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。     

Serendipitous identification of a new Iflavirus‐like virus infecting tomato and its subsequent characterization

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive‐sense ssRNA molecule of 8506 nucleotides. It is predicted to encode a single polyprotein of 314·5 kDa that is subsequently processed into three coat protein components of 13·7, 17·9 and 13·5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease and RNA‐dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19–47% identity). Evidence of replication in plants was detected by RT‐PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. The name Tomato matilda virus (TMaV) is proposed, and furthermore, that the genus Tomavirus (Tomato matilda virus) be created within the family Iflaviridae. This is the first report of a plant‐infecting virus resembling members of the Iflaviridae.     

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002     

稻瘟菌双分病毒MoPV2特性研究

稻瘟病是稻瘟菌引起的一种世界性病害,每年造成水稻大幅减产。真菌病毒是指能够在真菌体内进行复制和繁殖的病毒,可以作为生物防治的资源。本实验从发病的水稻叶片上采用单孢分离的方法获得纯化培养的稻瘟菌菌株YC13。菌株YC13携带多条dsRNA片段,其中片段F1与Magnaporthe oryzae virus 2(MoV2)的Coat Protein(CP)和RNA-dependent RNA polymerase(RdRp)的氨基酸序列均具有99%的同源性,确定为MoV2的基因组;片段F4、F5序列与双分病毒Penicillium stoloniferum virus F(Ps V-F)的RdRp和CP的氨基酸序列分别具有69%和63%的同源性,命名为Magnaporthe oryzae partitivirus virus 2(MoPV2)。MoPV2的基因组含有2条dsRNA:dsRNA 1编码RdRp,dsRNA 2编码CP。根据MoPV2的RdRp编码的氨基酸序列将MoPV2归属于双分病毒科(Partitiviridae)G ammapartitivirus属,是一种新的真菌病毒。通过病毒粒子转染,将MoPV2转到稻瘟菌菌株RB11。与菌株RB11相比,带毒菌株RB11T25的分生孢子产生量显著减少,并且对大麦活体的致病性也有所降低,但其他生物学特征均无明显的影响。本研究在稻瘟菌中发现了一种新的真菌病毒,该病毒能够使稻瘟菌致病力下降,为稻瘟病的防治提供了潜在的病毒资源。     

北方四省区番茄褪绿病毒的分子鉴定

2015-2016年间,在山西省、内蒙古自治区、辽宁省、吉林省采集到疑似感染番茄褪绿病毒Tomato chlorosis virus(ToCV)的番茄植株。利用扩增ToCV外壳蛋白(coat protein,CP)和类热激蛋白(heat shock protein 70homolog,HSP70h)基因片段的两对特异性引物,通过RT-PCR对疑似样品进行分子检测,得到970bp和1 864bp的特异条带,经测序、比对确定为ToCV。序列分析表明,山西晋中分离物SXJZ(KX853540)的CP核苷酸序列与河南安阳分离物HNAYHX(KP264983)相似性为99.7%,内蒙古呼和浩特分离物NMHHHT(KU204709)和辽宁大连分离物LNDL(KU204707)的CP核苷酸序列与国内已报道的山东寿光分离物SDSG(KC709510)相似性分别为99.5%和99.7%,吉林长春分离物JLCC(KU306111)的CP核苷酸序列与山东聊城分离物SDLC(KC812622)的相似性为99.8%。而HSP70h核苷酸序列的相似性分析表明,山西晋中分离物SXJZ(KX853539)与日本分离物Tochigi(AB513442)相似性为99.4%,内蒙古呼和浩特分离物NMHHHT(KU204710)、辽宁大连分离物LNDL(KU204708)和吉林长春分离物JLCC(KX880384)与山东寿光分离物SDSG(KC709510)相似性分别为99.7%、99.8%和99.7%。试验结果明确了番茄褪绿病毒已蔓延传播到我国山西、内蒙古及东北地区。     

小西葫芦黄花叶病毒北京分离物的鉴定及其基因组3''端特性分析

在北京东郊自然感病的南瓜Cucurbita moschata上获得一病毒分离物（BJ-1），经生物学、血清学和分子生物学鉴定，确定为小西葫芦黄花叶病毒（Zucchini yellow mosaic virus，ZYMV）。为分析其基因组3’端特性，以发病叶片中提取的总RNA为模板，对基因组3’端进行RT-PCR扩增，产物克隆到pMD18-T栽体上进行序列分析，共测定了该病毒分离物包括全部CP基因在内的1269bp。该分离物CP基因由837个核苷酸组成，编码279个氨基酸。对包括该分离物在内的30个序列的760bp（含NIb基因3’端56bp和CP基因中的704bp）片段、NIb蛋白与CP蛋白的切割位点、蚜传必需基序的变异、寄主来源及地域来源进行了分析。结果表明，ZYMV不同分离物的基因分型与上述五个因素无明显关系。     

