4株SARS冠状病毒基因组5′端序列的分析与比较

利用5'RACE试剂盒对从中国不同地区、不同SARS患者体中分离的SARS-CoV基因组5'端序列进行RT-PCR扩增,并将扩增产物克隆至T easy vector.扩增片段的序列测定结果表明所分离的4株SARS-CoV基因组5'端非编码区的核苷酸序列和其他国家和地区报道的序列基本一致,而且所形成二级结构也完全相同,但与已知普通冠状病毒的差别较大.同时发现在依赖于RNA的RNA聚合酶起始密码子上游-197 nt处有冠状病毒典型的转录调控核心保守序列5'-CUAAAC-3'.     

4株SARS冠状病毒基因组5′端序列的分析与比较

利用5′RACE试剂盒对从中国不同地区、不同SARS患者体中分离的SARS—CoV基因组5′端序列进行RT-PCR扩增,并将扩增产物克隆至Teasy vector。扩增片段的序列测定结果表明：所分离的4株SARS—CoV基因组5′端非编码区的核苷酸序列和其他国家和地区报道的序列基本一致,而且所形成二级结构也完全相同,但与已知普通冠状病毒的差别较大。同时发现在依赖于RNA的RNA聚合酶起始密码子上游—197nt处有冠状病毒典型的转录调控核心保守序列5′-CUAAAC-3′。     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

水稻条纹病毒中国分离物和日本分离物RNA2节段序列比较

测定了来源于我国水稻条纹叶枯病常年流行区的云南楚雄(CX)及病害暴发区的江苏洪泽(HZ)的水稻条纹病毒(RSV)2个分离物RNA2全长序列,其长度分别为3506bp和3514 bp.与已报道的日本T和O分离物RNA2序列进行比较的结果表明,这4个分离物可分为两组,其中,HZ、T和O分离物为一组,组内分离物之间,RNA2的毒义链(vRNA2)及RNA2的毒义互补链(vcRNA2)上的ORF的核苷酸一致性分别为97.2%～98.0%和96.8%～97.1%,5′端和3′端非编码区的序列则完全一致.但HZ分离物与T分离物的亲缘关系更为密切,其基因间隔区(IR)与T和O分离物的等长.另一组为我国CX分离物,组与组之间,vRNA2及vcRNA2上的ORF的核苷酸一致性分别为95.0%～95.7%和93.9%～94.4%.CX分离物的IR与HZ分离物相比缺失了一段8 nt的片段.5′端非编码区的序列完全一致,但3′末端非编码区有一个碱基的差异.这些结果表明,RSV在自然界的分子变异与其地理分布具有密切的关系.此外,非编码区序列的高度保守性暗示着它们在病毒基因转录和复制的调控方面具有重要的功能.本文还讨论了RSV的分子流行病学.     

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%。     

普通冠状病毒229E株的分子流行病学分析

目的明确哈尔滨地区普通冠状病毒229E(Human Coronavirus-229E,HCOV-229E)株的流行和变异情况及其与SARS-CoV的异同,为进一步掌握该地区常见上呼吸道病毒的流行规律及预防,乃至疫苗的制备打下基础。方法利用RT—PCR法对2003年上半年采集的部分发热病人血清及血细胞进行筛选,同时采用基因测序、序列分析等手段对扩增的HCOV-229E N gene片断进行蛋白和基因分析。结果55例标本中HCOV-229E RNA阳性病例5例,占9．09％;测序结果看出哈尔滨地区检出229E株N基因的序列与已公布的HCOV-229E Ngene(295—802)序列完全相同,所测片断为HCOV-229E N gene的部分序列。该基因与猪流行性腹泻病毒、犬冠状病毒、TGEV及猫感染性腹膜炎病毒有一定同源性;但与已公布的SARS-CoV N gene序列比较,相似性小于1％。结论(1)该地区发热病人普通冠状病毒229E株阳性率为9．09％;(2)哈尔滨地区流行的HCOVN基因无变异发生;(3)HCOV-229E与猪流行性腹泻病毒、犬冠状病毒、TGEV和猫感染性腹膜炎病毒有一定的同源性;(4)哈尔滨地区HCOV-229E N gene与SARS-CoV N gene相似性小于1％。     

