番茄黄化曲叶病毒北京分离物侵染性克隆的构建与其致病性

为明确番茄黄化曲叶病毒北京分离物（Beijing isolate of tomato yellow leaf curl virus,TYLCV-BJ）致病性的强弱,以感染TYLCV-BJ的番茄叶片DNA为模板PCR扩增获得该分离物基因组全长序列,并构建该分离物的侵染性克隆,将其分别接种到番茄、烟草和拟南芥植株上,比较该分离物和TYLCV上海分离物2(TYLCV-Shanghai 2,TYLCV-SH2)致病性的差异。结果显示,该分离物基因组全长序列同TYLCV-SH2的相似度为99.03%,在番茄和烟草植株上TYLCV-BJ比TYLCV-SH2发病更早,症状更重,TYLCV DNA和外壳蛋白积累量更高。TYLCV-BJ可以通过农杆菌Agrobacterium tumefaciens注射法在拟南芥中复制和系统侵染,而TYLCV-SH2不能有效侵染拟南芥。表明TYLCV-BJ的致病性强于TYLCV-SH2,所建立的侵染性克隆有广泛的研究和应用价值。     

烟草花叶病毒丁香分离物的分离与鉴定

 从表现花叶症状的丁香病株上获得一病毒分离物,其在电镜下为约300 nm×18nm的杆状粒子;电泳分析表明感病组织中ds RNA大约为6.4kbp,而其外壳蛋白分子量约为17.6k Da。以上实验结果初步将该病毒分离物鉴定为烟草花叶病毒属(Tobamovirus)。根据该属病毒复制酶基因序列设计通用引物,进行RT-PCR检测,扩增出约1000 bp的预期特异片段(Gen Bank AY566703)。将PCR产物克隆后测序,序列分析表明,与从蚕豆中分离的TMV-B株系序列(Gen Bank AJ011933.1)同源性为99.90%。根据烟草花叶病毒(Tobacco mosaic virus,TMV)的RNA CP基因序列设计引物,进行RT-PCR,扩增出约800 bp的预期特异片段(Gen Bank AY56672),序列分析表明,与TMV-B株系序列(Gen Bank AJ011933.1)同源性达99%,上述实验结果表明,该病毒分离物为TMV。由于该分离物与TMV-B在指示植物上的症状存在明显差异,所以,作者把该分离物暂命名为TMV-S。     

葡萄病毒E分子检测及基因序列分析

 皱木复合病(rugose wood complex disease)是一种发生普遍、危害严重的葡萄病毒病,该病与葡萄病毒属(Vitivirus)病毒的侵染密切相关,葡萄病毒E(Grapevine virus E,GVE)是葡萄病毒属的新成员。为明确我国GVE侵染状况以及不同分离物的基因变异情况,本研究针对GVE的MP和CP基因设计了两对引物,RT-PCR结果表明,两对引物均能从带毒样品中扩增到目的基因片段。采用上述引物检测了211株葡萄样品,GVE检出率为5.7%。对7个GVE分离物的外壳蛋白基因进行测序和系统进化树分析,结果显示,上述分离物克隆分别处于2个明显分化的组群(Group I 和Group II)。研究测定了GVE中国分离物GFMG-1的全基因组序列,该分离物与日本分离物TvAQ7核苷酸序列相似度高达98%,但与日本分离物TvP15、南非分离物SA94、美国分离物WAHH2及波兰分离物59PE核苷酸序列相似性仅为70%~75%。本研究为进一步明确我国GVE发生、分布及分子检测提供了依据。     

芝麻坏死花叶病毒的鉴定

 自武昌芝麻坏死花叶病株得到1个病毒分离物,根据该分离物生物学特性、粒体形状、血清学性质、外壳蛋白分子量和核酸组分、大小,明确为自然侵染芝麻的一种新病毒,定名为芝麻坏死花叶病毒(Sesame necrotic mosaic virus,SNMV)。在人工接种7科38种植物中,SNMV侵染6科23种;体外稳定性状:稀释限点10^-4~10^-5,致死温度80~85℃,体外存活期限40d;病毒可通过土壤和接触传播。SNMV病毒粒体球状,表面有粒状突起,直径约32nm。制备病毒抗血清用琼脂双扩散方法测定效价为1:128。SNMV和红三叶草坏死花叶病毒(RCNMV)、甜三叶草坏死花叶病毒(SCNMV)、黄瓜花叶病毒(CMV)和花生矮化病毒(PSV)抗血清没有反应。病毒外壳蛋白亚基分子量为38 100道尔顿;核酸1个组分,大小约4700nt,另含RNA片段,大小约600nt,可能为卫星RNA。依据上述病毒特性,SNMV可能归属于番茄丛矮病毒科(Tombusviridae)。     

