芜菁花叶病毒温州分离物HC-Pro基因序列分析*

从浙江温州盘菜上获得芜菁花叶病毒温州分离物（TuMV-WZ）,病毒提纯后,经电镜观察,可发现大量长约700nm的线形粒子。利用免疫捕捉RT-PCR,通过特异性引物对TuMV-WZ的HC-Pro基因进行PCR扩增,经电泳可见1.5kb的特异条带。PCR扩增产物经过纯化后进行序列测定,序列长度为1449个核苷酸,编码482个氨基酸。其核苷酸序列与已报道的TuMV的其他分离物（Canada,Ukl和Japanese）同源率分别为83.9％、96.5％和94.0％,氨基酸同源率分别为97.3％、98.8％和99.     

香蕉束顶病毒NS株DNA组分5的克隆和序列分析

本文利用PCR技术扩增得到香蕉束顶病毒(BBTV)NS株DNA组分5的全基因,该基因全长为1014nt,具有一个开放阅读框,编码146个氨基酸,蛋白质二级结构包括6个α螺旋,7个β折叠。NS株系与南太平洋组澳大利亚、夏威夷、埃及分离物DNA组分5核苷酸和编码的氨基酸序列相比较,核苷酸序列同源率介于88%～89%之间,氨基酸序列同源率介于80%～88%之间。NS株系与其它分离物在编码成视网膜细胞瘤蛋白(Retinoblastomaprotein,Rb)的基元序列“LXCDE”附近的二级结构上表现明显的差异,推测这种差异可能影响与植物中Rb蛋白的结合效率。     

香蕉束顶病毒NS株DNA组分5的克隆和序列分析

本文利用PCR技术扩增得到香蕉束顶病毒(BBTV)NS株DNA组分5的全基因,该基因全长为1014nt,具有一个开放阅读框,编码146个氨基酸,蛋白质二级结构包括6个α-螺旋,7个β-折叠.NS株系与南太平洋组澳大利亚、夏威夷、埃及分离物DNA组分5核苷酸和编码的氨基酸序列相比较,核苷酸序列同源率介于88%～89%之间,氨基酸序列同源率介于80%～88%之间.NS株系与其它分离物在编码成视网膜细胞瘤蛋白(Retinoblastoma protein,Rb)的基元序列"LXCDE"附近的二级结构上表现明显的差异,推测这种差异可能影响与植物中Rb蛋白的结合效率.     

黄瓜花叶病毒亚组Ⅰ和Ⅱ分离物外壳蛋白基因的序列分析与比较

对我国分离的经生物学和血清学鉴定为黄瓜花叶病毒（ＣＭＶ）亚组Ⅰ和Ⅱ的各一分离物（ＧＢ、ＸＢ）的外壳蛋白（ＣＰ）基因进行了序列分析和比较。以提纯病毒ＲＮＡ为模板,进行逆转录及ＰＣＲ扩增,并通过常规基因克隆方法得到插入ＣＰ基因片段的重组克隆。对插入ＧＢ和ＸＢ两个分离物ＣＰ基因片段的重组克隆进行全序列测定,结果表明重组克隆序列长分别为７７７ｂｐ和７９２ｂｐ,均只含一个开放读框（ＯＲＦ）,长度为６５７ｎｔ,可编码２１８个氨基酸;两个分离物的ＣＰ基因核苷酸序列同源率为７７５％,氨基酸序列同源率为８２６％。与我国已报道的７个ＣＭＶ分离物的ＣＰ基因序列比较,分离物ＧＢ的同源率为９１３％～９７３％,分离物ＸＢ的同源率为７６６％～７８４％。与国际上已报道部分ＣＭＶ株系的ＣＰ基因序列相比较,分离物ＧＢ与亚组Ⅰ株系有更密切的亲缘关系,而分离物ＸＢ则与亚组Ⅱ株系的亲缘关系更密切。     

