酶联免疫吸附法（ELISA）检测四川“榨菜”病毒种类

1990至1991年在“榨菜”病毒病发生的始期和盛期,从四川九个县(市)近郊、远郊采集样品492份,用TuMV、CMV、TMV、PVX、PVY和CaMV六种抗血清,经酶联免疫吸附间接法进行检测,属于TuMV和TuMV与其它五种病毒之一或之二复合感染的有422份,占总样品数的85.77％,其中由TuMV单一感染的305份,占61.99％。此外,还有PVX、PVY、CaMV单一感染病样27份,占5.49％,而情况不明的43份,占8.74％,总检出率91.26％。检测结果表明,发病始期或盛期,近郊或远郊均以TuMV和TuMV与CMV复合感染的病毒为主,是四川“榨菜”生产上主要的病毒种类。     

芜菁花叶病毒(TuMV)特性的研究进展*

芜菁花叶病毒 (TuMV)是世界范围内广泛分布的重要病毒。从生物学和理化特性、基因组结构、侵染和繁殖、变异及利用转基因技术提高病毒抗性等方面对TuMV的国内外研究现状进行了阐述。     

芜菁花叶病毒单克隆抗体的制备及检测应用

先以芜菁花叶病毒（TuMV）免疫BAL B/C小鼠,然后取其脾细胞使之与SP2/0鼠骨髓瘤细胞融合,经筛选、克隆,获得4株能稳定传代并分泌抗TuMV单克隆抗体（Mab）的杂交瘤细胞,并以之制备腹水单抗。4株单克隆抗体腹水ELISA效价在10-5～10-6之间,仅对TuMV起特异性反应。Western blot分析表明,4株单抗都能与TuMV 34kD的外壳蛋白亚基起特异反应。利用TuMV的多抗兔血清和单抗腹水建立了三抗体夹心ELISA检测TuMV的方法,检测病叶的灵敏度为1∶5120倍,检测提纯TuMV病毒绝对量为21.9 ng。利用三抗体夹心ELISA测定出7种作物上有TuMV侵染。      

芜菁花叶病毒的3’末端序列克隆与CP基因序列分析

目的:克隆芜菁花叶病毒(Turnip mosaic virus,TuMV)的3'末端序列,并进行CP基因序列分析.方法:以TuMV杭州榨菜分离物(TuMv-HZZC)接种病叶为材料,利用病毒粒子吸附法制备病毒RNA模板,经RT-PCR扩增获得了TuMV-HZZC 3'末端序列,将其克隆到PMD 18-T质粒上进行序列分析.结果:TuMV-HZZC分离物3'末端序列包括部分的Nib基因、完整的TuMVCP基因和3'-UTR,CP基因为864bp,分别编码288个氨基酸,3'-UTR序列(不包括PolyA尾巴)为213bp.经过与其他TuMV分离物的CP基因核苷酸和氨基酸比较,同源性分别达到88.0%～97.6%和91.0%-96.5%.结论:TuMV的系统进化具有典型的地域和寄主关联性.     

分泌抗芜菁花叶病毒株系特异性单克隆抗体杂交瘤细胞株的建立及抗体理化性质测定

用经芜菁花叶病毒(TuMV)免疫的BALB／c小鼠脾细胞与骨髓瘤细胞(Sp2／0－Agl4)融合,经3次克隆化培养和ELISA筛选,建立了5类分泌抗TuMV的单克隆抗体(McAb)的杂交瘤细胞株。其中4类分别对TuMV－CI株系、TuMV—C3株系、TuMV—C4株系和TuMV－c5株系具有特异性反应,第5类对TuMV 5个株系均有反应。以双抗体夹心ELISA和间接ELISA检测,上述McAb同CaMV、CMV、TMV、PVX和PVY等均不产生交叉反应,小鼠腹水McAb的滴度在1：256000--2048000之间,多为1：1024000,比常规多抗血清高400倍左右。对上述杂交瘤细胞系和McAb的生物学特性及理化性质进行了鉴定。用SDS—PAGE、Western—blotting对TuMV外壳蛋白亚基、McAb识别位点进行了分析,并就McAb区分TuMV株系进行了讨。     

芜菁花叶病毒粒体及寄主细胞内内含体的超薄切片电镜观察

从沈阳地区分离到侵染大白菜(Brassicapekinensis)及萝卜(Raphanussativus)的芜菁花叶病毒(TurnipMosaicVirus,TuMV)纯化繁殖于大白菜(小白口)及萝卜(长白品种)上。经约25天采收纯化繁殖的毒原材料,进行病毒粗提纯,并做病毒粒子的电镜观察。看到病毒粒体较为疏散清晰,丝状,长度多分布在720nm之间。另将接种TuMV的感病寄主有关部位,切成小块经固定、系列脱水、环氮树脂包埋后,制作超薄切片并用电镜观察,看到病叶、病茎和病根韧皮部细胞内,均有清晰的风轮状、环状及带状的内含体,这是马铃薯Y病毒组(Potyvirus)成员特征之一。     