油桃茎痘相关病毒中国分离物基因组的分子特征分析

为明确我国油桃茎痘相关病毒(nectarine stem-pitting-associated virus,NSPaV)基因组的分子特征,利用RT-PCR和RACE技术对NSPaV中国分离物NSPaV-T04基因组进行克隆,采用最大似然法对得到的NSPaV基因组序列和GenBank中的5条NSPaV基因组序列构建系统发育树,应用RDP软件对NSPaV基因组序列进行重组分析。结果表明:中国分离物NSPaV-T04基因组序列全长为4 991 nt,包括4个开放阅读框(open reading frame,ORF),其中ORF1与ORF2共同编码1个RdRp P1-P2融合蛋白,ORF3编码1个CP,ORF5与ORF3共同编码CP通读蛋白。系统发育树和序列比较分析结果显示,中国分离物NSPaV-T04(MN095353)与美国分离物NSPaV/12P42(KT273410)的亲缘关系最近,核苷酸序列同源性最高,为96.4%;NSPaV-T04的RdRp P1变异较大,与GenBank中5条NSPaV基因组核苷酸序列的同源性为90.5%~96.1%,CP较为保守,核苷酸序列的同源性为96.6%~98.7%。重组分析结果显示,中国分离物NSPaV-T04为鉴定的一个重组体(韩国分离物SK)的亲本序列,表明中国分离物NSPaV-T04可能是一个实际的重组体。     

A novel member of the genus Nepovirus isolated from Cucumis melo in Japan

An unusual virus was isolated from a Japanese Cucumis melo cv. Prince melon plant showing mild mottling of the leaves. The virus had a broad experimental host range including at least 19 plant species in five families, with most infected plants showing no symptoms on inoculated and uninoculated systemically infected leaves. The virus particles were spherical, approximately 28 nm in diameter, and the coat protein (CP) had an apparent molecular mass of about 55 kDa. The virus possessed a bi-partite genome with two RNA species, of approximately 8,000 and 4,000 nucleotides. Both genome components for the new virus were sequenced. Amino acid sequence identities in CP between the new virus and previously characterized nepoviruses were found to be low (less than 27%); however, in phylogenetic reconstructions the closest relationship was revealed between the new virus and subgroup A nepoviruses. These results suggest that the new virus represents a novel member of the genus Nepovirus. A new name, Melon mild mottle virus, has been proposed for this new virus.     

陕西杨凌番茄褪绿病毒的分子检测

近年来在陕西番茄生产区出现了一种新病害,病株表现为叶片褪绿,叶脉颜色变深及叶片增厚,果实变小发白,严重影响番茄产量及经济效益,2016年发病极为严重,部分产区甚至绝收。该病疑似由番茄褪绿病毒Tomato chlorosis virus(ToCV)引起。利用ToCV特异引物对感病番茄样品进行RT-PCR检测,结果从所采集的4份病样中均检测到ToCV预期大小的特异片段。对ToCV外壳蛋白CP基因和类热激蛋白HSP70h基因进行克隆与序列分析,ToCV-YL1 CP基因774个核苷酸序列与已报道的ToCVCP基因有较高的一致性,与山东青岛和山西晋中分离物同源性为100%。ToCV-YL1 HSP70h基因1 665个核苷酸序列与已报道的ToCV HSP70h基因有较高的同源性,与中国、韩国、日本、美国、希腊分离物同源性达到99%以上。研究表明ToCV已传播至陕西地区。     

基于全基因组序列的黄瓜绿斑驳花叶病毒重组和系统发育分析

 通过RT-PCR扩增了黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)济南分离物(CGMMV-JN)的基因组片段。序列测定结果表明CGMMV-JN基因组全长6 424核苷酸(nt),5′-和3′-UTR分别为60和176 nt,含有4个ORF,分别编码129 kDa和186 kDa复制酶相关蛋白、29 kDa移动蛋白及17.4 kDa外壳蛋白。CGMMV-JN与另外29个CGMMV分离物全基因组核苷酸序列一致率为90.0%~99.7%。重组分析发现韩国KOM(AF417243)、以色列EC(KF155231)、印度(DQ767631)和我国河北的CHB(KJ658958)4个分离物在RdRp编码区存在重组。系统发育分析结果表明,这些分离物可分成3个组。选择压力分析结果表明cp基因处于正选择,其它基因处于负选择。本文研究的结果为黄瓜绿斑驳花叶病毒的监测及防控提供了理论依据。     