侵染万寿菊的黄瓜花叶病毒外壳蛋白基因的克隆与序列分析

应用ELISA的方法检测到感染了黄瓜花叶病毒(Cucumber mosaic virus,CMV)的万寿菊。根据已报道的CMV外壳蛋白(CP)基因的保守序列设计并合成引物,提取万寿菊叶片总RNA为模板,进行cDNA合成和PCR扩增,得到约875bp的片断,与预期片断大小相符。序列分析显示:该分离物CMV-WSJ CP基因全长657bp,编码218个氨基酸,其核苷酸和氨基酸序列与CMV亚组Ⅱ的分离物有很高的同源率,分别达到98.33%～99.24%和98.63%～100%;与CMV亚组I分离物的同源率分别仅为74.43%～76.26%和77.17%～78.99%。因此,从万寿菊叶片上检测出的黄瓜花叶病毒分离物CMV-WSJ应归属于亚组Ⅱ。     

水稻条叶枯病毒(RStV)基因组组分4的克隆与序列分析

利用RTPCR技术合成并扩增了水稻条叶枯病毒(RStV)中国云南分离物基因组组分4的全长cDNA,将PCR产物克隆在载体pCRII上,并进行全序列测定,所得核苷酸序列及推测的氨基酸序列与日本分离物T进行比较。结果表明,在核苷酸水平,两分离物的vORF、vcORF及基因间非编码区序列的同源性分别为94.9%、94.1%、86.1%,5’端非编码区序列相同,而3’非编码区同源性为96.1%,仅有两个核苷酸不同;在氨基酸水平,vORF及vcORF编码蛋白的同源性分别为99.4%和98.3%。可见,编码区的大小及其氨基酸序列和两末端序列都是很保守的。因此,中国云南分离物Y与日本分离物T可能有很近的亲缘关系。     

人乙型肝炎病毒前表面抗原preS基因的克隆与序列分析

从杭州、兰州两地各一例乙型肝炎病毒(HBV)表面抗原阳性血清中提取病毒DNA,采取PCR技术扩增出前表面抗原(preS)基因片段,重组到质粒载体上,对该基因进行了全序列测定[GenBank索取号CpreS-HZAF325674;preS-LZAF325675].克隆的HBVpreS基因杭州分离物(preS-HZ)和兰州分离物(preS-LZ)全长522个核苷酸,编码174个氨基酸.preS-HZ与已发表的HBVadr亚型上海分离物[8]、北京分离物[9]、日本分离物[5]、HBVadw亚型[10]和ayw亚型[11]preS基因的核苷酸序列同源性分别为96.7%、96.2%、97.3%、88.7%和84.1%,氨基酸序列同源性分别为96.0%、94.9%、97.1%、85.1%和85.4%;preS-LZ与相应序列的核苷酸序列同源性分别为96.4%、96.2%、96.9%、88.7%、83.7%,氨基酸序列同源性分别为94.9%、94.9%、96.0%、85.1%、84.1%.分子进化分析(DNASTAR,1999)表明,克隆的两例HBVpreS基因属于adr亚型.preS-LZ与preS-HZ之间有两个核苷酸变异(对应两个氨基酸变异),相对于以上报道的序列二者含有四个特异的氨基酸突变位点.在免疫保护区内二者具有较好的保守性,可用于表达乙肝重组亚单位疫苗.     

人乙型肝炎病毒前表面抗原preS基因的克隆与序列分析

从杭州、兰州两地各一例乙型肝炎病毒（HBV）表面抗原阳性血清中提取病毒DNA,采取PCR技术扩增出前表面抗原（preS)基因片段,重组到质粒载体上,对该基因进行了全序列测定[GenBank索取号CpreS-HZ:AF 325674;preS-LZ:325675].克隆的HBVpreS基因杭州分离物（preS－HZ）和兰州分离物（preS－LZ）全长522个核苷酸,编码174个氨基酸。preS－HZ与已发表的HBV adr亚型上海分离物、北京分离物、日本分离物、HBVadw亚型和ayw亚型preS基因的核苷酸序列同源性分别为96．7％、96．2％、97．3％、88．7％和84．1％,氨基酸序列同源性分别为96．0％、94．9％、97．1％、85．1％和85．4％;preS－LZ与相应序列的核苷酸序列同源性分别为96．4％、96．2％、96．9％、88．7％、83．7％,氨基酸序列同源性分别为94．9％、94．9％、96．0％、85．1％、84．1％,分子进化分析（ADNASTAR,1999）表明,相对于以上报道的序列二者含有四个特异的氨基酸突变位点,在免疫保护区内二者具有较好的保守性,可用于表达乙肝重组亚单位疫苗。     

