Biological and molecular characterization of a novel carmovirus isolated from angelonia

ABSTRACT A new carmovirus was isolated from Angelonia plants (Angelonia angustifolia), with flower break and mild foliar symptoms, grown in the United States and Israel. The virus, for which the name Angelonia flower break virus (AnFBV) is proposed, has isometric particles, approximately 30 nm in diameter. The experimental host range was limited to Nicotiana species, Schizanthus pinnatus, Myosotis sylvatica, Phlox drummondii, and Digitalis purpurea. Virions were isolated from systemically infected N. benthamiana leaves, and directly from naturally infected Angelonia leaves, using typical carmovirus protocols. Koch's postulates were completed by mechanical inoculation of uninfected Angelonia seedlings with purified virions. Isometric particles were observed in leaf dips and virion preparations from both Angelonia and N. benthamiana, and in thin sections of Angelonia flower tissue by electron microscopy. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 38 kDa was observed. Virion preparations were used to produce virus-specific polyclonal antisera in both Israel and the United States. The antisera did not react with Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), or Saguaro cactus virus (SgCV) by either enzyme-linked immunosorbent assay or immunoblotting. In reciprocal tests, antisera against PFBV, CarMV, and SgCV reacted only with the homologous viruses. The complete nucleotide sequence of a Florida isolate of AnFBV and the coat protein (CP) gene sequences of Israeli and Maryland isolates were determined. The genomic RNA is 3,964 nucleotides and contains four open reading frames arranged in a manner typical of carmoviruses. The AnFBV CP is most closely related to PFBV, whereas the AnFBV replicase is most closely related to PFBV, CarMV, and SgCV. Particle morphology, serological properties, genome organization, and phylogenetic analysis are all consistent with assignment of AnFBV to the genus Carmovirus.     

The coat protein of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> L<Subscript>11</Subscript>Y is associated with virus-induced chlorosis on infected tobacco plants

The L₁₁Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L₁₁Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L₁₁Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L₁₁Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L₁₁Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L₁₁Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L₁₁Y. Fewer virus particles accumulated in L₁₁Y-infected protoplasts, and many of them were shorter-than-full-length. The nucleotide sequence data reported is available in DDBJ/EMBL/GenBank databases as accession AB355139.     

烟草坏死病毒A大豆分离物的分子鉴定

 对曹琦和濮祖芹早期分离到的烟草坏死病毒大豆分离物的生物学、血清学和外壳蛋白的序列进行了进一步研究。该分离物能侵染8科29种植物, 除系统侵染大豆和本生烟外, 其余寄主均为局部侵染。电镜下病毒粒子呈球状, 直径约28nm。基因组为单组分RNA, 大小约为3.7 kb, 具有2条亚基因组, 分别约为1.6 kb和1.3 kb。外壳蛋白亚基的分子量约为30 kDa。血清学试验表明, 该分离物与TNV柳树分离物的抗血清呈特异反应, 与同属坏死病毒属(Necrovirus)的烟草坏死病毒D(TNV-D)和甜菜黑色焦枯病毒(BBSV)无血清学关系。利用简并引物通过RT-PCR克隆了该分离物的外壳蛋白基因。序列分析表明, 该分离物与烟草坏死病毒A(TNV-A)、TNV-D和TNV-D^H的外壳蛋白分别具有88.77%、45.13%和45.49%的氨基酸序列一致性。因此, 该大豆分离物属于TNV-A的一个新株系, 命名为TNV-A^C。     

侵染昆明玫瑰的李坏死环斑病毒的鉴定及其分子检测

采集昆明地区种植的花叶症状明显的玫瑰样品,运用现代分子生物学技术与常规技术相结合的方法,初步鉴定引起玫瑰花叶病的主要病毒病原为李坏死环斑病毒.该病毒引起玫瑰植株的系统花叶、畸形和皱缩等症状;电镜下病毒粒体为球形,直径为22～23nm;ELISA检测发现该病毒在植株芽、花粉和顶部叶片的浓度最高;同时,根据外壳蛋白的保守区利用Primer 5.0设计该病毒的特异引物,对该病毒进行分子检测,得到450bp的预期DNA片断,并在此基础上,进行了巢式RT-PCR的分子检测,表明巢式RT-PCR的检测能力最强.并通过序列的同源性分析得知该病毒的外壳蛋白与已知PNRSV的同源性为98.0%,进一步证明了该病毒为李坏死环斑病毒.     

