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。     

黄瓜绿斑驳病毒河北分离物基因组克隆及序列分析

为揭示河北省黄瓜绿斑驳病毒(Cucumber green mottle mosaic virus,CGMMV)分类地位及系统进化情况,通过分子克隆测定了来自河北省西瓜产区分离获得的分离物CGMMV-chb的近全基因组序列,分析其基因组结构特征;并依据近全基因组核苷酸序列及外壳蛋白氨基酸序列进行了系统进化关系分析。结果表明,CGMMV-chb近全基因组大小为6 383 nts,Gen Bank登录号为KJ658958,含4个开放阅读框,编码4种蛋白;与Gen Bank库中的其它12个CGMMV分离物的核苷酸相似性为92%~100%,氨基酸相似性为98%~100%。CGMMV-chb与韩国分离物CGMMV-KW基因序列相似性最高,为98%~100%,与以色列分离物CGMMV isolate Ec基因序列相似性最低,为92%~99%;系统进化关系中CGMMV-chb与其它中国分离物、日韩分离物亲缘关系较近,处于同一亚组的不同分支中,与以色列分离物亲缘关系相对较远。     

瓜类褪绿黄化病毒新疆分离物基因组分析

2016年秋季,新疆维吾尔自治区吐鲁番市哈密瓜大面积发生叶片黄化和褪绿的病害,症状呈瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)引起的典型特征。通过CCYV的特异性引物对该疑似样本进行了RT-PCR检测,获得了预期大小的扩增片段。随后,对新疆的CCYV分离物(CCYV-Xinjiang)全长基因组进行克隆并测序,获得了CCYV-Xinjiang分离物的RNA1和RNA2全长序列信息。对CCYV-Xinjiang和来自Gen Bank数据库的其他CCYV分离物的基因组序列分析,不同分离物RNA1和RNA2两条链的一致性分别是99.65%~99.93%和99.68%~99.89%。单独对基因组的各个非翻译区(UTRs)和编码区的核苷酸变异分析,数据表明不同区域其变异程度不同,其中RNA2的3'UTR区域变异最大,而RNA1的2个UTRs区域变异最小。对已登录Gen Bank的所有CCYV分离物基因组系统进化树分析结果表明,所有分离物聚在同一个组。另外,对来自Gen Bank的18个分离物的外壳蛋白(CP)氨基酸序列进行系统进化树分析,表明来自伊朗的3个分离物分在组2(GroupⅡ),而来自亚洲其他地区的分离物均被分到组1(GroupⅠ)。基于CCYV基因组的信息,发现来自亚洲地区的CCYV的群体遗传变异很小。     

芜菁花叶病毒欧亚分离物HC-Pro基因的克隆和序列分析

 16个芜菁花叶病毒(Turnip mosaic virus,TuMV)欧亚分离物分别来自奥地利、丹麦、德国、匈牙利、尼泊尔和英国6国。利用免疫捕获反转录PCR(Immunocapture RT-PCR,IC-RT-PCR)对16个分离物的HC-Pro(Helper component pro-teinase)基因进行PCR扩增,扩增产物克隆后进行序列测定,HC-Pro基因序列长度均为1374个核苷酸,编码458个氨基酸。16个分离物的HC-Pro基因核苷酸序列同源性为79.5%~99.8%,所编码的氨基酸同源性为94.1%~99.8%。对16个分离物及GenBank上已报道的其它14个TuMV的HC-Pro基因核苷酸的系统进化树分析表明:在16个TuMV欧亚分离物中,除了来自亚洲的分离物N23属Asian-BR组,其余15个来自欧洲的分离物都属于world-B组,其中分离物H1归属world-wide亚组,另外14个分离物则归属New World亚组。     

甘薯病毒C中国分离物全基因组序列测定及比较分析

利用RT-PCR结合RACE方法,从采自河南南阳的甘薯样品上获得甘薯病毒C中国分离物(SPVC-Ch1)的全长基因组序列。序列分析结果表明,SPVC-Ch1基因组由1 0846个核苷酸组成,3'末端包含poly(A)尾序。基因组含有1个由10 446个核苷酸构成的开放阅读框,编码一个由3 481个氨基酸残基构成的393 k Da多聚蛋白。将SPVC-Ch1与Gen Bank中登录的其他SPVC分离物序列进行比较分析发现,SPVC不同分离物间全基因组核苷酸序列相似性为92.7%~98.9%,多聚蛋白的氨基酸序列相似性为95.1%~99.2%,SPVC-Ch1与Bungo分离物的相似性最高,与C1分离物的相似性最低。系统进化树分析结果表明,SPVC-Ch1与日本的Bungo、以色列的IL、韩国的CW135和UN202等分离物形成一个分支,亲缘关系较近。这是SPVC中国分离物全基因组序列的首次报道,研究结果丰富了SPVC全基因组序列信息,有助于全面了解SPVC种群的遗传进化关系。     

