Sequence Analysis and Detection Using Immunocapture-PCR of Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus in Hawaiian Orchids

A Hawaiian isolate of Cymbidium mosaic virus (CyMV-H) was purified from Dendrobium orchid, and a cDNA library was constructed. Clones containing the coat protein (CP) gene and movement protein (MP) gene were identified by colony hybridization and polymerase chain reaction (PCR). The Hawaiian isolate of Odontoglossum ringspot virus (ORSVH) was purified from Cattleya orchid. The CyMV CP gene was PCR amplified from a cDNA done. The ORSV CP and 54 kDa putative replicase genes and CyMV-MP gene were cloned by RT-PCR Sequences of these genes of CyMV-H and ORSV-H were compared with those of CyMV and ORSV from Singapore, Japan. Korea, and Germany. The high degree of sequence identity (91–99%) at the nucleotide level for all gene sequences analysed, shows that CyMV and ORSV from different countries are closely related. Sequence comparison results show that CyMV strains can be divided into two groups based on differences in amino acid sequences of the coat protein gene: CyMV-H closely resembles CyMV-SI while CyMV-S2 resembles CyMV-K, A sensitive, rapid, and reliable immunocapture PCR (ICPCR) assay was developed to detect both viruses, CyMV was detected from dilutions equivalent to 100 mg of orchid material and ORSV was detected from dilutions equivalent to 10 μg of orchid material. IC-PCR was compared with direct binding PCR (DB-PCR) and ELISA for their sensitivities.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Coat Protein Gene of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island

Nucleotide and amino acid sequences of the coat protein (CP) of 12 isolates of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island (CyMV‐R) were compared with each other and with those of previously described Asian strains. Alignment revealed that CyMV‐R isolates were highly homologous, suggesting that one strain is prevalent in Reunion. This strain also showed high homology with the Korean CyMV‐K2 and Singapore CyMV‐S2 strains, but nucleotide additions resulted in the carboxy‐terminal ends of the CP sequences differing from those of the Korean CyMV‐K1 and Singapore CyMV‐K1 strains.     

侵染厦门地区蝴蝶兰的建兰花叶病毒的分子鉴定

根据已公布的建兰花叶病毒（Cymbidium mosaic virus,CyMV）序列,设计特异引物,利用RT-PCR方法分离克隆了厦门蝴蝶兰CyMV的病毒分离物,获得完整的CP基因序列,并与来自中国和其它亚洲国家的病毒分离物进行同源性分析,其同源性均达96%以上。系统进化树分析结果表明,厦门的病毒分离物与中国台湾、韩国、印度的分离物成簇,而福建、北京和新加坡的病毒分离物独立形成另一簇。     

建兰花叶病毒运动蛋白基因克隆及序列分析

从建兰花叶病毒（CyMV）石斛兰分离物中提取病毒RNA,用反转录——聚合酶链式反应(RT-PCR)方法获得约500bp的运动蛋白基因片断,插入pGEM-T载体克隆并测序。序列分析表明,该基因片断由474个核苷酸组成,和CyMV美国夏威夷分离物、新加坡分离物相应基因核甘酸序列分别具有97.8%同源性;根据核酸序列推导该片断含有3个部分重叠的开放阅读框架（ORF）,分别编码14kD、12kD和10kD的多肽。     

广东地区两种兰花病毒病害的分子鉴定及检测

根据已报道的建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)基因组核苷酸序列,在其cp基因上下游设计PCR引物。CyMV预计扩增产物784bp,ORSV预计扩增产物604bp。以采集自广东省顺德的墨兰和文心兰表现病毒病症状的病株叶组织总RNA为模板,进行RT—PCR扩增。对预期大小的5个扩增产物进行克隆和测序,结果表明,来源于不同兰种或同一兰种不同兰场的病样CyMV引物扩增产物核苷酸序列存在少量差异,但均与世界各地的CyMV分离物cp基因高度同源;而来源于不同兰种的病样ORSV引物扩增产物核苷酸序列完全相同,与世界各地的ORSV分离物cp基因高度同源。因此可将侵染广东兰花的两种病毒鉴定为CyMV和ORSV。混合上述两种病毒的PCR引物,采用双重RT—PCR扩增,对采自广东顺德23个兰场共153份样品进行病毒检测,76份(49．7％)检出CyMV,52份(34．0％)检出ORSV,2份(1．3％)同时检出CyMV和ORSV。     

