Biological and molecular characterization of a new cucurbit-infecting tobamovirus

ABSTRACT An uncharacterized virus was isolated from greenhouse-grown cucumber plants. Biological and serological data described in the present study indicated that the virus belonged in the genus Tobamovirus. The host range of the virus included several plant species within the family Cucurbitaceae. The virus designated Cucumber fruit mottle mosaic virus (CFMMV) causes severe mottling or mosaic on cucumber fruits, and its fast spread within greenhouses could lead to significant economic losses in cucumber crops. The genome of CFMMV has been completely sequenced and its genome organization was typical of a Tobamovirus. However, its sequence was distinct from other described viruses within the group of cucurbit-infecting Tobamoviruses. Comparisons of sequences and phylogenetic analysis suggested that the cucurbit-infecting Tobamoviruses be separated into two subgroups: subgroup I comprising the strains and isolates referred to in the literature as Cucumber green mottle mosaic virus (CGMMV) (CV3, CV4, CGMMV-W, CGMMV-SH, and CGMMV-Is) and subgroup II comprising CFMMV, Kyuri green mottle mosaic virus (KGMMV), and the Yodo strain of CGMMV, which is closely related to KGMMV and may be considered a strain of it.     

Characterization and Detection of Several Filamentous Viruses of Cherry: Adaptation of an Alternative Cloning Method (DOP-PCR), and Modification of an RNA Extraction Protocol

For the identification and analysis of new RNA plant viruses infecting fruit trees, an initial step often involves the laborious procedure of isolation and cDNA synthesis and cloning from purified viral dsRNA. For subsequent RT-PCR detection of these and other viruses from tissue with high phenolic and polysaccharide concentrations, a simple and efficient extraction protocol for viral nucleic acid is also important. A method for rapid cDNA cloning from small amounts of purified dsRNA using a modification of degenerate oligo primed polymerase chain reaction mbox(DOP-PCR), and a modification of a protocol for effective extraction of viral RNA for use in RT-PCR are presented. Both methods were used to analyze a number of mottling diseases described in cherry. The causal agents for two of these diseases have been previously described, Cherry green ring mottle virus, a tentative member of the foveaviruses, and Cherry mottle leaf virus, a member of the trichoviruses. For the diseases cherry rusty mottle and cherry necrotic rusty mottle, data are presented identifying viruses associated with each disease. Viruses associated with cherry rusty mottle, cherry necrotic rusty mottle and European isolates of cherry mottle leaf diseases, are closely related to Cherry green ring mottle virus and can be tentatively included in the foveavirus genus. An additional virus, related to cherry green ring mottle virus, was discovered by RT-PCR cloning and appears to be a common latent virus of cherry. Finally, isolates of cherry necrotic mottle disease could be assayed positive by RT-PCR for a virus     

黄瓜绿斑驳花叶病毒低风险参照物质的研制

采用RT-PCR方法扩增黄瓜绿斑驳花叶病毒的外壳蛋白基因与3,非编码区,并将其构建到马铃薯X病毒(PVX)载体中.重组质粒经线性化及体外转录后接种烟草,获得含有病毒外壳蛋白基因的感病植株.感病组织可用于分子检测的质控,也可作为毒源来繁殖阳性参照物质.基于PVX载体制备的参照物质可降低检疫性病毒的生物安全风险,尤其对稳定性强、可造成严重经济损失的高风险病毒的检疫更具实用价值.     

小西葫芦绿斑驳花叶病毒生物学特性及传播方式

小西葫芦绿斑驳花叶病毒(Zucchini green mottle mosaic virus,ZGMMV)是我国新报道的侵染葫芦科作物的烟草花叶病毒属病毒.从感染病毒的葫芦叶片中分离出ZGMMV,摩擦接种西瓜(Citrullus lanatus)、葫芦(Lagenaria siceraria)、西葫芦(Cucurbit...     

