猪繁殖与呼吸综合征病毒S1株基因组序列测定和分析

应用RT-PCR方法分段扩增出PRRSV上海分离株S1毒株的4条基因大片段,扩增后的产物分别克隆于pCR-XL-TOPO载体鉴定后测序,同时应用RACE方法对S1毒株的3′和5′基因末端进行了成功的扩增并克隆于pMD-18T载体进行测序,按顺序将这些序列进行拼接得到PRRSVS1株全基因组cDNA序列。测序结果表明PRRSVS1株基因组全长15441bp,包含9个开放式阅读框,5′UTR含有189nt,3′端UTR含有181nt,其中包含30ntPoly(A)。基因组序列分析结果显示该病毒与ATCCVR-2332和BJ-4分离株的核苷酸同源性分别99.5%和99.6%。与另一国内分离株CH-1a的核苷酸同源性为90.8%。     

中国6株狂犬病病毒街毒株全基因组测序与分析

实验研究中对分离于中国的6株狂犬病病毒街毒株进行了全基因组测序,对基因组的5个结构基因(N、P、M、G和L)的核苷酸和推断的氨基酸序列以及非编码区序列进行了分析与比较,并与来自GenBank的40株毒株从全基因组水平进行了分子进化分析。所测6株中国狂犬病病毒街毒株的全基因组核苷酸序列长度介于11 907 nt(CQ92)和11 924 nt(SH06和gg4)之间,基因组结构相同,用全基因组和不同的结构基因构建的进化树拓扑结构相似,基因组3′和5′末端高度保守而且末端11个核苷酸互补配对,5个结构基因的保守性依次是NLMGP,核苷酸同源性的最小值依次分别是81.9%、81.7%、80.7%、78.3%和76.7%。     

猪繁殖与呼吸综合征病毒S1株基因组序列测定和分析

应用RT-PCR方法分段扩增出PRRSV上海分离株S1毒株的4条基因大片段,扩增后的产物分别克隆于pCR-XL-TOPO载体鉴定后测序,同时应用RACE方法对S1毒株的3'和5'基因末端进行了成功的扩增并克隆于pMD-18T载体进行测序,按顺序将这些序列进行拼接得到PRRSV S1株全基因组cDNA序列.测序结果表明PRRSV S1株基因组全长15441 bp,包含9个开放式阅读框,5'UTR含有189nt,3'端UTR含有181nt,其中包含30nt Poly (A).基因组序列分析结果显示该病毒与ATCC VR-2332和BJ-4分离株的核苷酸同源性分别99.5%和99.6%.与另一国内分离株CH-1a的核苷酸同源性为90.8%.     

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 .     

兔出血症病毒（RHDV）WHNRH株的分离鉴定及基因组全序列的测定与分析

从发病长毛兔中分离鉴定了兔病毒性出血症病毒WHNRH株。参考GenBank中已登录的RHDV毒株序列对RHDV WHNRH分离株进行了全基因组序列测定与分析。设计5对扩增区段相互重叠的RHDV特异性引物,扩增除5′和3′末端以外的序列,采用设计锚引物的5′RACE方法以及针对RHDV 3′末端的polyA结构设计引物获得了RHDV WHNRH株的5′和3′末端序列。胶回收各PCR产物,连接pMD 18-T克隆载体,测得RHDV WHNRH分离株的基因组全长为7437nt(不包括polyA),与GenBank公布的全部共6株RHDV全基因序列进行同源性比较分析,同源性在89.0%~97.1%之间,ORF1同源性为89.0%~97.1%,编码氨基酸序列的同源性为95.2%~98.7%;ORF2的核酸苷序列同源性为92.1%~97.7%,编码氨基酸序列的同源性为94.1%~96.6%。     

我国登革 4型病毒 B5株基因组全序列的测定及分析(英文)

 对我国登革 4型病毒 B5株 (D4- B5)基因组进行全序列测定及分析 ,为研究病毒基因组结构与功能的关系及研制新型登革疫苗奠定基础 .根据登革 4型病毒 81 4669株的序列设计特异引物 ,通过 RT- PCR扩增出 D4- B5株不同长度的片段 ,分别克隆到 p GEM- T载体 ,将挑取的阳性克隆进行 PCR、酶切鉴定及序列测定 .结果显示 ,D4- B5株的基因组全长 1 0 665nt,5′和 3′非编码区分别为 1 0 1 nt和 40 3nt,中间一个长 1 0 1 61 nt的开放读码框架 ,编码 3387个氨基酸 .与 D4- 81 4669株比较 ,两者核苷酸序列同源性为 93.0 8% ,氨基酸序列同源性为 96.58% .D4- B5株的基因组全序列与 D4- 81 4669株类似 ,但也有较大差异 .同源进化分析表明 ,D4- B5株的基因型为 型 ,与登革 4型病毒菲律宾分离株亲缘关系较近 .这是首次报道的我国登革 4型病毒分离株基因组全序列 ,对研究病毒基因组结构与功能的关系 ,探讨我国毒株的地理来源及研制适合我国人群的新型登革疫苗具有一定的意义 .     

