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 yellowmosaic 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载体上进行序列分析,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。     

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。     

甘薯脉花叶病毒外壳蛋白基因在大肠杆菌中的表达及其抗血清的制备

 根据已报道的甘薯脉花叶病毒(Sweet potato vein mosaic virus,SPVMV)外壳蛋白（CP）基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPVMV河南分离物（SPVMV-HN）基因组3′端1.8 kb的基因片段,包括部分NIb 基因序列和完整的CP基因及3′端非编码区序列(3′UTR)。序列分析表明,SPVMV-HN的CP基因由996个核苷酸组成（GenBank登录号为FJ687211）,编码332个氨基酸残基。与已发表的SPVMV其他分离物相比,其推导的氨基酸序列一致性为95.2%～98.5%,与 SPVMV广东分离物的氨基酸序列一致性为97.9%。将CP基因克隆到原核表达载体pET-28a（+）上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3) pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPVMV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。利用SPVMV的抗血清,对采自全国14个省（市）的田间甘薯样品以及嫁接的巴西牵牛样品进行了检测,结果表明,SPVMV在我国甘薯上普遍存在。     

合肥地区发生的西瓜花叶病的病原鉴定

 从合肥市郊区的西瓜病叶上分离出一病毒分离物,该分离物的热钝化温度为60~65℃,稀释终点为10^-4~10^-5,寄主体外存活期为20~23 d,电镜观察病毒粒子约750 nm×13 nm。参考已发表的小西葫芦黄化花叶病毒(Zucchini yellow mosaic virus,ZYMV) CP基因序列设计引物,RT-PCR扩增得到CP基因片段。将该CP片段克隆到pGEM-3Zf (+)中,测序结果表明,该CP基因全长840 nt,共编码279个氨基酸,与国内报道的ZYMV CP基因的核苷酸序列同源性为83.0%~97.3%,氨基酸序列的同源性为91.8%~99.8%,证明该分离物为ZYMV。     

甜菜花叶病毒新疆分离物基因组3'末端序列分析

甜菜花叶病毒（Beet mosaic virus，BtMV）属马铃薯Y病毒科、马铃薯Y病毒属，可经多种蚜虫以非持久性方式传播，病毒粒子为弯曲线状，核酸为单分子正义ssRNA。目前只有美国华盛顿分离物的全序列以及斯洛伐克和英国少数几个分离物3’端的部分序列被报道。美国分离物全长9591 nt，3’端具有PolyA尾，编码一个由3086个氨基酸组成的多聚蛋白，与其它Potyvirus病毒一样可切割成10个蛋白，从N到C端依次为P1、HC—Pro、P3、6K1、CI、6K2、NIa—Vpg、NIa～Pro、NIb和CP。对于我国发生的BtMV，1981年Liu等报道了发生于北京地区菠菜上的BtMV，之后研究人员相继报道了黑龙江、内蒙古和新疆等甜菜主产区甜菜花叶病的发生及危害情况，并陆续开展了对BtMV的生物学特性、外壳蛋白分子量测定和氨基酸组分分析、细胞病理学等研究，目前对于我国发生的BtMV的分子结构特征还未见报道。本文报道了甜菜花叶病毒新疆分离物（BtMV—XJ）3’端的核酸序列，并与国外已报道序列进行了比较分析，为从分子水平上明确我国BtMV的分子结构特点、深入研究其编码蛋白的功能打下了基础。     

白草花叶病毒承德玉米分离物3′-cDNA 片段序列分析

 采自河北承德11 个表现矮花叶症状的玉米样品,用甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)和白草花叶病毒
(Pennisetum mosaic virus, PenMV)简并引物扩增了基因组3′ 端约2. 1 kb 的片段并进行测序。Blast 结果表明其中8 个样
品含有PenMV。扩增到的PenMV 序列均为2 135 nt,包括部分NIb 基因(985 nt)、完整的CP 基因(909 nt)和3′-UTR(241
nt)。这8 个分离物CP 基因和3′-UTR 与GenBank 上其他PenMV 分离物相应序列的核苷酸一致率分别为89. 8% ~ 93． 4%
和95. 9% ~ 97. 9% 。根据扩增的2 135 nt 序列和CP 基因序列构建系统发育树,8 个分离物与GenBank 上其他PenMV 分离
物都分为2 个组:山西组和承德组。重组分析表明CD9 的CP 基因存在重组。     