烟草脉带花叶病毒cp基因的原核表达及抗血清制备

 利用马铃薯Y病毒属病毒的简并引物, 通过RT-PCR技术获得了云南烟草病毒分离物YND的3'端约1.7 kb片段, 序列分析证明该分离物为烟草脉带花叶病毒(Tobacco vein banding mosaic virus, TVBMV)。将TVBMVcp基因克隆到表达载体pET-22b (+)上, 在大肠杆菌BL21(DE3)中诱导表达出分子量为35.0 kD的融合蛋白。利用该融合蛋白制备了TVBMV的多克隆抗体, ELISA测定抗血清效价为1/4 096。Western blotting和DIBA分析结果表明, 获得的抗血清和原核表达的病毒蛋白及植物病汁液中的病毒均有特异性反应, 可用于TVBMV的快速检测。     

一个马铃薯Y病毒山东分离物的分离与鉴定

 从具有典型花叶症状的马铃薯叶片中分离到马铃薯Y病毒(Potato virus Y,PVY)(本文称PVY-SD-TA分离物),扩繁后,提纯病毒,电镜下可观察到700~900 nm×11 nm的病毒粒体,病组织超薄切片观察可见风轮状的内含体结构,寄主反应特性研究表明其能侵染2科13种植物。SDS-PAGE电泳检测病毒编码的外壳蛋白亚基的分子量为33 kDa。以PVY-SD-TA基因组RNA为模板,应用RT-PCR方法和特异引物合成了外壳蛋白基因。对cDNA全序列分析表明,PVY-SD-TA CP基因核苷酸序列与N株系的同源性为96%,与GenBank中登录序列号为AJ390306的O株系分离物的同源性最高,为99%;与国内不同学者报道的PVY中国流行株的同源性分别为96%,97%和98%。通过以上生物学特性和分子水平的研究将PVY-SD-TA鉴定为普通株系(PVY^O株系)。     

侵染番茄的番茄褪绿病毒山东泰安分离物的分子鉴定和序列分析

2012年秋季, 在山东泰安番茄主要种植区中采集到叶片褪绿, 叶脉颜色变深的疑似番茄褪绿病毒病和番茄侵染性褪绿病毒病的番茄样品。利用番茄褪绿病毒(Tomato chlorosis virus, ToCV)的特异引物 ToCV1/ToCV2和番茄侵染性褪绿病毒(Tomato infectious chlorosis virus, TICV)的特异引物TICV1/TICV2分别对样品进行扩增, 最后仅得到利用引物 ToCV1/ToCV2 扩增的101 bp 的核苷酸序列, 对该核苷酸序列克隆并测序。序列比对表明, 山东泰安地区分离物与已登录的番茄褪绿病毒(ToCV)分离物相似性都在99%以上。随后, 对山东泰安种植区ToCV番茄分离物进行外壳蛋白(CP)及热激蛋白(HSP70)序列的扩增、克隆和测序(GenBank登录号KC812620/KC812625), 经NCBI BLAST比对发现, 目的序列与番茄褪绿病毒日本番茄分离物ToCV Japan/Tochigi (GenBank登录号AB513442/AB513443)相似性最高为99%, 同属于毛型病毒属的番茄褪绿病毒, 这是首次明确山东地区番茄受到番茄褪绿病毒的侵染。     

烟草蚀纹病毒山东分离物全基因组序列的克隆和保守性分析

为利用RNA介导的病毒抗性策略,培育抗性稳定或抗多烟草蚀纹病毒（Tobacco etch virus,TEV）株系的转基因植株,采用RT-PCR及5＇-RACE方法克隆了烟草蚀纹病毒山东分离物TEV-SD1的全基因组序列。TEV-SD1全基因组核苷酸序列长度为9494 bp,包含1个9165 bp的开放阅读框架（open reading frame,ORF）,编码3054个氨基酸。将TEV-SD1基因组序列与GenBank中已公布的4个TEV全基因组序列和11个外壳蛋白（coat protein,CP）基因序列比对分析发现,各分离物CP基因间的核苷酸和氨基酸序列平均相似性分别为96.65%和98.31%,高于其它功能基因间的相似性;各分离物CP基因3＇端核苷酸序列相似性平均为96.55%,高于5＇端序列。聚类分析发现TEV在自然界中的分子变异与其寄主关系密切。     