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     

Nucleotide sequence of the coat protein gene of Mirafiori lettuce virus

 The coat protein (CP) gene of Mirafiori lettuce virus (MiLV), a tentative member of the genus Ophiovirus was isolated and sequenced. The established sequence consists of 1514 nucleotides including one open reading frame (ORF) with 1311 nucleotides that encodes 437 amino acids with a relative molecular mass 48 543. When the ORF was expressed in Escherichia coli, the obtained protein was confirmed as CP by Western blotting using an antiserum against MiLV. Database searches showed that the CP gene of MiLV has a sequence similar to that of Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus. The comparison between MiLV and CPsV CP genes revealed that the identities of the nucleotide and amino acid sequences were 46.5% and 30.9%, respectively. Received: July 29, 2002 / Accepted: October 2, 2002     

Molecular variability of Spanish and Hungarian isolates of Tomato torrado virus

The population structure and genetic variation of Tomato torrado virus (ToTV) were estimated from 19 Spanish isolates collected from 2001 to 2009 in different tomato‐production areas by analyses of the partial nucleotide sequences of five regions of the virus genome: the protease cofactor (Pro‐Co) and the RNA‐dependent RNA polymerase (RdRp) in RNA1, and the movement protein (MP) and two subunits of the coat protein (CP; viz. Vp35 and Vp23) in RNA2. Three Hungarian isolates of the virus were also included in the analyses. All the ToTV isolates clustered together in the phylogenetic analysis of the nucleotide sequences of the different regions. However, some genetic diversity was observed in the case of the two CP subunits among the Gran Canaria isolates and the remaining ToTV‐isolates analysed, which grouped together. A high similarity was observed among all the isolates and the two published ToTV isolates: the ToTV type isolate (PRI‐ToTV0301) and the Polish isolate Wal03. The most variable encoding regions studied were those on RNA2. In general, no correlation was found between genetic diversity and collection date. Studying the genetic distances between pairs of sequences, the ratio between nonsynonymous (amino‐acid‐replacing) and synonymous (silent mutational) substitutions was low, indicating a strong negative selective pressure in the studied regions. Nine negatively selected sites (distributed in Pro‐Co, MP, Vp23 and Vp35) and just one positively selected one (in Pro‐Co) were found for all the genome regions studied.     

Partial characterisation of a novel <Emphasis Type="Italic">Tobamovirus</Emphasis> infecting <Emphasis Type="Italic">Actinidia chinensis</Emphasis> and <Emphasis Type="Italic">A. deliciosa</Emphasis> (Actinidiaceae) from China

Actinidia chinensis and A. deliciosa plants from China, showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots, were found to contain ~300 nm rod-shaped virus particles. The virus was mechanically transmitted to several herbaceous indicators causing systemic infections in Nicotiana benthamiana, N. clevelandii, and N. occidentalis, and local lesions in Chenopodium quinoa. Systemically- infected leaves reacted with a Tobacco mosaic virus polyclonal antibody in indirect ELISA. PCR using generic and specific Tobamovirus primers produced a 1,526 bp sequence spanning the coat protein (CP), movement protein (MP), and partial RNA replicase genes which showed a maximum nucleotide identity (88%) with Turnip vein clearing virus and Penstemon ringspot virus. However, when the CP sequence alone was considered the highest CP sequence identity (96% nt and 98% aa) was to Ribgrass mosaic virus strain Kons 1105. The morphological, transmission, serological and molecular properties indicate that the virus is a member of subgroup 3 of the genus Tobamovirus.     

Characterization and detection of a novel idaeovirus infecting blackcurrant

A novel virus was discovered in a blackcurrant accession (Ribes nigrum L.) at the USDA genebank in Oregon, USA. The genome consists of two positive-sense, single-stranded RNAs with the first encoding a 197 kDa multifunctional protein with methyl transferase, helicase and RNA-dependent RNA polymerase enzymatic motifs. The second molecule encodes two putative proteins; the 39 kDa movement and 30 kDa coat proteins. Both RNAs have conserved sequences and structures at the 5′ and 3′ termini. The genome organization, sequence and phylogenetic analyses indicate that the virus is a putative new member of the genus Idaeovirus, as it consistently groups with privet leaf blotch-associated virus and raspberry bushy dwarf virus. A duplex RT-PCR assay was developed for rapid detection of both genomic RNAs simultaneously. The work presented in this communication will assure the health status of blackcurrant plants in mother blocks, nurseries and production fields alike.     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.