论　YN87448为一株新的辛德毕斯病毒株。     

中国分离的甲属病毒YN87448毒株非结构区基因的？…

目的 研究我国首例从发热病人血清中分离的ＹＮ８７４４８病毒的分子致病基因。方法 甲属病毒基因序列保地区设计引物,用逆转录聚合酶链反应９ＲＴ－ＰＣＲ）方法扩增,再次扩增片段克隆到ｐＧＥＭ－Ｔ载体。测定ＹＮ８７４４８毒株非结构基因序列。结果 ＹＮ８７４４８病毒非结构区基因序列为７６１３个核苷酸,编码非结构蛋白,非结构蛋白２,非结构蛋白３,非结构蛋白４;有一个起始密码子位于第６０位碱基;含有２个终止密码     

我国分离的甲病毒YN87448株基因组序列的初步分析

我国分离的甲病毒ＹＮ８７４４８株基因组序列的初步分析周国林梁国栋李蕾付士红金奇张海林黄文丽侯云德作者单位：１０００５２北京中国预防医学科学院病毒基因工程国家重点实验室（周国林梁国栋李蕾付士红金奇侯云德）；云南省流行病防治研究所（张海林黄文丽）。１９９...     

中国人4型登革病毒E基因部分片段的cDNA克隆与序列分析

１９９３年１０月广东省发生登革热的流行，我们从其中一个病人血清中扩增到４型登革病毒（ＤＶ４）包膜蛋白基因（Ｅ）的部分ｃＤＮＡ片段，对其进行了分子克隆及序列测定。ＤＶＲＮＡ经随机引物逆转录后用ＤＶ４特异的引物扩增，获得４２１ｂｐ的产物，补齐和提纯后插入ｐＵＣ１８和ｐＵＣ１９质粒载体，采用双脱氧链末端终止法测定核苷酸序列，与同型ＤＶ代表株比较，同源性达到９３７２％，与不同型ＤＶ的同源性介乎６１２６％～６４４０％，而与同属的乙型脑炎病毒比较，同源性只有４０３１％。比较推导的氨基酸序列后发现一个含有１２个氨基酸的保守区，可能是黄病毒属中具有重要生物学功能的部位。     

我国分离的两株病毒为重组甲病毒

目的 明确我国分离的XJ－90260和XJ－91006病毒的分类地位、种系发生和遗传型。方法 特异引物逆转录－聚合酶链反应（RT－PCR）扩增两株病毒的NSP4、E1基因区和3′端非编码区,测序,进行核苷酸序列同源性比较和3′端非编码区核苷酸序列分析,并结合同属其他病毒这些基因区的核苷酸序列进行种系进化分析。结果 两株病毒的核苷酸序列同源性为100％,与西方马脑炎病毒的核种酸序列同源性最高,具有西方马脑炎病毒3′端非编码区结构特征,其NSP4基因区与东方马脑炎病毒同源,E1基因区与辛德毕斯病毒同源,两株病毒均位于西方马脑炎病毒B组,与西方马脑炎病毒俄罗斯分离株进化关系最近。结论 我国分离的XJ－90260和91006病毒属于西方马脑炎病毒同一遗传型,均为重组甲病毒。     

从海南岛蚊和蜱分离出28株甲病毒属病毒

１９８３～１９８８年在海南岛从自然界捕蚊和蜱分离出非登革病毒和非日本脑炎病毒的２８株病毒，其中２７株是从不同种的蚊分离出的，１株是从蜱分离出的。９０年代初期在美国疾病控制中心的媒介传播的病毒性疾病分部，用全套国际标准化的抗节肢动物携带的病毒的抗体，对这２８株病毒进行了病毒科的鉴别和病毒属的鉴定，结果证明这２８株病毒都属于披膜病毒科的甲病毒属。     

赖型钩端螺旋体毒力株新基因差异片段筛选和核苷酸序列分析

目的:筛选致病钩端螺旋体赖型017株毒力相关基因DNA差异片段,并进行核苷酸及基因信息学分析。方法:提取钩端螺旋体017株基因组DNA,根据抑制消减杂交(SSH)筛选的017株特有的153bp核苷酸短片段,采用盒式连接半巢式PCR技术,扩增相邻未知序列,进行序列测定、分析及同源性检索,并进行蛋白二级结构预测。结果:克隆获得580bp核苷酸长度的基因片段,包含4个重叠开放阅读框架(ORF),在氨基酸水平上与A型化脓性链球菌,肺炎链球菌保守的假想蛋白高度同源。被GenBank收录,收录号为AF495587。结论:本研究筛选并分析了可能是钩体毒力相关的基因片段,为进一步探讨该基因的生物学功能及阐明赖型钩端螺旋体致病分子机制打下了基础。     

北京地区TT病毒分离株全基因的克隆及序列测定

目的 克隆和测定北京地区ＴＴ病毒（ＴＴＶ）分离株全基因序列。方法 采用聚合酶链反应（ＰＣＲ）技术从１例临床非甲￣庚型肝炎病人血清中分段扩增ＴＴＶ全基因,获得５个互相重叠的片段,分别长１９９ｂｐ（１－１９９）,１２６７ｂｐ（９０－１３５６,８７８ｂｐ（１３５４－２２３１,１１２９ｂｐ（２１７８－３３０６,４３４ｂｐ（３３０６－３７３９）,用标准的分子生物学方法测定了这５个序列,从而得到全长ＴＴＶ基因     

7株TT病毒部分核苷酸及氨基酸序列分析

目的 了解TT病毒（Transfusion transmitted virus,TTV）在我国的流行情况,研究我国TTV株序列特点。方法 采用半巢式聚合酶链反应（PCR）扩增TTVDNA,PCR阳性扩增产物直接测序。结果 我国7例TTVDNA株部分序列与日本株相比,核苷酸及氨基酸同源性分别为64．7％－98．4％及62．7％－96．4％;7株间的核苷酸及氨基酸同源性分别为63．5％－98．4％及60．2％－96．4％,分别属于两种型及其中一型中的两种亚型。结论 我国存在TTV并可能存在多种TTV基因型。     

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3′ untranslated region (10 nucleotides in length) and 2 insertions in the 3′ untranslated region involving a total of 5 nucleotides. Interestingly, from the 5′ UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3′ UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus. Jin-Sheng Wen and Wen-Zhong Zhao equally contributed to this work. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number: EF011023.     