黄瓜花叶病毒cDNA载体在烟草细胞中表达外源基因

为了探讨利用黄瓜花叶病毒(CMV)构建表达载体的可行性,分离了山东株(SD) CMV RNA 3的全长cDNA。测定其全序列后,采用定点突变的方法在衣壳蛋白(CP)基因起始密码子处改造出一个NsiⅠ位点,可将外源基因引入NsiⅠ位点和CP基因终止密码子上游附近的XhoⅠ或SalⅠ位点而置换掉CP基因。分别用绿色荧光蛋白(GFP)基因、β-葡糖醛酸酶(GUS)基因以及小鼠二氢叶酸还原酶(DHFR)基因3种报告基因置换CP基因。将Fny株CMV RNA 1、RNA 2和SDCMV嵌合RNA 3的cDNA分别插入pCass载体的35S启动子和终止子之间,将构建的置换型载体直接以质粒的方式转染烟草原生质体,表达了3种报告基因。     

香蕉束顶病毒基因克隆和序列分析

对香蕉束顶病毒（ＢＢＴＶ）中国分离株ＤＮＡ组份Ｉ（ＤＮＡ－１）、外壳蛋白（ＣＰ）和运转蛋白（ＭＰ）基因进行了克隆和序列分析。ＢＢＴＶＤＮＡ－１含有１１０３个核苷酸,与南太平洋和亚洲分离株分别有８７％－８８％ ９６．９－９８％的核苷酸序列同源性。由ＤＮＡ－１编码的复制酶含有１８６个在酸残基。与南太平洋和亚洲分离株分别有８４．４％－９５．８％和９７．６％、９８．０％的氨基酸序列同源性。外壳蛋白基因由５     

番木瓜环斑病毒畸叶株系的CP基因克隆和序列分析

采用RT－PCR方法合成了本研究室保存的番木瓜畸叶病毒（PMaLV）的外壳蛋白（CP）基因,将其CP基因克隆进Promega公司的pGEM-T and pGEM-T Easy Vector System（简称T－载体）,并进行了序列分析。结果表明,PMaLV CP基因核苷酸序列全长为861nt,推导其编码287个氨基酸。与番木瓜环斑病毒（PRSV）美国夏威HA株系和澳大利亚W株系的CP基因相比,在第66nt处开始连续缺失3个核苷酸。与PRSV的华南Ys、Sm和G株系以及夏威夷的HA和澳大利亚的W株系相比,其CP基因序列同源率分别为96％、98％、95％、89％和89％。其的氨基酸序列同源率分别为98％、97％、97％、96％和95％。此结果表明,PMaLV属于PRSV的一个株系,不是一种新病毒。因此,我们称其为番木瓜环斑病毒畸叶株系（ML株系）。     

Sequences of Tobacco Rattle Viruses from Potato

The RNA2 of the tobacco rattle virus (TRV) isolate 'Rostock' (TRV-R), and the coat protein gene (CP) of TRV isolate 'Mirow' (TRV-M) were sequenced. Both were isolated from potato. Sequence analysis revealed a 5' noncoding-region (NCR) and a CP gene, which are among the shortest of any tobravirus RNA2 sequenced so far. Downstream of the CP open reading frame (ORF) there was an ORF for a 8 k protein, that is probably a truncated 9 k protein, followed by an ORF for a 16 k protein and the 3' NCR. The 16 k protein and the 3' NCR showed a considerable sequence identity with the 3' end of TRV RNA1 and RNA2 from other TRV isolates. The high identity in the amino acid sequences of the CPs from TRV-R and TRV-M as well as other TRV isolates suggested that they are related to the TCM group of the genus Tobravirus and belong serologically to the RQ-serotype. This could be confirmed using a rabbit antiserum that was prepared against recombinant TRV-R CP expressed in bacteria. This serum was found to be suitable for differentiation of TRV-isolates.     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

Potato Virus Y-Like Particles as a New Carrier for the Presentation of Foreign Protein Stretches