甘肃省南瓜及西葫芦小西葫芦黄花叶病毒病鉴定

利用双抗夹心酶联免疫吸附测定(DAS-ELISA)的方法对甘肃出入境南瓜、西葫芦种子及采自河西地区显症病株叶片进行检测,在种子及病叶组织中均检测到ZYMV病毒,其中南瓜种子带毒批次占12.5%,西葫芦种子带毒批次占11.8%。根据已报道的小西葫芦黄花叶病毒(Zucchini yellow mosaic virus)基因组核苷酸序列,设计引物扩增其外壳蛋白(CP)基因,以ELISA阳性种子或病叶组织总RNA为模板,进行RT-PCR扩增,对预期大小的扩增产物进行测序,结果表明扩增获得的核苷酸序列与世界各地的ZYMV分离物CP基因具有高度一致性,综合ELISA检测和RT-PCR的结果,确定南瓜、西葫芦种子可携带ZYMV,且ZYMV是侵染甘肃瓜类作物的重要病毒种类。     

小西葫芦黄花叶病毒外壳蛋白基因的克隆及序列分析

 以小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)的中国分离物(CH-87)接种发病的叶片中提取的总RNA为模板,经RT-PCR扩增获得ZYMV CP基因,将其克隆到pUCm-T质粒上进行序列分析。结果表明该CP基因由837个核苷酸组成,编码279个氨基酸。与已发表序列相比较,该CP基因与国际上已报道的4个基因型不同,应属于新的基因型,暂命名为基因型Ⅴ。     

菜豆普通花叶病毒RT-KR检测及3＇末端序列分析

根据哈尔滨地区豇豆感病植株的症状,初步鉴定感染豇豆的病毒为菜豆普通花叶病毒（Bean common mosaic, virus,SCMV）。利用马铃薯Y病毒属通用引物扩增出病毒基因组3’末端序列,经BLAST检索表明该病毒为BCMV,将该序列与GenBank上的21个BCMV分离物的3＇末端序列进行比较,显示其核苷酸序列与其他分离物的序列同源性为91.7％-97.3％。系统进化分析显示不同分离物可聚为5个类群,并显示出一定的寄主相关性。哈尔滨分离物BCMV-X与2个浙江分离物、1个澳大利亚分离物聚为一支,且该4株分离物的寄主均为豇豆。RNA二级结构分析显示BCMV基因组3’末端非编码区形成4个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化。     

黄瓜绿斑驳花叶病毒安徽分离物全基因组序列测定及分析

通过胶体金免疫层析试纸条和RT-PCR等手段对采自安徽和县的西瓜病株进行检测,确定其病原为黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)。为明确CGMMV安徽分离物CGMMV-Anhui的分类地位,进一步克隆了该病毒的全基因组序列,分析了其基因组结构特征。结果表明,CGMMV-Anhui基因组全长为6 423bp(GenBank登录号KT236095),与已报道的CGMMV的编码区的基因结构一致,仅5′和3′端非编码区核苷酸数目略有差异。将CGMMV-Anhui与已报道的分离物的全基因组序列和外壳蛋白基因序列进行系统发育分析,显示CGMMV不同分离物可分为亚洲和欧洲两个组,安徽分离物CGMMV-Anhui与亚洲分离物亲缘关系较近,可能具有共同的侵染源。     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

一种侵染滇重楼的Potexvirus病毒分离物分子鉴定及其3'末端序列分析

 从云南武定的滇重楼上得到一个病毒分离物Paris-YN,病毒粒体为弯曲线状。利用RT-PCR扩增获得一条1074bp的片段,序列比较分析发现其与马铃薯X病毒属(Potexvirus)病毒3'末端的结构最为相似,且与属内的白三叶草花叶病毒等20个不同分离物3'末端有36.7%~58.9%的同源性;该病毒cp基因长639个核苷酸,编码212个氨基酸(22.8kDa),与20个Potexvirus病毒分离物的CP氨基酸序列比较发现,Paris-YN与白三叶草花叶病毒的CP氨基酸同源性最高(60.1%)。证据表明,该分离物可能为Potexvirus的新成员,暂命名为重楼X病毒(Paris polyphylla virus X)。     