水稻条纹病毒中国分离物和日本分离物RNA2节段序列比较

测定了来源于我国水稻条纹叶枯病常年流行区的云南楚雄(CX)及病害暴发区的江苏洪泽(HZ)的水稻条纹病毒(RSV)2个分离物RNA2全长序列,其长度分别为3506bp和3514bp。与已报道的日本T和O分离物RNA2序列进行比较的结果表明,这4个分离物可分为两组,其中,HZ、T和O分离物为一组,组内分离物之间,RNA2的毒义链(vRNA2)及RNA2的毒义互补链(vcRNA2)上的ORF的核苷酸一致性分别为97．2％～98．0％和96．8％～97．1％,5′端和3′端非编码区的序列则完全一致。但：HZ分离物与T分离物的亲缘关系更为密切,其基因间隔区(IR)与T和O分离物的等长。另一组为我国CX分离物,组与组之间,vRNA2及vcRNA2上的ORF的核苷酸一致性分别为95．0％～95．7％和93．9％～94．4％。CX分离物的IR与HZ分离物相比缺失了一段8nt的片段。5′端非编码区的序列完全一致,但3′末端非编码区有一个碱基的差异。这些结果表明,RSV在自然界的分子变异与其地理分布具有密切的关系。此外,非编码区序列的高度保守性暗示着它们在病毒基因转录和复制的调控方面具有重要的功能。本文还讨论了RSV的分子流行病学。     

牛β-酪蛋白5′端上游调控序列的克隆和序列分析

该文用PCR扩增了牛β－酪蛋白基因5′－端上游调控序列,并对其进行了克隆和序列分析。采集成年母牛肝,提取DNA。在牛β－酪蛋白基因外显子1和上游调控区内设计引物,扩增其上游调控序列。两条引物长均为19个核苷酸,引物间跨度为635bp。以牛肝DNA为模板,进行PCR扩增,扩增产物在2％琼脂糖凝胶上电泳,可见特异的目的条带。从凝胶中回收目的片段,克隆到pGEM－T载体中。重组质粒提取DNA,进行序列分析。测序结果与文献发表的类似序列相比,仅有4个碱基不同,同源性达99．4％。表明获得了牛β－酪蛋白基因5′－端上游调控序列的克隆。     

小西葫芦黄化花叶病毒分离物的3′末端序列多态性研究

研究了来自中国大陆9个小西葫芦黄化花叶病毒（ZYMV）分离物的基因组3′末端核苷酸序列及所推导的外壳蛋白（CP）氨基酸序列以及3′末端非编码区（UTR）序列,并与其它地区所报道的16个ZMYV分离物进行了同源性比较。ZYMV CP基因核苷酸序列具有一定的寄主相关性和地域相关性,但总体上其关联程度不明显;同时,CP氨基酸序列的寄主适应性程度明显高于地域相关性。25个ZYMV分离物的CP氨基酸序列根据其变异程度分为2个区： N端约41个氨基酸为高度变异区,CP核心区和C端氨基酸序列为保守区。研究结果初步揭示了ZYMV作为单链RNA病毒通过与寄主相互作用而表现寄主适应性变异的趋势。     

南方菜豆花叶病毒外壳蛋白基因序列的分析

用重组DNA技术及序列分析法测定了南方菜豆花叶病毒RNA基因组3′端1,000个碱基的序列,以及由此序列推导出的整个外壳蛋白的氨基酸顺序,它与巳报导的基本上一致。介绍了用DNA的寡核苷酸水解混合物作为起始引物,以3′端不含PolyA尾巴且不能加上PolyA的病毒RNA作为模板合成互补DNA,及进一步无性繁殖此cDNA的方法。     

SARS-Cov及其他冠状病毒基因组比较分析

摘要：对病毒种内和种间基因组的比较分析能获得很多关于病毒起源与演化的信息。对17株SARS-CoV的种内基因组变异分析发现共有137个变异位点,估算出SARS-CoV的突变率为8.04×10-3核苷酸替换/位点/年。变异位点在基因组上的分布不均匀,变异位点最多的是基因组中编码S1蛋白的区域,而在编码依赖于RNA的RNA聚合酶区域中几乎没有变异位点。核苷酸和氨基酸替换的偏性预示变异可能不仅仅是由随机漂变产生。对冠状病毒种间基因组结构比较分析发现,SARS-CoV的基因组结构与IBV很相似;而保守基因系统发育分析表明,SARS-CoV属于冠状病毒的一个新分支,并且与血清型第二组冠状病毒进化关系较近。对其他某些分子特征的分析发现,在不同的方面SARS-CoV和不同组冠状病毒有不同的相似点。进一步对基因组非保守开放阅读框(ORF)的基序(motif)和跨膜区分析发现,各组冠状病毒基因组中位于基因S-E间的非保守ORF可能是同源的,但不是绝对必要的;而IBV和SARS-CoV的基因组中位于基因M-N间ORF可能不是同源的。综合分析SARS-CoV与3组血清型冠状病毒进化关系、宿主分布,以及SARS-CoV和IBV的s2m的进化关系,可以推测SARS-CoV有可能来自禽类。 Abstract:The genome comparison of inter-species and intra-species can give us much information about the origin and evolution of viruses.There are 137 mutation sites in the 17 genomes of SARS-CoV,and the mutation rate is about 8.04×10-3 substitution/site/year.The distribution of the segregating sites is not steady,the most variable region appears in S1 protein,and the nucleotide sequence of RNA-dependent RNA polymerase has very few mutation sites.The substitution bias of nucleotide acids and amino acids indicates the non-random drift products.The comparison of genome structures of SARS-CoV and other coronaviruses shows that SARS-CoV and IBV share the same genome structure.Phylogenetic analyses of conserved genes of coronaviruses indicate that SARS-CoV is a new branch of coronaviruses and appears more close to the group II coronaviruses.Interestingly,SARS-CoV shares some different features with different groups of coronaviruses.Additional analyses show that the first ORFs between S and E genes of some coronaviruses are transmembrane proteins and share the common motif,indicating the possible common ancestor.From the host distribution of different groups of coronaviruses and the phylogeny of s2m,we can deduce that avian is the probable natural host of SARS-CoV.     

Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication

The 5' portion of the Sindbis virus (SIN) genome RNA is multifunctional. Besides initiating translation of the nonstructural polyprotein, RNA elements in the 5' 200 bases of the SIN genome RNA, or its complement at the 3' end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand RNAs. We used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. Replacement of the SIN 5' end in defective-interfering (DI) and genome RNAs with sequences from a distantly related alphavirus, Semliki Forest virus (SFV), resulted in nonviable chimeras. The addition of five nucleotides from the 5' terminus of SIN restored negative-strand RNA synthesis in DI genomes but not their replication in vivo. Pseudorevertants of various SFV-SIN chimeras were isolated, and suppressor mutations were mapped to AU-rich sequences added to the 5' end of the original SFV 5' sequence or its "deleted" versions. Early pseudorevertants had heterogeneous 5' termini that were inefficient for replication relative to the parental SIN 5' sequence. In contrast, passaging of these pseudorevertant viral populations in BHK cells under competitive conditions yielded evolved, more homogeneous 5'-terminal sequences that were highly efficient for negative-strand synthesis and replication. These 5'-terminal sequences always began with 5'-AU, followed by one or more AU repeats or short stretches of oligo(A). Further analysis demonstrated a positive correlation between the number of repeat units and replication efficiency. Interestingly, some 5' modifications restored high-level viral replication in BHK-21 cells, but these viruses were impaired for replication in the cells of mosquito origin. These studies provide new information on sequence determinants required for SIN RNA replication and suggest new strategies for restricting cell tropism and optimizing the packaging of alphavirus vectors.     

Alphavirus RNA genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome

The 3' nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3'CSE). The 3'CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3'CSEs were studied in detail to evaluate the role of the 3'CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3'CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3' terminal motifs of the SIN genome; iii) the 5' 1-kb regions of all SIN genomes, regardless of the differences in 3' terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3'CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3' terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3'CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.     

4株SARS冠状病毒中国分离株3′非编码区的序列测定及分析

分别对 4株SARS冠状病毒中国株基因组 3′非编码区序列进行测定和分析 ,然后应用Blast、DNAstarGeneQuest等软件对中国株的这段序列与其它SARS和非SARS冠状病毒的序列进行比较 .结果显示 ,所测 4株SARS冠状病毒中国分离株的 3′非编码区长度均为 339nt,与其它已测序SARS冠状病毒的相应序列完全一致 .SARS冠状病毒的非编码区含有冠状病毒保守的“伪结”结构 .在距末端 138nt处还存在 1个长约 32nt的Ⅱ型茎 环结构的序列 ,该序列在其它已知人冠状病毒中并不存在 ,而与禽传染性支气管炎病毒和星状病毒中的对应序列高度同源 .     

From SARS-CoV to SARS-CoV-2: safety and broad-spectrum are important for coronavirus vaccine development

The global pandemic of COVID-19 caused by SARS-CoV-2 (also known as 2019-nCoV and HCoV-19) has posed serious threats to public health and economic stability worldwide, thus calling for development of vaccines against SARS-CoV-2 and other emerging and reemerging coronaviruses. Since SARS-CoV-2 and SARS-CoV have high similarity of their genomic sequences and share the same cellular receptor (ACE2), it is essential to learn the lessons and experiences from the development of SARS-CoV vaccines for the development of SARS-CoV-2 vaccines. In this review, we summarized the current knowledge on the advantages and disadvantages of the SARS-CoV vaccine candidates and prospected the strategies for the development of safe, effective and broad-spectrum coronavirus vaccines for prevention of infection by currently circulating SARS-CoV-2 and other emerging and reemerging coronaviruses that may cause future epidemics or pandemics.