汉坦病毒K24株M和S片段基因的结构特征

为研究汉坦病毒K24株M和S片段基因的结构特征,从病毒感染细胞提取细胞总RNA,经35个循环的一次性PCR得到了与理论值相符合产物,将RT-PCR产物直接克隆T载体,经K24毒株M片段的全基因序列共3 653个核苷酸,四种核苷酸的酶切和PCR鉴定正确的克隆通过柱纯化后进行序列测定,结果比例分别为A 30.44%,T30.14%,G20.72%,C 18.70%,GC含量为39.42%,AT含量为60.58%,编码1 133个氨基酸.S片段的全基因序列共1 772个核苷酸,四种核苷酸的比例分别为A31.30%,G26.05%,T22.79%,C19.77%,GC含量为45.81%,AT含量为54.19%,共编码429个氨基酸.K24株M片段核苷酸全基因和氨基酸与HTN型毒株同源率分别为70.7%～71.2%和75.9%～76.9%,与SEO型为95.2%～99.2%和95.9%～99.4%.S片段与HTN 76～118和SEO SR-11病毒的比较,核苷酸同源率分别为74.0%和95.6%,氨基酸同源率为83.3%和97.9%,与M片段的结果相类似.M片段氨基酸与SEO型10个毒株存在4个氨基酸替代.应用DNASTAR软件的系统发生分析方法分别绘出了基于M片段核苷酸及推导氨基酸的系统发生树.     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系（PVY－C）HC－Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K326(Nicotina tabacum cv.k326)。PCR和Southern blot分析证明了HC－Pro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HC－Pro中心区域介导转基因烟草中PVC－C和黄瓜花叶病毒（CMV）、PVY－C和马铃薯X病毒（PVX）之间的协生作用,从而明确了PVY－C HC－Pro中心区域为病毒协生作用的功能区域。     

香蕉束顶病毒基因克隆和序列分析

对香蕉束顶病毒（ＢＢＴＶ）中国分离株ＤＮＡ组份Ｉ（ＤＮＡ－１）、外壳蛋白（ＣＰ）和运转蛋白（ＭＰ）基因进行了克隆和序列分析。ＢＢＴＶＤＮＡ－１含有１１０３个核苷酸,与南太平洋和亚洲分离株分别有８７％－８８％ ９６．９－９８％的核苷酸序列同源性。由ＤＮＡ－１编码的复制酶含有１８６个在酸残基。与南太平洋和亚洲分离株分别有８４．４％－９５．８％和９７．６％、９８．０％的氨基酸序列同源性。外壳蛋白基因由５     

Whole Genome Sequence and Characterization of a Novel Isolate of PVY Inducing Tuber Necrotic Ringspot in Potato and Leaf Mosaic in Tobacco

In a previous study on a Syrian isolate of Potato virus Y (PVY), namely PVY-12, a point mutation in the coat protein (CP) was detected. This mutation caused the double reactivity of this isolate to monoclonal antibodies specific to O and N serotypes. We report here the biological and molecular characteristics of PVY-12. In potato, PVY-12 behaved like a PVY^NTN isolate inducing potato tuber necrotic ringspot disease although it induced mosaic in tobacco like PVY^O. The genomic analysis grouped PVY-12 with the recombinant PVY^NTN isolates, which is consistent with the phenotype in potato. PVY-12 HC-Pro had the two amino acids K400 and E419 that were previously reported as determinant keys of the tobacco necrotic response. This indicates the involvement of other determinants in this phenotype yet to be determined. This is the first report on a PVY^NTN isolate that induces mosaic in tobacco, implying that the induction of potato tuber necrosis does not require the ability to induce the tobacco necrosis. PVY-12 genome had four recombinant points in the P1, HC-Pro/P3, 6K2/NIa and C terminal region of the CP gene identical to those of PVY^NTN isolates 12–94 and 34/01. The PVY-12 central genomic part flanked by nucleotide positions 2414 and 8604 had highest similarity with that of the Syrian isolate SYR-NB-16 suggesting a common origin of these isolates. This common origin was supported using the phylogenetic analysis of this region. In addition, the phylogenetic analysis of the whole genome of the reported North American PVY^N:O and the European PVY^NW along with other PVY isolates suggests that PVY^N:O might have descended from PVY^NW with the isolate SASA-207 as a nearest-known relative.     