Analysis of the coat protein gene of Indian <Emphasis Type="Italic">Potato virus X</Emphasis> isolates for identification of strain groups and determination of the complete genome sequence of two isolates

Complete coat protein (CP) gene sequences of 66 Potato virus X (PVX) isolates were sequenced and compared with other PVX isolates. The CP gene of these isolates shared 93.9–100.0 % and 97.0–100.0 % identities among them at nucleotide and amino acid sequence level, respectively. Phylogenetic analysis with isolates of known PVX strain groups showed that all 66 isolates were found in clade I (strain groups 1, 3 and 4) and none of them in Clade II (strain groups 2 and 4). The Indian isolates had the 714 bp coat protein gene and were closer to clade I isolates with 92.9–99.5 % identities and distantly related to Clade II isolates (74.2 to 80.0 % identities). Hence, these isolates may belong to either of the strain groups 1, 3 and 4. A threonine residue at position 122 and glutamine residue at position 78 were found conserved in all the Indian isolates suggesting that these isolates cannot overcome Rx1gene and Nx gene mediated resistance, characteristic of group 1 and 3. However, unique amino acid substitutions were observed in Indian isolates and further studies are required to ascertain their role in symptom expression, virulence and host range. In addition, whole genome sequences of two isolates one each from Jalandhar (Punjab) and Kufri (Himachal Pradesh) were also determined. They were 6435 nts long with five ORFs and shared 81.4–97.2 % identities to clade I isolates from USA, Russia, India, Iran, China, Japan, Taiwan and 77.0 to 77.5 % identities with clade II isolates from Peru.     

甘蔗花叶病毒福建分离物外壳蛋白基因的克隆及序列分析

 A fujian isolate of Sugarcane mosaic virus named SCMV-FJ was isolated from infected sugarcane. Cloning and sequence analysis of the coat protein gene of this isolate was carried out. A pair of primers was designed and synthesized based on the nucleotide sequences of coat protein genes of sugarcane mosaic viruses reported. The coat protein gene of SCMV-FJ was amplified from the extracted total RNA of the infected sugarcane by using RT-PCR, and cloned into the pMD18-T vector. The sequencing result indicated that the cloned segment included a 1137 bp open reading frame(ORF) and a 228 bp 3' untranslated region, in which the ORF comprised the whole coat protein and part of the nuclear inclusion b. The nucleotide and the deduced amino acid sequences of the coat protein gene were compared with those of the other isolates or strains of SCMV subgroup reported in GenBank. The result showed that it shares 56.8%-97.1% and 55.3%-99.4% homology in nucleotide and the putative amino acid sequences, respectively, with the highest amino acid homology of 99.4% with SCMV-D. Thus it was identified as a SCMV-D. This experiment provided a rapid, sensitive and relatively inexpensive method for RT-PCR detection of SCMV. At the same time, the cloning of SCMV-FJ coat protein gene provided the foundation for plant gene engineering against SCMV.     

草莓轻型黄边病毒3'末端序列多态性研究

 The 930 bp segment in 3' terminal region of Strawberry mild yellow edge virus(SMYEV) genome was amplified by 3' rapid amplification of cDNA ends(RACE).Ten Chinese isolates were sequenced,and 7 of them were the same.Nucleotide and amino acid identities and phylogenesis were analyzed between Chinese isolates and 24 isolates from other regions of the world.Sequence analysis of the 878 nt stretch within 3' terminal region of SMYEV genome showed that nucleotide acid identities ranged from 79.5% to 100%,deduced amino acid sequences of coat protein gene identity were 86.4% to 100%.Phylogenetic analysis showed that all isolates of SMYEV fell into four clades.To a certain extent,the clades were related with the geological distribution of SMYEV.Chinese isolates SY01 and SY04 lay in the same clade with European and American isolates,but formed a small separate branch.Isolates SY03 and SY02,derived from Fragaria×ananassa cv.Changhong-2 and F.pentaphylla respectively,had a far relationship with other isolates and fell into one clade.They were likely to be the special isolates that existed only in China.     