芜菁花叶病毒分子生物学研究进展

芜菁花叶病毒(Turnip mosaic virus,TuMV)是马铃薯Y病毒属(Potyvirus)的一个重要成员, 在世界上分布广泛,主要危害十字花科植物。本文对芜菁花叶病毒的分子生物学相关研究,如基因组结构和功能、株系变异与进化、寄主植物抗性以及抗病毒转基因工程等的研究进展作一阐述。     

芜菁花叶病毒（广州油菜株）生化性质的研究

我们对芜菁花叶病毒广州油菜株(TuMV—G.Z)进行了生化性质的研究。结果表明,纯化的病毒外壳蛋白经10％SDS-聚丙烯胺酰胶电泳出,现一个组份,其分子量为1.4×10~4d;氨基酸组份分析表明,TuMV—G.Z外壳蛋白亚基由约107个氨基酸残基组成,其中包括1个色氨酸和5个酩氨酸;DNS—C1末端法测其N端,结果显示为苏氨酸,且其氨基是游离的。     

基于相对密码子频率的芜菁花叶病毒基因组序列的进化分析

现有92株芜菁花叶病毒(TuMV)的全基因组序列已在GenBank报道,据分析报道其中58株不含重组序列。利用系统聚类法对92株TuMV的全基因组序列和58株TuMV全基因组序列的相对密码子频率RSCU值进行聚类分析。同时利用系统发育分析方法分析了这92株和58株TuMV全基因组序列。结果发现,92株芜菁花叶病毒株的密码子偏性聚类树与其系统进化树的一致度很低;而不含重组序列的58株芜菁花叶病毒株的密码子偏性聚类树与其系统进化树的一致度却非常高,且与寄生宿主类型基本对应。这表明在不存在重组的情况下,TuMV密码子频率的偏性可能是宿主内的一种选择压力,影响TuMV基因组的点突变进化方向,促使TuMV适应宿主内环境。     

不结球白菜种质资源对TuMV的抗性鉴定与评价

分别利用苗期人工接种鉴定及ELISA检测方法,对127份不结球白菜种质资源进行芜菁花叶病毒(TuMV)的抗性鉴定。结果显示,苗期人工接种鉴定的病情指数(DI)分布在3.55~95.68之间,不同种质间表现出较大的抗性差异。不同抗性级别的次数分布图基本符合正态分布,略向感病区域偏离。基于病情指数的聚类分析结果与抗病性分级基本一致。通过苗期鉴定共筛选获得高抗TuMV的不结球白菜种质6份,抗病种质13份,其主要农艺性状表现出一定的多样性。其中叶面皱缩和具有刺毛的抗病材料所占比例较高,可能与TuMV抗性存在一定的相关性。ELISA检测结果显示,117份供试种质的P/N值分布在3.10~25.37之间,不同种质间表现出病毒含量的差异,但与DI值未呈现明显相关性,可作为抗病材料筛选的辅助指标。     

Genetic control of immunity to turnip mosaic virus in winter oilseed rape (Brassica napus ssp. oleifera) and the effect of foreign isolates of the virus

Immunity to a UK isolate (UK 1) of turnip mosaic virus (TuMV) was studied in eight lines of oilseed rape selected from cv Rafal. Six of these lines were uniformly immune and two segregated. Segregation ratios in the F₂ generation of reciprocal crosses between two uniformly immune lines and two uniformly susceptible cultivars (Mikado and Yeoman) showed that immunity was controlled by a dominant nuclear allele. The immunity was confirmed by the inability to detect virus particles in mechanically inoculated plants by back inoculations, ELISA and ISEM tests. Plants were immune to repeated inoculations and aphid transmissions. The immunity was effective against one other UK isolate and two German isolates of TuMV. Another UK isolate (UK 3) and a Greek isolate partially overcame the immunity causing local infection and a Canadian and a Danish isolate overcame it completely causing systemic mosaic-type symptoms. When these immunity-breaking strains were tested against swede line 165 which is also immune to UK 1 TuMV, the Canadian isolate overcame the immunity whereas the Danish isolate did not. Using this swede line, one susceptible and one immune line of oilseed rape as differentials, four distinct groups of TuMV isolates could be identified. Selections of oilseed rape immune to UK TuMV isolates were more severely affected by the Canadian TuMV than UK TuMV susceptible selections. The gene determining immunity to TuMV had no pleiotropic effect on susceptibility to cauliflower mosaic virus. The implications on these findings in relation to breeding for virus resistance are discussed.     

A plant RNA virus hijacks endocytic proteins to establish its infection in plants

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor‐mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin‐related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co‐opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV‐infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1‐dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.     