高通量测序发现河南开封、中牟西瓜病毒病由多种病毒复合侵染导致

 调查发现河南省开封市祥符区和郑州市中牟县西瓜田病毒病严重发生,症状表现为叶片黄化、蕨叶、皱缩、卷曲,枝梢翘起,果实变小和瓜纹不清。为明确其病毒种类,通过小RNA测序及生物信息学分析发现采集的西瓜样本中存在8种病毒。其中包括5种已知病毒:小西葫芦黄花叶病毒(Zucchini yellow mosaic virus, ZYMV)、甜瓜蚜传黄化病毒(Melon aphid-borne yellows virus, MABYV)、西瓜花叶病毒(Watermelon mosaic virus, WMV)、黄瓜绿斑驳花叶病毒(Cucumber green mottle mo-saic virus, CGMMV)和西瓜隐潜病毒(Citrullus lanatus cryptic virus, CiLCV);3种为新检出的RNA病毒:西瓜病毒A(Watermelon virus A, WVA)、西瓜皱叶相关病毒1号(Watermelon crinkle leaf-associated virus 1, WCLaV-1)和西瓜皱叶相关病毒2号(Watermelon crinkle leaf-associated virus 2, WCLaV-2)。RT-PCR检测田间66个西瓜标样发现,WMV、CiLCV和ZYMV是侵染两地西瓜的主要病毒,其阳性检出率分别为61%、56%和52%,两种及以上病毒复合侵染的比率达68%。     

侵染葫芦的黄瓜绿斑驳花叶病毒广西分离物分子鉴定

从广西南宁市郊温室大棚中的葫芦[Lagenaria siceraria(Molina)Stand.]上采集到一个表现脉绿、花叶症状的病毒样品,ELISA检测表明,该样品与黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)有密切的血清学关系,利用RT-PCR方法从样品中扩增获得约500bp的DNA片段,序列分析表明,该片段是CGMMV的外壳蛋白基因,暂将该病毒分离物定名为GX-BG。外壳蛋白基因核苷酸序列系统进化树分析表明,已报道的CGMMV主要分为3大群体,GX-BG与中国辽宁分离物(CGMMV-LN)分别属于不同的群体。     

辽中地区西瓜花叶病病原的分子鉴定

 利用ELISA和RT-PCR方法对采自我国辽中地区的西瓜花叶病样品进行检测,表明其病原为黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)。将此分离物(CGMMV-Wcn)的外壳蛋白基因克隆后进行序列分析,结果表明其cp基因由486个碱基组成,编码161个氨基酸,与已报道的其它分离物一致;CGMMV-Wcn所致症状与西瓜株系(CGMMV-W)相同,且二者cp基因的氨基酸序列完全一致,因此该分离物应为CGMMV-W。     

葫芦种子传黄瓜绿斑驳花叶病毒的检测*

从感染黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus, CGMMV）的葫芦植株上收取种子,通过苗期症状观察法、双抗体夹心酶联免疫吸附法（DAS-ELISA）、免疫捕获反转录PCR（IC-RT-PCR）法测定葫芦种子的带毒情况,并用生物学接种方法测定葫芦种子携带病毒的侵染活性。苗期症状观察法结果表明,199株幼苗有2株表现花叶斑驳症状,种子传毒率为1.01%;而利用DAS-ELISA和IC-RT-PCR法随机检测30粒葫芦病株种子,CGMMV检出率为100%。种子各部位携带的CGMMV接种葫芦表现典型的花叶斑驳症状,表明葫芦种子携带的CGMMV具有侵染活性。DAS-ELISA检测葫芦种子CGMMV的灵敏度为1/5120种子研磨液。     

中国甘薯病毒种类的血清学和分子检测

 2009～2010年,从我国18个省（市）采集了176份表现病毒病症状的甘薯样品。利用血清学、PCR和核苷酸序列测定的方法,对上述样品中的病毒种类进行了鉴定。血清学检测结果表明,供试样品中甘薯羽状斑驳病毒（SPFMV）的阳性率最高,达56.3%,其次为甘薯G病毒（SPVG）和甘薯类花椰菜花叶病毒(SPCaLV),阳性率分别为34.1%和33.5%。PCR和核苷酸序列测定结果表明,我国甘薯上至少存在SPFMV、SPVG、甘薯潜隐病毒（SPLV）、甘薯褪绿斑病毒（SPCFV）、甘薯褪绿矮化病毒（SPCSV）、黄瓜花叶病毒（CMV）、甘薯脉花叶病毒（SPVMV）和甘薯卷叶病毒（SPLCV）8种病毒。此外,供试样品中没有检测出甘薯轻斑驳病毒（SPMMV）,是否存在甘薯轻斑点病毒(SPMSV)、SPCaLV和C 6病毒尚不能确定。     

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.     