Book Review

To study the variability and to identify the species of Begomovirus associated with yellow mosaic disease of blackgram in Andhra Pradesh, India, infected blackgram samples were collected from six districts belonging to three regions of Andhra Pradesh. The total DNA was isolated by modified CTAB method and amplified with coat protein gene-specific primers (RHA-F and AC abut) resulting in 900?bp gene product. The PCR products were cloned, sequenced and deposited in GenBank. The sequence analysis of six clones showed that the size of amplified CP gene of YMV was 920?bp. Based on nucleotide sequence identity of six isolates representing three regions of Andhra Pradesh, the isolates from Rayalaseema and Telangana region are the same variant of YMV (>99.5% identity) and isolate from coastal Andhra is another variant of YMV (>95.4%) when compared with other region isolates. Comparison of CP gene sequence of YMV-TPT isolate with 27 other isolates in database revealed more than 93.2 and 86.2% identity with MYMIV isolates and less than 80 and 64% identity with MYMY isolates that originate from Indian sub-continent and South-East Asia at nucleotide and amino acid level, respectively. Phylogenetic tree based on CP gene sequences of six isolates with other isolates from GenBank formed unique cluster with MYMIV. Hence the YMV infecting blackgram in Andhra Pradesh is caused by MYMIV rather than MYMY as reported in Tamil Nadu which is adjoining state in southern India.     

Konjac mosaic virus Naturally Infecting Three Aroid Plant Species in Andhra Pradesh,India

The genomes of three potyvirus isolates from, respectively, naturally infected Colocasia esculenta, Caladium spp. and Dieffenbachia spp. in Andhra Pradesh, India, were amplified by RT‐PCR using degenerate potyvirus primers. Sequence analysis of RT‐PCR amplicons (1599 nucleotides) showed maximum identity of 97% with the KoMV‐Zan isolate of Konjac mosaic virus (KoMV) from Taiwan (A/C AF332872). The three isolates had a maximum identity of 99.4%. The length of coat protein (CP) gene of three isolates was 846 nucleotides encoding 282 amino acids with a deduced size of 32.25 kDa. The CP gene of the isolates had, respectively, 78.1–95.7% and 88.2–96.4% identity at nucleotide and amino acid levels with KoMV isolates. The CP gene of the three isolates had 93.1–100% (nucleotide) and 98.2–100% (amino acid) identity. The 3′‐UTR of the three isolates showed maximum identity of 91.1–100% identity between and with other KoMV isolates. In the CP amino acid–based phylogenetic analyses, the isolates branched as a distinct cluster along with known KoMV isolates. The three potyvirus isolates associated with mosaic, chlorotic feathery mottling, chlorotic spots, leaf deformation and chlorotic ring spots on three aroids were identified as isolates of KoMV for the first time from Andhra Pradesh, India.     

First Report of Soil‐Borne Wheat Mosaic Virus (SBWMV)‐Infecting Triticale in Poland

The virus in naturally infected, stunted triticale plants was identified as soil‐borne wheat mosaic virus (SBWMV). The infected plants were collected in the Southern Wielkopolska region (Western Poland). Molecular analysis including RT‐PCR, and sequencing of the complete coding sequence of coat protein gene, was performed. The sequence of the Polish isolate of SBWMV (SBWMV‐Pol1) shared 100, 99 and 98% identities with the corresponding regions of De1 (AF519799), OKL‐1 (X81639) and US‐Nebraska (L07938) isolates of SBWMV, respectively. Phylogenetic analyses showed that the Polish isolate, SBWMV‐Pol1, clustered together with other SBWMV isolates. This is the first report of the occurrence of SBWMV in Poland and the second of its presence in Europe.     