Maize fine streak virus, a New Leafhopper-Transmitted Rhabdovirus

ABSTRACT A previously uncharacterized virus was isolated from fall-planted sweet corn (Zea mays L., Syngenta GSS 0966) leaves showing fine chlorotic streaks. Symptomatic plants were negative in enzyme-linked immunosorbent assay against many maize viruses, but reacted weakly with antisera to Sorghum stunt mosaic virus suggesting a distant relationship between the viruses. The virus was readily transmitted by vascular puncture inoculation (VPI), but not by leaf-rub inoculation. Symptoms on maize included dwarfing and fine chlorotic streaks along intermediate and small veins that developed 12 to 17 days post-VPI. The isolated virus was bacilliform (231 +/- 5 nm long and 71 +/- 2 nm wide), with a knobby surface, and obvious helical structure typical of rhabdovirus morphology. Nucleorhabdovirus virions were observed by transmission electron microscopy of infected maize leaf tissue sections. Proteins unique to infected plants were observed in extracts of infected leaves, and the isolated virion contained three proteins with molecular masses 82 +/- 2, 50 +/- 3, and 32 +/- 2 kDa. Preliminary sequence analysis indicated the virus had similarity to members of the family Rhabdoviridae. The virus was transmitted by Graminella nigrifrons under persistent conditions. The data indicate the virus, provisionally designated Maize fine streak virus, is a new species in the genus Nucleorhabdovirus.     

Characterization of a New Nepovirus Infecting Apricot in Southeastern France: Apricot Latent Ringspot Virus

A pathogen was transmitted from apricot trees showing symptoms of viral infection to GF305 peach seedlings which reacted by stunting, shortened internodes and chlorotic mottling. The agent was transmitted to cherry, apricot, peach and plum by grafting and to several herbaceous hosts by mechanical inoculation. Isometric nepovirus-like particles of 30–31 nm diameter extracted from infected Chenopodium quinoa sedimented as two peaks in sucrose gradients. These particles contained two single stranded RNAs of approximately 5.9 and 7.9 kb, and a single coat protein subunit of 53.7 kDa. No cross-reactions were observed with a number of nepoviruses infecting fruit trees. Inoculation of purified particles to herbaceous or woody hosts reproduced the same symptoms caused by the original isolate. Sequencing of a 2.2 kbp cDNA clone covering the 3 end of the small genomic RNA identified an open reading frame encoding a 317 aa N-truncated protein exhibiting significant similarities with the coat protein of nepoviruses. The 1257 nt long 3 non-coding region showed up to about 65% homology to the equivalent region of members of the subgroup C of nepoviruses. The properties of this pathogen do not match those of any previously described nepovirus. It should therefore be considered as a new member of the subgroup C of nepoviruses, for which the name of Apricot latent ringspot virus (ALRSV) is proposed.The nucleotide sequence reported in this work has been deposited in the EMBL databank under the accession number AJ278875.     

Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants

ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins.     

A potyvirus isolated from solanaceous hosts

A potyvirus was isolated from Datura stramonium, Lycopersicon esculentum (tomato) and Solanum nigrum in the Yemen. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. Its flexuous rod-shaped particles had a mean length of 719 nm and some of its pinwheel inclusion bodies in infected Nicotiana clevelandii leaves were unusual in that they were dichotomously branched. The virus infected various solanaceous species, but the symptoms it induced were distinct from those of pepper veinal mottle (PVMV) and potato Y viruses. Its particles were purified from N. glutinosa and their coat protein had an atypically high molecular mass a potyvirus of 41·5 kDa. They showed a distant serological relationship to those of PVMV and potato virus V in ISEM decoration tests, but did not react with antisera to particles of any other potyvirus tested. The virus has been tentatively named tomato mild mottle virus.     