黄瓜绿斑驳花叶病毒cp基因的原核表达及抗血清制备

 采用RT-PCR方法克隆了黄瓜绿斑驳花叶病毒辽宁分离物(Cucumber green mottle mosaic virus Liaoning isolate, CGMMV-LN)的cp基因并连接到原核表达载体pGEX-4T-3和pET-22b (+)上, 将获得的重组子pGEX-4T-3-CGMMV CP和pET-22b (+)-CGMMV CP转化大肠杆菌BL21后用IPTG进行诱导表达。SDS-PAGE和W estern blot分析表明, cp基因在大肠杆菌中获得了高效表达, 融合蛋白分子量分别为43.8 kDa和17.3 kDa。将17.3 kDa融合蛋白纯化后免疫家兔, 制备了CGMMV特异性抗血清, 抗原包被间接ELISA法(ACP-ELISA)测定抗血清的效价为1/20 000。     

南瓜果实中黄瓜绿斑驳花叶病毒的RT-PCR检测及cp基因序列分析

以来自甘肃的南瓜果实样品中提取的总RNA为模板进行RT-PCR扩增，序列测定和分析结果表明：扩增片段包含CGMMV的外壳蛋白基因和3’非编码区，证明南瓜果实中存在黄瓜绿斑驳花叶病毒，将其印基因序列与来自国内不同地区的其他CGMMV分离物进行序列比对，发现国内各分离物间的同源率高，有很近的亲缘关系。     

A Cucumber mosaic virus isolated from Momordica charantia L.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.     

侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用

 根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR (mRT-PCR)扩增的引物浓度、Mg²⁺浓度、Taq DNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。     

黄瓜绿斑驳花叶病毒基因组全序列分析及病害的田间调查

为明确近年来在浙江省葫芦科作物上发生的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)基因组特征及其发生分布情况,从浙江省及上海地区的甜瓜、西瓜和瓠瓜上采集疑似样品进行RT-PCR鉴定,通过分段扩增测序的方法拼接获得基因组全序列并进行系统进化分析,利用特异性引物扩增获得CGMMV外壳蛋白(coat protein,CP)基因序列,制备CGMMV CP抗血清进行Western-blot和Dot-ELISA检测。结果显示,来自甜瓜、西瓜和瓠瓜的3个CGMMV分离物基因组全序列均具有烟草花叶病毒属典型基因组结构特征,全部由6 423 nt构成;3个全序列间的核苷酸同源性高达99.11%~99.67%,编码的CP氨基酸同源性为100%。系统进化分析发现,CGMMV不同分离物形成2个进化相关群体,3个浙江的CGMMV分离物均位于第I组内,与已报道的中国CGMMV分离物和韩国CGMMV分离物亲缘性较高。Western-blot检测表明CGMMV CP抗血清可以与感病植株中的病毒发生特异性反应,可用于CGMMV鉴定;Dot-ELISA检测发现CGMMV在浙江省和上海市的葫芦科作物上普遍存在。     

番茄斑驳花叶病毒在广东省的首次报道

番茄斑驳花叶病毒(tomato mottle mosaic virus, ToMMV)是2013年发现的烟草花叶病毒属一个新种,目前在多国(地区)有发生。本文采用小RNA深度测序及RT-PCR检测方法在广东省广州市南沙区辣椒疑似病样中检测到ToMMV,命名为番茄斑驳花叶病毒广东分离物(ToMMV-GD-2020)。采用RT-PCR分段扩增获得了ToMMV-GD-2020的基因组全序列,该分离物基因组全长6 399 nt,包含4个开放阅读框,分别编码4个蛋白。序列相似性分析表明,ToMMV-GD-2020与已登录GenBank的14个ToMMV分离物基因组序列相似性分别为99.0%～99.7%,其中与中国辽宁分离物ToMMV-LN(GenBank登录号：MN853592)的相似性最高(99.7%),与危害我国番茄、同属烟草花叶病毒属的番茄花叶病毒(tomato mosaic virus, ToMV)、番茄褐色皱果病毒(tomato brown rugose fruit virus, ToBRFV)代表分离物的相似性分别为84.6%和81.0%。系统进化分析表明,ToMMV-GD-2020...