烟台市富士苹果上苹果锈果类病毒分子变异分析

为明确苹果锈果类病毒(Apple scar skin viroid,ASSVd)在烟台市富士苹果上的变异情况,通过特异性引物对携带苹果锈果类病毒的富士嫩叶进行ASSVd全长扩增,利用生物学软件DNAMAN对所得变异序列进行分析并构建系统进化树。结果表明,从130个样品中筛选到36个阳性样品,36个阳性样品中共克隆获得52条329~333 nt的ASSVd变异序列,其中30个阳性样品含2条或2条以上的ASSVd序列。对所得变异序列进行序列比对发现,同一样品不同克隆间核苷酸序列相似性为94.0%~100.0%,所有变异序列核苷酸序列相似性为94.0%~99.7%,与Gen Bank中来自不同国家或地区的10条ASSVd分离物核苷酸序列相似性为88.3%~99.7%。将序列相似性低于97.0%的12条变异序列进行系统进化分析,结果显示除了333 nt变异序列ZS2-6与伊朗苹果分离物在同一分支外,其余11条变异序列位于同一分支。52条变异序列多序列比对发现,变异位点主要集中在TL区、P区和C区。研究表明烟台市富士苹果上ASSVd存在一定的分子变异。     

芜菁花叶病毒对油菜致病力差异及壳蛋白基因序列分析

 2004年春季参试的湖北、安徽2省11个芜菁花叶病毒(Turnip mosaic virus,TuMV)分离物感染4个油菜品种,病情指数幅度为26.2~76.0;秋季参试5个TuMV分离物感染14个油菜品种,病情指数幅度为38.3~55.9,均值方差分析表明,致病力差异分别达到极显著和显著水平。壳蛋白(CP)基因序列分析表明,来源于2省油菜、白菜、红菜薹、芝麻和萝卜的17个TuMV分离物与浙江分离物ZJB3序列同源性在97%以上,同属于MB类群;而另一个萝卜分离物WRS1与ZJR1、CH1和CH2分离物序列同源性在95.4%~98.7%之间,属于MR类群。类群间分离物序列同源性仅为88.0%~92.2%。遗传进化树分析表明,萝卜分离物WRS2在MB类群中单独构成一个分支,可能是MR类群和MB类群发生重组的后代。     

北京地区茄子感染番茄褪绿病毒的分子鉴定

为明确北京地区茄子感染番茄褪绿病毒(Tomato chlorosis virus,To CV)的情况,于2015年3—6月收集了10份疑似感染To CV的设施茄子样品,通过PCR分子鉴定法进行检测,并进一步对扩增阳性样品To CV的CP基因采用邻接法进行了系统进化分析。结果表明,10个检测样品中有4个扩增得到约463 bp的特异条带,经测序与Gen Bank中To CV序列相似性达到99.0%以上,表明这4个样品均携带有该病毒,检出率为40%;To CV茄子分离物的CP基因序列大小为774 bp,Gen Bank登录号为KT751008,与To CV北京番茄分离物(KC311375)同源性最高,达到99.9%,确认该序列为To CV片段;系统进化树显示,To CV茄子分离物与To CV日本分离物(AB513443)处于同一分支,具有密切的亲缘关系,表明不同国家To CV分离物之间的亲缘关系与地理距离具有一定的相关性。     

Analysis of genetic relations between <Emphasis Type="Italic">Broad bean wilt virus 1</Emphasis> and <Emphasis Type="Italic">Broad bean wilt virus 2</Emphasis>

We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)     

Tobacco rattle virus serotypes and associated nematode vector species of Trichodoridae in the bulb-growing areas in the Netherlands