Lineage replacement accompanying duplication and rapid fixation of an RNA element in the nsP3 gene in a species of alphavirus

A sequence of thirty-six nucleotides in the nsP3 gene of Ross River virus (RRV), coding for the amino acid sequence HADTVSLDSTVS, was duplicated some time between 1969 and 1979 coinciding with the appearance of a new lineage of this virus and with a major outbreak of Epidemic Polyarthritis among residents of the Pacific Islands. This lineage of RRV continues to circulate throughout Australia and both earlier lineages, which lacked the duplicated element, now are extinct. Multiple copies of several other elements also were observed in this region of the nsP3 gene in all lineages of RRV. Multiple copies of one of these, coding for the amino acid sequence P*P*PR, were detected in the C-terminal region of the nsP3 protein of all alphaviruses except those of African origin. The fixation of duplications and insertions in 3′ region of nsP3 genes from all lineages of alphaviruses, suggests they provide some fitness advantage.     

中国庚型肝炎病毒河南株非结构基因（NS）3区cDNA的克隆 …

目的 为了研究中国庚型肝炎病毒（ＨＧＶ）非结构（ＮＳ３）区基因结构特征。方法 利用逆转录－半巢式－聚合酶链反应从河南１份ＨＧＶ ＲＮＡ阳性血清获得覆盖ＨＢＶ ＮＳ３全长ｃＤＮＡ的４个片段，并克隆到ｐｃＤＮＡⅡ载体中，采用Ｓａｎｇｅｒ双脱氧末端终止法测定全部ｃＤＮＡ序列。结果 发现克隆到的包括ＨＢＶ ＮＳ３区在内的ｃＤＮＡ序列和度为２１３７个核苷酸，编码７１１个氨基酸。与国内外已测定的５株全序列的相     

Molecular epidemiology and evolution of mosquito-borne flaviviruses and alphaviruses enzootic in Australia

Three distinct patterns in the molecular epidemiology and evolution are evident among the alphaviruses and flaviviruses enzootic in Australia. One pattern, exemplified by MVE and KUN viruses, is of a single genetic type evolving slowly and uniformly in geographically widely separated regions of Australia with no evidence of independent divergence. The second pattern, exemplified by RR virus, is of separate genotypes evolving in different geographic regions with significant nucleotide divergence between genotypes. The third pattern, exemplified by SIN virus, is of a succession of temporally related genotypes that extend over most of the Australian continent, with relatively low levels of nucleotide divergence within a genotype, and which are each replaced by the subsequent genotype. These patterns are associated in part due to the nature and dispersal of their vertebrate hosts. Nucleotide divergence rates for Australian alphaviruses are similar to those reported elsewhere. Genomic relationships between Australian flavivirus members of the JE virus serological complex and between Australian alphaviruses are discussed, and evidence is presented for a possible new genomic lineage of SIN virus.     

湖南3县(市)狂犬病病毒分子生物学研究

目的研究湖南省狂犬病高发区和无病例区动物携带狂犬病的分子生物学特征。方法用直接免疫荧光法检测犬唾液及犬、猫脑标本，以RT．PCR法复核阳性进行遗传学分析。结果武冈市和洞口县送检的82只和17只犬中，分别有12只和1只检测到狂犬病毒抗原与核苷酸阳性，阳性率分别为14．63％和5．88％。凤凰县67份大脑标本未检测出病毒。28份猫脑组织标本也未检测出狂犬病毒。用RT-PCR法扩增阳性大脑组织（编号为Wg13，Dk13）的狂犬病毒N基因，两株病毒之间核苷酸与氨基酸的同源性分别为99、4％及99．1％；Wg13株与中国疫苗株CTN株和aG株的核苷酸同源性（氨基酸）分别为89．4％（98．2％）、86．1％（95．1％）；Dk13株与中国疫苗株CTN株和aG株的核苷酸同源性分别为89．1％（98．0％）、86．1％（94．9％）。与其他国家分离到的狂犬病毒相比，两株病毒与印度尼西亚的同源性最大，分别为92．8％、93．2％，而与印度、日本及斯里兰卡等其他国家同源性相对较小。结论两株狂犬病毒均为I型狂犬病毒。其N基因的核苷酸序列与当前使用的疫苗株相比，两株病毒与CTN疫苗株分在同一组，同源性较大。     

Molecular cloning and primary structure analysis of the mouse mammary tumor virus-related element from dwarf hamster genome

Eleven recombinant bacteriophages carrying mouse mammary tumor virus (MMTV)-related sequences (MRS) were isolated from the genomic library of the dwarf hamster (Phodopus sungorus) using radioactively labeled DNA of MMTV as a hybridization probe. There are approximately 50 copies of MRS-Ps per genome, as estimated by the number of positive signals on the autoradiogram of the primary screening plate. MRS of Phodopus sungorus (MRS-Ps) contain regions homologous to the LTR, pol, and, probably, env, but not gag genes of MMTV. A 0.9 kb MRS-Ps fragment was sequenced and proved to be 63% identical to the MMTV pol gene sequence. However, all absolute frames contain multiple stop codons and do not seem to be expressed.