黄瓜绿斑驳花叶病毒北京和山东分离物的生物学测定及其基因组比较

为揭示黄瓜绿斑驳花叶病毒的分子流行学特征,通过RT-PCR和cDNA克隆技术获得并分析了从北京甜瓜和山东西瓜上分离的2个黄瓜绿斑驳花叶病毒(CGMMV)分离物CGMMV-Beijing和CGMMV-Shandong的全序列.结果表明CGMMV-Beijing和CGMMV-Shandong的基因组分别由6423和6427个核苷酸组成,包括5'及3'端非编码区和4个开放性的阅读框,分别编码129kDa和186kDa复制相关蛋白、29kDa的移动蛋白及17.4kDa的外壳蛋白.寄主范围测定显示在供试的6科11种植物中CGMMV-Beijing和CGMMV-Shandong均可侵染葫芦科的葫芦、南瓜和西瓜以及茄科的本氏烟和藜科的苋色藜,不侵染供试的其它植物.CGMMV-Beijing和CGMMV-Shandong基因组的核苷酸同源性为99.5%,186kDa蛋白的核酸和氨基酸同源性分别为99.7%和99.8%,移动蛋白的核酸和氨基酸同源性分别为99.3%和98.1%,外壳蛋白的核苷酸同源性为100%.多序列比对分析结果显示,CGMMV的各分离物之间的分子差异并不明显,核苷酸同源性均在98%以上.将CGMMV-Beijing和CGMMV-Shandong与侵染葫芦科植物的烟草花叶病毒属其它成员比较,结果显示其亲缘关系较远,基因组的核苷酸同源性介于56.7%～59.6%,外壳蛋白的氨基酸同源性在44.7%～54.0%之间.综合分析显示,CGMMV分离物之间的差异并不明显,可能具有共同的侵染来源.     

胜红蓟上一种与双生病毒伴随的重组β卫星分子的鉴定

双生病毒是一类在全球范围内危害严重的单链环状DNA病毒。本研究从2017年采自云南的胜红蓟样品中(YN2017)获得了一条菜豆金色花叶病毒属病毒和一条β卫星的全基因组序列。该双生病毒的全长序列与烟草曲茎病毒的相似性最高,为99.60%,确定为烟草曲茎病毒的分离物。YN2017β卫星与中国胜红蓟黄脉β卫星的同源性最高,为90.8%。进一步分析发现,该β卫星的卫星保守区域(SCR)至富含腺嘌呤区(A-rich区)之间约1 kb的序列与中国胜红蓟黄脉β卫星相应序列的相似性高达97.2%;其包含的A-rich区上游与SCR之间约300 bp的序列与中国胜红蓟黄脉β卫星相应序列的相似性仅为70.2%,而与烟草曲茎β卫星相应序列的相似性最高,为97.3%。重组分析发现,所分离的β卫星是由中国胜红蓟黄脉β卫星和烟草曲茎β卫星重组产生。这是首次在中国发现由不同的β卫星重组产生的β卫星分子。     

Molecular identification of a begomovirus causing yellow vein disease on Calendula officinalis in India

A whitefly transmitted begomovirus was detected by PCR using begomovirus-specific primers from naturally infected Calendula officinalis plants showing yellow vein disease symptoms. An approximately 800 bp PCR amplicon was cloned and sequenced to identify the species of the virus isolate. Analysis of nucleotide sequence data resulted in its identification as the complete coat protein gene open reading frame (CP ORF) of 771 bp, which encoded 256 amino acid residues. The coat protein of the virus isolate shared maximum identities of 96–97% with four strains of Tobacco curly shoot virus (ToCSV) and an Ageratum enation virus (AgEV) during BLAST analysis of sequence data. Nucleotide- and amino acid-based phylogenetic analysis revealed the close relationship of the isolate with ToCSV strains, therefore it has been identified as an isolate of ToCSV and C. officinalis is considered to be a new host of ToCSV begomovirus. Association of a DNA-β molecule with the virus isolate was also detected by PCR and Southern hybridization tests using DNA-β specific primers and probe.     