Soil samples from the coastal bulb-growing areas in the provinces of North- and South-Holland and the North-East Polder in the Netherlands were examined for trichodorid nematodes and tobacco rattle virus (TRV) serotypes. At least one of a total of eight species of Trichodoridae, of whichParatrichodorus pachydermus was most prevalent, was found in 93% of the samples from the provinces of North- and South-Holland and TRV, including four serotypes, was obtained from 49% of these samples. In the North-East Polder one of three species of trichodorids, of whichP. teres occurred most frequently, was present in 72% of the samples, and TRV of one serotype was obtained from 28% of these samples. The TRV isolates recovered from these samples reacted serologically with one of four antisera to strains of TRV. Virus transmitted byP. pachydermus reacted to the PRN-, byTrichodorus viruliferus to the RQ-, byP. teres to the N5- and byT. similis, to the TS-antiserum, respectively.     

Characterization and genetic status of Banana bunchy top virus isolated from Okinawa, Japan

Isolates of Banana bunchy top virus (BBTV), which causes bunchy top disease in bananas, were collected in field surveys on seven islands in Okinawa Prefecture, Japan. From 44 banana samples, one isolate from each island was selected, and the DNA-1 and DNA-3 components were sequenced. Analysis of the major common region of DNA-1 showed that BBTV in Okinawa belongs to the Asian group of BBTV. DNA-1 and DNA-3 analysis revealed that Okinawan BBTV had a closer relationship with isolates from Taiwan and the Philippines than with some isolates from China and Vietnam. All the Okinawan BBTV isolates had high homology in the nucleotide sequences of DNA-1 and DNA-3 (%) because of a single, recent BBTV invasion of this area.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB108449 – AB108458     

Characterization and vector relation of a serologically distinct isolate of tobacco rattle tobravirus (TRV) transmitted byTrichodorus similis in northern Greece

A virus isolated from diseased tobacco plants growing in Macedonia, northern Greece, had host range and physico-chemical properties typical of a tobravirus. Although it was serologically unrelated to any of the ten tobravirus isolates tested, it reacted in spot hybridization tests with a probe derived from RNA-1 of tobacco rattle virus (TRV) strain SYM. Therefore, the isolate probably represents a previously undescribed serotype of TRV. Male, female and juvenileTrichodorus similis nematodes recovered from the rhizosphere of the diseased tobacco plants transmitted TRV in each of three laboratory experiments. In two of these experiments 50% and 54%, respectively, of the nematodes transmitted virus toPetunia hybrida bait plants, whereas only 18% transmitted virus toNicotiana tabacum plants in a third test. Ultrathin sections of the feeding apparatus of individual nematodes, which had transmitted virus, were examined by electron microscopy. Virus particles were observed, retained as a monolayer in the apices of the oesophageal lumen and as a group of particles within a matrix in the open part of the lumen.     

Biological and molecular characterization of a distinct Citrus tristeza virus isolate originating from a lemon tree in Greece

A Citrus tristeza virus (CTV) isolate (L192GR) naturally occurring in lemon trees of more than 100 years old in Greece was fully characterized. Virus‐derived small interfering RNAs, induced by Dicer processing of dsRNAs formed during RNA virus replication, were isolated and used as targets for sequencing. Next‐generation high‐throughput sequencing using the Ion Torrent platform was performed. A total of 432 632 sequences, 94·05% of which corresponded to L192GR, were determined. Subsequent bioinformatics analysis enabled the determination of the full‐length 19 251 nt genome of the L192GR isolate (GenBank no. KC262793 ). Comparative analysis of complete genomes revealed molecular homology with CTV‐VT isolate FS2‐2 from Florida (GenBank no. EU937519 ) with 98·2% nucleotide sequence identity. Recombination events were detected in L192GR and they probably contribute to its unique characteristics. Specifically, although most isolates of the CTV‐VT group induce the seedling yellows syndrome and react positively with the monoclonal antibody MCA13, which is typically associated with severe CTV isolates, the MCA13‐positive L192GR gave very mild or even no symptoms in the seedling yellows indicator plants. Furthermore, experimental aphid transmissibility studies revealed a poor transmission efficiency of 20%. This is the first report of a CTV isolate originating from a lemon tree being fully characterized at biological, serological and molecular levels. The present study further confirms that, when the goal is the risk assessment associated with a new pathogen or isolate in a particular area, molecular data have to be combined with the biological properties of the pathogen.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454