Virus-like particle (VLP) technology represents a promising approach for the creation of efficient vaccines and materials for use in nanotechnological applications. For construction of a new carrier for foreign protein sequences, the coat protein (CP) gene from potato virus Y (PVY) was cloned and expressed in Escherichia coli cells. The PVY CP self-assembles into PVY-like particles, as demonstrated by electron microscopy analysis of purified VLP preparations. The PVY CP with an N-terminal insertion of a foreign epitope (preS1) or of a whole protein (rubredoxin) retains its ability to form filamentous particles, whereas adding a foreign sequence to the C-terminus of the PVY CP generates mostly unstructured protein aggregates. This new filamentous plant virus-derived VLP carrier accommodates a foreign protein sequence that is up to 71 amino acids in length on the VLP surface and can be produced in E. coli in preparative amounts. The PVY CP VLPs are stable in physiological conditions, but they are sensitive to EDTA, high salt, and extreme pH. The presence of the preS1 epitope decreases the stability of the chimeric PVY CP particles at elevated temperatures. Mice that are immunized with chimeric PVY CP particles carrying preS1 epitopes exhibit a strong anti-preS1 immune response, even in the absence of adjuvants.     

Resistance to Vanilla Necrosis Potyvirus in Transgenic Nicotiana benthamiana Plants Containing the Virus Coat Protein Gene

The full-length vanilla necrosis potyvirus (VNV) coat protein (CP) gene was introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens-mediated transformation. Four constructs contained either: sense (+) CP sequence, antisense (-) CP sequence, sense CP sequence with a Kozak's consensus ATG resulting in a change in the first amino acid, or antisense CP sequence with the Kozak's modification. When mechanically inoculated with a high concentration of VNV, one of the plant lines containing the full-length sense CP gene was highly resistant to virus infection. Plants from the resistant lines expressed the CP at a relatively low level compared to susceptible lines containing the same construct. Plants containing the other three constructs were either susceptible or showed delayed symptom expression.     

烟草花叶病毒蚕豆株系外壳蛋白基因及3‘端非编码区的克隆与序列分析

根据烟草花叶病毒Ｕ１株系序列,人工合成引物,用ＲＴ法合成了ｃＤＮＡ后,通过ＰＣＲ技术扩增并克隆了烟草花叶病毒蚕豆株系的外壳蛋白的基因和３‘端非编码区。ＤＮＡ序列测定结果表明,外壳蛋白基因全长４８０个碱基,编码１５８个氨基酸,３’端非编码区全长２０４个碱基,与ＴＭＶ－Ｕ１株系的同源率为１００％。     

小西葫芦黄化花叶病毒分离物的3′末端序列多态性研究

研究了来自中国大陆9个小西葫芦黄化花叶病毒（ZYMV）分离物的基因组3′末端核苷酸序列及所推导的外壳蛋白（CP）氨基酸序列以及3′末端非编码区（UTR）序列,并与其它地区所报道的16个ZMYV分离物进行了同源性比较。ZYMV CP基因核苷酸序列具有一定的寄主相关性和地域相关性,但总体上其关联程度不明显;同时,CP氨基酸序列的寄主适应性程度明显高于地域相关性。25个ZYMV分离物的CP氨基酸序列根据其变异程度分为2个区： N端约41个氨基酸为高度变异区,CP核心区和C端氨基酸序列为保守区。研究结果初步揭示了ZYMV作为单链RNA病毒通过与寄主相互作用而表现寄主适应性变异的趋势。     

侵染节瓜的小西葫芦黄花叶病毒CP基因的克隆、表达及其抗血清的制备

通过鉴别寄主反应、病毒部分序列测定确定了采自广州白云区表现花叶、斑驳症状的节瓜上的病毒为ZYMV。采用RT PCR方法扩增和克隆了该病毒的外壳蛋白基因 ,连接到原核表达载体pET 2 2b( )上。获得的重组子pET ZCP转化大肠杆菌BL2 1(DE3)后 ,用IPTG进行诱导表达。SDS PAGE和Westernblot分析表明 ,CP基因在大肠杆菌中获得了高效表达 ,融合蛋白分子量约为 33 0kD。将融合蛋白纯化后免疫兔子 ,获得了特异性较高的抗血清。ELISA测定其效价为 1 4 0 96