从新疆哈密瓜分离到的烟草坏死病毒及其理化性质的研究

 从自然感病的哈密瓜上分离到的烟草坏死病毒(TNV),其寄主范围较广,在藜科、苋科、豆科和茄科一些植物上呈局部坏死反应,并在葫芦科一些植物上呈局部坏死斑和系统花叶症状。病毒的热灭活点为90~95℃,稀释限点10-5~10-6,体外保毒期在10天以上。病毒颗粒呈六角形,直径约25毫微米。提纯病毒的紫外吸收,最高260毫微米,最低为245毫微米;沉降常数为94s。从病毒提取的核酸的紫外吸收,最高为260毫微米,最低234毫微米;经聚丙烯酰胺凝胶电泳测定,核酸分子量为1.4×10⁶;病毒蛋白的分子量为2.75×10⁴。经血清学试验,该病毒与烟草花叶病毒的A型和D型的抗血清发生沉淀反应,而与黄瓜坏死病毒的抗血清无沉淀反应。     

Complete nucleotide sequence and latency of a novel blueberry-infecting closterovirus

A novel latent closterovirus was detected from highbush blueberry in Japan and provisionally named blueberry virus A (BVA). The BVA genome (17,798 nucleotides) contains 10 open reading frames, but no minor coat protein could be identified in the virus genome. The BVA RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h), and major coat protein (CP) shared the highest amino acid sequence identities with those of viruses in the genus Closterovirus (61.2, 27.6, and 20.9 %, respectively). In a phylogenetic analysis of the RdRp, HSP70h, and CP, BVA did not cluster with any genus in the family Closteroviridae.     

Properties of a citrus isolate of olive latent virus 1, a new necrovirus

A virus was recovered by sap transmission from plants of several citrus species exhibiting or not symptoms of chlorotic dwarf (CCD), a disease recently reported from Eastern Mediterranean Turkey. The virus was identified as an isolate of olive latent virus 1 (OLV-1), originally described as a possible sobemovirus. The citrus isolate of OLV-1 (OLV-1/Tk) possesses biological, morphological, physico-chemical, and ultrastructural properties similar, if not identical to those of the OLV-1 type strain and is also serologically indistinguishable from it. In addition, OLV-1/Tk has many properties, especially physico-chemical, in common with serotypes A and D of tobacco necrosis necrovirus (TNV-A and TNV-D). However, OLV-1/Tk is only very distantly related serologically to both TNV-A and D, suggesting that it can be regarded as a distinct species in the genusNecrovirus. OLV-1/Tk could not be detected in citrus tissues by ELIS A or dot-blot molecular hybridization, probably because of the extremely low virus concentration. By contrast, limited virus recovery was obtained by sap inoculation and fair detection rates were afforded by PCR. OLV-1/Tk was identified in 54 of 92 (59%) citrus plants affected by CCD and in 14 of 49 (28%) symptomless plants. These results do not support the notion that there is a cause-effect relationship between OLV-1/Tk and CCD, even though the more frequent association of this virus with diseased plants remains intriguing.     

Nucleotide Sequence of the 3′ -terminal Region of Carnation vein mottle virus RNA

The 2326 nucleotides of the 3′-terminal region of Carnation vein mottle virus (CVMV) RNA, which included part of the nuclear inclusion b gene, the complete coat protein (CP) gene and the entire 3′-noncoding region (3′-NCR) were determined. The region encoding the CP gene is 843 nucleotides long and the deduced protein consists of 280 amino acids. A search of the EMBL and PIR databases showed that the amino acid sequence of CVMV CP most resembled that of Plum pox virus with a similarity of 67.9%. The 3′-NCR of CVMV RNA is 541 nucleotides long, second longest in the genus Potyvirus. These results indicate that CVMV is closely related to Plum pox virus but is a distinct species in the genus Potyvirus. Received 8 October 1999/ Accepted in revised form 9 January 2000     

玉米小斑病菌中一种维多利亚病毒的分离及其基因组全序列分析

 从玉米小斑病菌(Bipolaris maydis)中检测发现一种dsRNA (double-stranded RNA)病毒,暂命名为Bipolaris maydis victorivirus 2 (BmV2)。电子显微镜下观察到病毒粒子为二十面体球状,直径为40 nm左右、无包膜;病毒基因组为单条dsRNA核酸分子,长度为5 222 bp,其基因组结构与其他维多利亚病毒相似,包含两个开放阅读框(ORFs)。序列BLASTx分析发现,BmV2的基因组核酸序列与Coniothyrium minitans RNA virus Illinois isolate (CmRV-IL)具有较高的同源性(78.15%),后者CmRV-IL是单分体病毒科(Totiviridae)维多利亚病毒属(Victorivirus)的暂定种,两者的外壳蛋白(CP)和RNA依赖性RNA聚合酶(RdRp)的氨基酸序列之间的同源性分别为88.02%和89.87%,说明BmV2、CmRV-IL是同一种病毒的不同分离物。基于BmV2和选择的单分体病毒的RdRp氨基酸序列的系统发育分析表明,BmV2与单分体病毒科维多利亚病毒属中的病毒形成了一个单系进化支。