Molecular identification of a begomovirus causing yellow vein disease on Calendula officinalis in India

A whitefly transmitted begomovirus was detected by PCR using begomovirus-specific primers from naturally infected Calendula officinalis plants showing yellow vein disease symptoms. An approximately 800 bp PCR amplicon was cloned and sequenced to identify the species of the virus isolate. Analysis of nucleotide sequence data resulted in its identification as the complete coat protein gene open reading frame (CP ORF) of 771 bp, which encoded 256 amino acid residues. The coat protein of the virus isolate shared maximum identities of 96–97% with four strains of Tobacco curly shoot virus (ToCSV) and an Ageratum enation virus (AgEV) during BLAST analysis of sequence data. Nucleotide- and amino acid-based phylogenetic analysis revealed the close relationship of the isolate with ToCSV strains, therefore it has been identified as an isolate of ToCSV and C. officinalis is considered to be a new host of ToCSV begomovirus. Association of a DNA-β molecule with the virus isolate was also detected by PCR and Southern hybridization tests using DNA-β specific primers and probe.     

苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒,能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清,建立了高效、准确的AMV间接ELISA检测方法,并应用于病害调查和抗性鉴定,结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍,血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片,不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测,有46份样品检测结果一致,符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明,病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定,结果显示40份大豆全部感染AMV,但是病毒载量存在差异,部分品种表现出AM...     

Development and use of detection methods specific for <Emphasis Type="Italic">Cucumber vein yellowing virus</Emphasis> (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.     

侵染番红花的芜菁花叶病毒

 从表现花叶症状的德国进口番红花上获得一线状病毒分离物SA,线状病毒粒子长度为700~800nm。其在人工接种的11科39种植物中能侵染6科14种植物,在克里芙兰烟上产生系统坏死,在小白菜、花椰菜等十字花科植物上产生系统花叶或为隐症,在昆诺藜等藜科植物上为局部侵染。在番红花病叶、球茎及克里芙兰烟病叶组织中观察到卷轴状和片层状聚集的风轮状内含体。免疫吸附和免疫修饰电镜观察结果显示,SA与芜菁花叶病毒((TuMV)具有紧密的血清学关系。根据这些特征将SA鉴定为TuMV的一个分离物。这是TuMV侵染番红花的首次报道。     

Characterization of the beet yellow stunt virus coat protein gene

ABSTRACT The beet yellow stunt virus (BYSV) genome contains at least nine open reading frames (ORFs) that code for proteins ranging from 6 to 66 kDa. Based on amino acid sequence comparisons, the coat protein (CP) was previously identified as the product of ORF7. We expressed the product of ORF7 in bacteria and confirmed that ORF7 codes for the BYSV CP by immunoblotting. BYSV is a phloem-limited virus, and virus CP antigen of a quality sufficient for diagnostic antisera production has not been available. To produce BYSV antigen free of plant host contaminants, ORF7 was cloned into a pMAL bacterial expression vector. The resulting fusion protein was affinity-purified and used as an antigen to raise anti-BYSV CP antisera in rabbits and guinea pigs. Using these antisera, an indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA)-based diagnostic system was developed. This indirect DAS-ELISA format enabled reliable detection of BYSV in tissue extracts from virus-infected lettuce diluted up to 5,000 times. The diagnostic system developed may enable large-scale epidemiological studies of BYSV using simple serological techniques. The antisera raised had a titer exceeding 1 x 10(5) in immunoblots and easily detected the 23.7-kDa BYSV CP in virus-infected lettuce and sowthistle plants. In these two plant species, BYSV CP was detected as two closely migrating bands during electrophoresis, which may suggest posttranslational CP modifications. To further characterize the BYSV CP gene, the 5'-untranslated region (UTR) of the BYSV CP subgenomic RNA (sgRNA) was cloned and sequenced. The CP-encoding, approximately 1.9-kb sgRNA has an AT-rich, 66-nucleotide-long 5'-UTR colinear to the genomic sequence upstream of ORF7.     