Biological, Serological and Coat Protein Properties of a Strain of Turnip Mosaic Virus Causing a Mosaic Disease of Brassica campestris and B. juncea in India

Biological, serological and coat protein properties of a potyvirus (Poty-Rape) causing a mosaic disease of Brassica campestris and B. juncea in India were investigated. The virus readily infected 4 of the 5 plant species in the family Brassicaceae in which it induced severe systemic mosaic symptoms; it also induced chlorotic and necrotic local lesions in Chenopodium amaranticolor , but failed to infect 4 other species of Chenopodiaceae or 20 species of Amaranthaceae, Apiaceae, Canabinaceae, Compositae, Cucurbitaceae, Euphorbiaceae, Leguminosae and Solanaceae. The virus was transmitted in a non-persistant manner by Myzus persicae, Brevicoryne brassicae and Aphis gossypii. The Average size, of the virus particles in a purified preparation was 740 nm × 12 nm. SDS-PAGE analysis of the viral coat protein showed two major bands of approximately 37 kDa and 31 kDa, a pattern very similar to that of a reference isolate of turnip mosaic virus (TuMV) from the U.S. In Western-blot immunoassay, an antiserum to TuMV reacted with both the coat protein bands of the Poty-Rape islate and the reference TuMV, but not with the coat proteins of four other potyviruses. The high performance liquid chromatographic profile of tryptic peptides from the coat protein of Poty-Rape was found to be very similar to that of the reference TuMV, but differed substantially from those of four other potyviruses. The Poty-Rape isolate is considered to be a distinct strain of, TuMV.     

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1–97), the core (amino acids 98–245), and the C-terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.     

The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.     

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.     

The Natural Occurrence of Turnip Mosaic Potyvirus in Allium ampeloprasum

An isolate of turnip mosaic potyvirus (TuMV) was obtained from Allium ampeloprasum grown in commercial greenhouses in Israel. Symptoms on infected plants include systemic chlorosis and yellow stripes, accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically, and in a non-persistent manner by aphids, among Allium, Chenopodium. Gomphrena and some Nicotiana spp. Purified preparations contained numerous filamentous particles similar to those observed in crude extracts of infected leaves. Particles from crude plant extracts had a normal length of 806 nm. Cells of infected plants contained cylindrical cytoplasmic inclusions with pinwheel, scrolls and laminated aggregates which indicated the presence of a potyvirus of Edwardson's subgroup III. and which resemble those of turnip mosaic virus (TuMV), The virus reacted strongly with antiserum to typical isolates of TuMV in immunoelectron microscopy and western blotting but not with antisera to several other potyviruses. Based on serological reactivity, electron microscopy, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV.     

Viruses infecting winter oilseed rape (Brassica napus ssp. oleifera)

Turnip mosaic virus (TuMV) and cauliflower mosaic virus (CaMV) have been found infecting field crops of winter oilseed rape (Brassica napus ssp. oleifera) in South Warwickshire. Other viruses found include broccoli necrotic yellows virus (BNYV) and a member of the beet western yellows virus group. Systemic leaf symptoms caused by TuMV varied within and between cultivars; the three predominant reaction types were classified as necrotic, mosaic and immune. Some recently introduced cultivars of oilseed rape were more severely affected by TuMV infection than older cultivars. Reactions to CaMV were less varied and immunity was not found. The seed yield from TuMV and CaMV-infected plants was less than that of healthy control plants. This effect was due to infected plants producing either fewer seeds, smaller seeds or both. Germination of seeds from infected plants was unaffected if sown soon after harvest. After storage for one year the germination of seed from a virus infected plant was significantly less than that of seed from a virus-free plant. All commercial cultivars tested were experimentally susceptible to turnip yellow mosaic virus (TYMV) and some American strains of cucumber mosaic virus (CMV).     

Effects of turnip mosaic virus and turnip yellow mosaic virus on the survival, growth and reproduction of wild cabbage (Brassica oleracea)

Wild plants of Brassica oleracea (wild cabbage) are commonly infected with turnip mosaic poty virus (TuMV), turnip yellow mosaic tymovirus (TYMV) and several other viruses. A field experiment in which plants were inoculated either with TuMV or TYMV showed that virus infection significantly reduced survival, growth and reproduction. Relative to water inoculated-controls, plants infected with TYMV had greater mortality, were shorter, had a smaller leaf area and number, showed a greater amount of damage from herbivory and chlorosis, were less likely to flower and produced fewer pods and lower total seed output. Plants infected with TuMV did not appear to be adversely affected at first; however, mortality after 18 months was higher than control plants. Although TuMV infection had no effect on the number of plants flowering, the infected plants did produce fewer pods and a lower total seed output. We conclude that both viruses can significantly affect vegetative and reproductive performance of wild cabbage and hence that introgression of virus resistance (particularly when conferred by a major gene or a transgene) from a crop might increase plant fitness in natural populations of this species. Ecological risk assessments of virus resistance transgenes must do more than survey adult plants in natural populations for the presence of the target virus. Failure to detect the virus could be due to high mortality on infection with the virus.