Genetic variability and population structure of <Emphasis Type="Italic">Grapevine virus A</Emphasis> coat protein gene from naturally infected Italian vines

Grapevine virus A (GVA) is considered one of the viruses associated with rugose wood (RW), one of the most economically important diseases of grapevine. Thirty-seven GVA isolates collected from grapevine cultivars from Marche (central-eastern Italy), Apulia and Campania (southern Italy), were subjected to molecular characterization. The genetic and population diversity was studied in the coat protein (CP) gene by RT-PCR-RFLP analysis with three restriction enzymes (MseI, AluI, and AciI), and nucleotide sequencing. A new primer pair (CP1F/R) allowing amplification of the whole CP gene (621 bp) was developed. RFLP with AciI yielded the highest number of variants in GVA isolates, showing seven different ‘simple’ profiles (A, B, C, D, E, F, and G). ‘Complex’ profiles were also found, and the most common variant combination was A + B in 39% of isolates. The analysis of GVA sequences confirmed the presence of plants infected with more than one GVA variant and suggested that RT-PCR-RFLP is suitable for evaluating population diversity of GVA enabling a screening of different haplotypes. The distribution of RFLP profiles and the phylogenetic analysis were not correlated with the location of infected plants, showing the presence of a GVA population with genetic diversity in the average with those of RNA viruses.     

亚洲玉米螟6-磷酸葡萄糖异构酶基因(Pgi)全序测定及分析

为进一步从核酸水平上研究亚洲玉米螟Pgi基因相关特性,采用反转录PCR及RACE等技术对该基因编码序列及DNA全序列进行了测定,与Gen Bank中其它昆虫Pgi基因相关信息进行比较分析,并构建了系统发育树。结果表明:亚洲玉米螟Pgi基因编码区序列长为1 671 bp,共编码556个氨基酸;其DNA序列全长为10 078 bp(短序列为9 311 bp),由12个外显子与11个内含子镶嵌而成;各外显子长度与鳞翅目大部分昆虫相同,介于95~188 bp之间,内含子序列总长度为8 407 bp(短序列为7 640 bp),各内含子长度介于418~1 547 bp之间,且在内含子3、4、5、11上均发现杂合现象。系统发育结果显示,大部分昆虫Pgi基因c DNA严格按物种聚类,除了双翅目的家蝇Musca domestica与黑森瘿蚊Mayetiola destructor出现一定的交叉现象外,其余没有出现交叉现象。     

Molecular characterization of the CP gene and 3'UTR of Chilli veinal mottle virus from South and Southeast Asia

Twenty-four isolates of Chilli veinal mottle virus (ChiVMV) from China, India, Indonesia, Taiwan and Thailand were analysed to determine their genetic relatedness. Pathogenicity of virus isolates was confirmed by induction of systemic mosaic and/or necrotic ringspot symptoms on Capsicum annuum after mechanical inoculation. The 3' terminal sequences of the viral genomic RNA were determined. The coat protein (CP) coding regions ranged from 858 to 864 nucleotides and the 3' untranslated regions (3'UTR) from 275 to 289 nucleotides in length. All isolates had the inverted repeat sequence GUGGNNNCCAC in the 3'UTR. The DAG motif, conserved in aphid-transmitted potyviruses, was observed in all isolates. All 24 isolates were considered as belonging to ChiVMV because of their high CP amino acid and nucleotide identity (more than 94·8 and 89·5%, respectively) with the reported ChiVMV isolates including the pepper vein banding virus (PVBV), the chilli vein-banding mottle virus (CVbMV) and the CVbMV Chiengmai isolate (CVbMV-CM1). Based on phylogenetic analysis, ChiVMV isolates including all 24 isolates tested, PVBV, CVbMV and CVbMV-CM1 can be classified into three groups. In addition, a conserved region of 204 amino acids with more than 90·2% identity was identified in the C terminal of the CP gene of ChiVMV and Pepper veinal mottle virus (PVMV), and may explain the serological cross reaction between these two viruses. The conserved region may also provide useful information for developing transgenic resistance to both ChiVMV and PVMV.     

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