The helper component proteinase cistron of Potato virus Y induces hypersensitivity and resistance in Potato genotypes carrying dominant resistance genes on chromosome IV

The Nc(tbr) and Ny(tbr) genes in Solanum tuberosum determine hypersensitive reactions, characterized by necrotic reactions and restriction of the virus systemic movement, toward isolates belonging to clade C and clade O of Potato virus Y (PVY), respectively. We describe a new resistance from S. sparsipilum which possesses the same phenotype and specificity as Nc(tbr) and is controlled by a dominant gene designated Nc(spl). Nc(spl) maps on potato chromosome IV close or allelic to Ny(tbr). The helper component proteinase (HC-Pro) cistron of PVY was shown to control necrotic reactions and resistance elicitation in plants carrying Nc(spl), Nc(tbr), and Ny(tbr). However, inductions of necrosis and of resistance to the systemic virus movement in plants carrying Nc(spl) reside in different regions of the HC-Pro cistron. Also, genomic determinants outside the HC-Pro cistron are involved in the systemic movement of PVY after induction of necroses on inoculated leaves of plants carrying Ny(tbr). These results suggest that the Ny(tbr) resistance may have been involved in the recent emergence of PVY isolates with a recombination breakpoint near the junction of HC-Pro and P3 cistrons in potato crops. Therefore, this emergence could constitute one of the rare examples of resistance breakdown by a virus which was caused by recombination instead of by successive accumulation of nucleotide substitutions.     

我国水稻条纹病毒一个强致病性分离物的RNA4序列测定与分析

对我国水稻条纹病毒(Rice Stripe Virus,RSV)一个强致病性分离物(辽宁PJ分离物)的RNA4区段进行扩增、克隆和测序,其核苷酸序列全长2157bp。与已报道的日本T和M分离物及我国云南CX分离物的RNA4序列进行比较分析,结果表明,这4个分离物可分为两组,其中,PJ、T和M分离物为一组,组内分离物之间,RNA4的毒义链(vRNA4)及RNA4的毒义互补链(vcRNA4)上的ORF的核苷酸一致性分别为970%和970%～975%,5′末端和3′末端非编码区的序列则完全一致。但PJ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分离物的等长,核苷酸一致性为930%,比M分离物的IR多了一段长19bp的插入序列,核苷酸一致性仅为850%。另一组为我国CX分离物,组与组之间,vRNA4及vcRNA4上的ORF的核苷酸一致性分别为940%和925%～935%,但在氨基酸水平上则没有明显的差异。CX分离物的IR与PJ分离物相比有一段长84bp的插入序列,组间,IR的核苷酸一致性仅为720%～750%,5′末端非编码区的序列完全一致,但3′末端非编码区有两个碱基的差异。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的分子流行学。     

马铃薯Y病毒蚜传辅助因子促进马铃薯X病毒长距离运输

采用PCR和定点突变法,对马铃薯Y病毒中国株系（Chyinese strain of potato Ypotyvirus,PVY-C)蚜传辅助成分（helper component proteinase,HC-Pro)基因中心区域的CCCT基序和PTK基序进行定点改造,获得了4种突变体。然后将突变体砍降到植物表达载体pBin438中,所得到的重组体通过根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)Conn）介导法转了烟草（Nicotiana tabacum L.cv.K326).Southern blotting和Western blotting分析表明4种突变体已经成功整合到烟草的基因组中,并在蛋白水平上得到了表达。马铃薯X病毒（potato X potexvirus,PVX)对转基因烟草的攻毒实验表明,4种突变体均使PVY－C HYC－Prog严重丧失了促进PVX病毒粒子在寄主体内积累和提高PVX致病性的功能,说明CCCT、PTK基序为PVY－C HYC－Pro介导PVX／PVY协生作用所必需。同时证明了HC-Pro具有增强PVX在寄主体内长距离运输的功能。