一个马铃薯Y病毒山东分离物的分离与鉴定

 从具有典型花叶症状的马铃薯叶片中分离到马铃薯Y病毒(Potato virus Y,PVY)(本文称PVY-SD-TA分离物),扩繁后,提纯病毒,电镜下可观察到700~900 nm×11 nm的病毒粒体,病组织超薄切片观察可见风轮状的内含体结构,寄主反应特性研究表明其能侵染2科13种植物。SDS-PAGE电泳检测病毒编码的外壳蛋白亚基的分子量为33 kDa。以PVY-SD-TA基因组RNA为模板,应用RT-PCR方法和特异引物合成了外壳蛋白基因。对cDNA全序列分析表明,PVY-SD-TA CP基因核苷酸序列与N株系的同源性为96%,与GenBank中登录序列号为AJ390306的O株系分离物的同源性最高,为99%;与国内不同学者报道的PVY中国流行株的同源性分别为96%,97%和98%。通过以上生物学特性和分子水平的研究将PVY-SD-TA鉴定为普通株系(PVY^O株系)。     

Biological and molecular characterization of Potato yellow blotch virus,a new species of the genus Potyvirus

A new species of the genus Potyvirus infecting potatoes, with the proposed name Potato yellow blotch virus (PYBV), was discovered in a breeding line 99m-022-026 in Scotland. The infected plants show isolated yellow blotches on the leaves. The genome of PYBV contains a large open reading frame encoding a single polyprotein of 3054 amino acids. Sequence analysis shows that PYBV is closely related to Potato virus A (PVA), with an overall 72% identity at the nucleotide level for the whole genome. The least conserved P1 protease gene shares only 50% nucleotide identity with PVA. The host range of PYBV was comparable to PVA on solanaceaous and non-solanaceous indicator plant species with the exception of Solanum demissum A and Y. Different symptoms were also observed for PYBV and PVA in Nicotiana benthamiana, Nicotiana hesperis and Nicotiana occidentalis P1. The susceptibility of potato (Solanum tuberosum) cultivars to PYBV and PVA was similar. In over 5 years of investigation, PYBV has not been found in commercial seed and ware potato crops in Scotland.     

中国水仙上一种球形病毒的研究Ⅰ.病毒分离及其生物学特性

 从水仙黄条病株上分离到一种球形病毒,直径约21nm,汁液摩擦接种35科(属)55(品)种植物,可侵染其中5科15(品)种,主要局限于豆种(Leguminosae)、藜科(Chenopodiaceae)以及茄科(Solanaceae)、苋科(Amaranthaceae)与番杏科(Aizoaceae)的个别种。在多数寄主上诱导局部坏死,豆科寄主上还伴随叶脉坏死症状产生。其中,昆诺阿藜(Chenopodium quinoa)、苋色藜(C.amaranticolor)、千日红(Gompherena globosa)、番杏(Tetragonia expansa)、大豆(Glycine max)、豇豆(Vigna sinensis)、克利夫兰烟(Nicotiana clevelandii)可作为诊断寄主;千日红、番杏与三叶草(Trifolium sibth)可作为毒源保存寄主。#br#在昆诺阿藜叶片汁液中,该病毒分离物的体外存活期在60天以上(25℃),热钝化点80-85℃(10分钟),稀释限点10^-5-10^-6。回接水仙健株,诱导产生系统褪绿条纹症状,不诱导水仙叶片产生具有特殊结构的内含体。     

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。