一株保存的波瓦生病毒全基因组序列测定及分析

目的：对保存的WJBC株波瓦生病毒进行全基因组序列测定和分析,阐明其与已报道毒株之间的关系。方法：将波瓦生病毒基因组编码区分11段进行RT－PCR扩增,扩增产物直接进行测序,非编码区采用RACE法进行扩增,扩增产物纯化并连接pGEM－Teasy载体后转化大肠杆菌DH5ct感受态细胞,挑取阳性克隆鉴定后进行测序,用DNAstar软件将测序结果拼接得到全基因组序列。下载波瓦生病毒全基因组核苷酸序列,利用MEGA5．0软件构建系统进化发生树。结果与结论：WJBC株波瓦生病毒全基因组共11839nt,编码3415个氨基酸残基,病毒基因组5’端和3’端分别有111、483nt的非编码区;进化树结果显示,WJBC株波瓦生病毒与LB株波瓦生病毒的亲缘性最高,可能为同一病毒株．．     

登革病毒基因组核苷酸序列与血清学分型的相关性研究

为证明登革病毒基因组核苷酸序列与病毒血清学分型的相关性,本文以病毒全基因组和病毒5′末端非编码区,E蛋白,NS1蛋白和3′末端非编码区四个基因区段分别进行病毒型间比较.比较结果表明病毒全基因组和病毒的各个基因区段均明显分为相互独立的4个分支,与病毒血清型分型完全吻合,不存在型间重组的现象.文章进一步分析了各型登革病毒型内核酸序列的相似性和型内病毒重组的可能性.     

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。     

J亚群禽白血病病毒蛋鸡分离株SD07LK1全基因组核苷酸序列的比较分析

[目的]分析我国ALV-J蛋鸡分离株的来源和进一步演变趋势.[方法]以J亚群禽白血病病毒(ALV-J)蛋鸡分离株SD07LK1感染的鸡胚成纤维细胞(CEF)基因组:DNA作为其前病毒基因组模板,根据已发表序列设计合成9对引物,经PCR扩增出9段连续的、相互部分重叠的DNA片段和闭合环形前病毒两末端LTR的连接区段,并分别连入T载体进行克隆、测序.[结果]用:DNAstar软件对测序结果进行剪辑和拼接,首次完成了ALV-J蛋鸡分离株SD07LK1的前病毒全基因组核苷酸序列.[结论]将该序列与另外已完成的全基因组序列的比较表明,ALV-J的整个基因组gag和pol基因相对保守,各毒株间对应基因的同源性分别在95.O%以上,env基因的同源性仅为88.6%～94.0%.     

Extreme ends of the genome are conserved and rearranged in the defective interfering RNAs of semliki forest virus

We have analyzed Semliki Forest virus defective interfering RNA molecules, generated by serial undiluted passaging of the virus in baby hamster kidney cells. The 42 S RNA genome (about 13 kb ²) has been greatly deleted to generate the DI RNAs, which are heterogeneous both in size (about 2 kb) and sequence content. The DI RNAs offer a system for exploring binding sites for RNA polymerase and encapsidation signals, which must have been conserved in them since they are replicated and packaged. In order to study the structural organization of DI RNAs, and to analyze which regions from the genome have been conserved, we have determined the nucleotide sequences of (1) a 2.3 kb long DI RNA molecule, DI309, (2) 3′-terminal sequences (each about 0.3 kb) of two other DI RNAs, and (3) the nucleotide sequence of 0.4 kb at the extreme 5′ end of the 42 S RNA genome.The DI309 molecule consists of a duplicated region with flanking unique terminal sequences. A 273-nucleotide sequence is present in four copies per molecule. The extreme 5′-terminal nucleotide sequence of the 42 S RNA genome is shown to contain domains that are conserved in the two DI RNAs of known structure: DI309, and the previously sequenced DI301 (Lehtovaara et al., 1981). Here we report which terminal genome sequences are conserved in the DI RNAs, and how they have been modified, rearranged or amplified.     

草鱼出血病病毒873株基因组外发现缺损性干扰颗粒的亚基因组成份

草鱼出血病病毒基因组由 11条dsRNA片段组成。最近在研究其基因组时发现 ,在病毒基因组外存在许多核酸成份 ,但在核苷酸数量上少于基因组成份 ,表现为较小分子量的RNA片段。在完整地克隆了这些片段的全长cDNA后 ,测定了其中两个克隆的序列组成 ,发现它们为病毒基因组经剪切后的部分片段 ,已经重新装配 ,而且都含有原基因组某一片段 3′端和 5′端的保守区和倒转重复区 ,缺失中间部分。根据其特点来看 ,它们应为目前病毒学研究的重要材料———缺损性干扰颗粒的亚基因组成份。     

简化cDNA末端快速扩增技术测定基因Ⅲ、Ⅵb和Ⅶ d型新城疫病毒基因组末端序列及分析

[目的]简化cDNA末端快速扩增技术(Rapid amplification of cDNA ends,fLAcE)流程,测定基因Ⅲ、VIb)和VIId型新城疫病毒(Newcastle disease virus,NDV)基因组两侧末端序列,并对NDV的leader和trailer进行分析.[方法]利用T4 RNA连接酶将特定寡聚核苷酸片段的连接于病毒基因组RNA和eDNA,再利用RT-PCR或PCR方法对病毒基因组的末端进行快速的扩增.[结果]建立一套操作简单、低成本、可重复性高的RACE方法,测定了三种基因型5株NDV 3'末端leader和5'末端trailer序列比对分析.[结论]本实验测定的鹅源VII型毒株JS/7/05/Ch基因组的15,184 nt由一个T变为了C,5'端trailer与3'端leader的连续互补序列由8 nt变为12 nt,而其它4株基因Ⅲ型和VI型NDV均未发现该突变.通过RNA的二级结构分析,NDV基因组和反向基因组RNA的3'末端形成一个发卡结构.JS/7/05/Ch等3株NDV U→C(T→C)的突变位于发卡环上,不影响二级结构的形成,发卡环的RNA序列突变为3'-UCUC-5',与基因组3'端发卡环的3'-UCUUA-5'相似,推测可能影响了基因组RNA的复制速度.     

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

赤狐线粒体全基因组及系统发育分析

测定了赤狐的线粒体基因组全序列,总长度为16 723 bp,碱基组成为：31.3% A、26.1% C、14.8% G、27.8% T。和大多数哺乳动物一样,赤狐的线粒体全基因组包含13个蛋白质编码基因、2个核糖体RNA基因、22个转运RNA基因和1个控制区。除ND3基因起始密码子为不常见的ATT外,赤狐与北极狐、狼、家犬、郊狼的线粒体蛋白质编码遵循相同模式。在控制区的保守序列区段1和2之间发现一段较长的富含AC的随机重复序列。为了验证赤狐与其他犬科动物的系统发育关系,利用12个重链蛋白质编码基因,分别通过邻接法和最大简约法构建了系统发育树。结果表明：赤狐与北极狐是姐妹群,它们在犬科中都属于赤狐型分支,而灰狼、家犬和郊狼则属于狼型分支,与现有的系统进化研究结果一致。     

A comparative analysis of MC4R gene sequence, polymorphism, and chromosomal localization in Chinese raccoon dog and Arctic fox

Numerous mutations of the human melanocortin receptor type 4 (MC4R) gene are responsible for monogenic obesity, and some of them appear to be associated with predisposition or resistance to polygenic obesity. Thus, this gene is considered a functional candidate for fat tissue accumulation and body weight in domestic mammals. The aim of the study was comparative analysis of chromosome localization, nucleotide sequence, and polymorphism of the MC4R gene in two farmed species of the Canidae family, namely the Chinese raccoon dog (Nycterutes procyonoides procyonoides) and the arctic fox (Alopex lagopus). The whole coding sequence, including fragments of 3'UTR and 5'UTR, shows 89% similarity between the arctic fox (1276 bp) and Chinese raccoon dog (1213 bp). Altogether, 30 farmed Chinese raccoon dogs and 30 farmed arctic foxes were searched for polymorphisms. In the Chinese raccoon dog, only one silent substitution in the coding sequence was identified; whereas in the arctic fox, four InDels and two single-nucleotide polymorphisms (SNPs) in the 5'UTR and six silent SNPs in the exon were found. The studied gene was mapped by FISH to the Chinese raccoon dog chromosome 9 (NPP9q1.2) and arctic fox chromosome 24 (ALA24q1.2-1.3). The obtained results are discussed in terms of genome evolution of species belonging to the family Canidae and their potential use in animal breeding.     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.