山东烟台小麦土传病毒病由小麦黄花叶病毒和土传小麦花叶病毒相关病毒复合侵染所致

在山东省烟台地区的小麦上发生一种由土壤中禾谷多粘菌Polymyxa graminis传播的病毒病,感病小麦植株表现矮化褪绿和花叶症状.我们于1997年4月从病区采集感病小麦植株,进行了病毒种类鉴定.直接电镜观察发现有二种病毒粒子,一种粒子呈棒状,占大多数,其长度约为300nm和150nm; 另一种粒子呈线状,数量较少,长度为500nm～700nm.免疫电镜结果表明,棒状病毒粒子仅与土传小麦花叶病毒(soil-borne wheat mosaic virus, SBWMV)抗血清反应,而不与小麦黄花叶病毒(wheat yellow mosaic virus,WYMV)抗血清和小麦梭条斑花叶病毒(wheat spindle streat mosaic virus,WSSMV)抗血清反应;反之,线状病毒仅与WYMV、WSSMV抗血清反应,而不与SBWMV抗血清反应.用WYMV和SBWMV两种抗血清同时进行修饰时,线状病毒粒子和棒状病毒粒子均发生反应.     

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.     

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。     

建兰花叶病毒单克隆抗体的制备及检测应用

用建兰花叶病毒(Cymbidium mosaic virus,CymMV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得3株能稳定传代并分泌抗CymMV单克隆抗体(McAb)的杂交瘤细胞(2C6、5B7和12G9),分别制备它们的单抗腹水。其中5B7和12G92株单克隆抗体腹水间接ELISA效价达10-6,3株单抗的抗体类型及亚类均为IgG1,轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测CymMV的方法。蝴蝶兰病叶作1∶10240倍稀释、提纯CymMV病毒浓度为4.87ng/mL(每孔的病毒绝对量为0.487ng)时,该方法仍能检测到病毒。利用ACP-ELISA方法检测了田间样品,发现CymMV在兰花上发病很普遍。     

单抗免疫斑点法和组织印迹法检测侵染蝴蝶兰的建兰花叶病毒

应用抗建兰花叶病毒(CymMV)的单克隆抗体, 建立了快速检测蝴蝶兰病样的免疫斑点法(Dot-ELISA)和组织印迹法(Tissue blot-ELISA)。CymMV单抗稀释8000倍时, Dot-ELISA可检出病毒粗汁液的最大稀释度为1:10240; Tissue blot-ELISA中样品1次平切后1~5次印迹与Dot-ELISA样品1:80稀释结果相当, 6~8次印迹与Dot-ELISA 1:320稀释结果相当, 前8次印迹均可以得到满意的检测效果。Tissue blot-ELISA的灵敏度略低     

一株貂肠炎病毒某些特性的研究

采用猫肾传代细胞FK增殖华东地区分离的貂传染性肠炎病毒(mink infectious enteritis virus,MEV-S_1)能产生明显的细胞病变。浓缩的病毒液经Sepharose-4B柱层析提纯处理后置电镜下观察,可见典型的病毒粒子。经蛋白酶K、SDS裂解病毒,用苯酚-氯仿法抽提病毒核酸,二苯胺试验和酶消化试验表明该病毒核酸为DNA类型。吖啶橙染色试验表明该DNA为单链。甲酰胺法进行核酸分子展层,病毒核酸呈线状,长度为1.5～2.0/μm。     

RBSDV在玉米叶脉细胞内的侵染状态与灰飞虱传毒活力的关系

根据水稻黑条矮缩病毒(RBSDV)侵染玉米(Zea mays L.)的症状发展过程先后取叶脉做超薄切片,在透射电镜下观察病毒在细胞内的侵染状态,并在取样前用灰飞虱无毒若虫进行饲毒和传毒试验.结果显示RBSDV侵入玉米叶细胞后先出现在细胞壁附近,个别粒子似与胞间连丝相连;细胞质内产生病毒基质,病毒粒子先增殖并分布其周边,后向病毒基质内扩展;当病毒粒子布满病毒基质后在细胞质中出现直径约90nm的管状结构,病毒成串排列在该管状结构中;随后管状结构逐渐消失,最终形成晶格状聚集排列.用灰飞虱无毒若虫在细胞内病毒基质出现和病毒增殖期饲毒的,到成虫时分别有2.93%和7.83%个体传毒率;在细胞内病毒成串分布于管状结构和晶格状聚集排列期饲毒的,到成虫时均不能传毒.     

5种虾脊兰菌根显微结构观察

采用石蜡切片法观察5种虾脊兰菌根的显微结构、菌根真菌的侵入途径与分布特征,为更好地保护和开发利用虾脊兰属植物资源提供理论依据。结果表明:(1)5种虾脊兰菌根的显微结构由根被、皮层和中柱组成,根被细胞3～6层,皮层由9～13层薄壁细胞组成,韧皮部与木质部8～12束,呈辐射状相间排列,中柱中央为薄壁细胞组成的髓。(2)菌丝通过外皮层中的通道细胞侵入皮层,且皮层是内生菌根真菌寄居的主要区域,菌丝和菌丝结在靠近外皮层的几层皮层细胞中数量居多,菌丝在细胞核附近被消解,细胞核变形膨大。研究认为,5种虾脊兰可能拥有广泛的侵染能力较强的菌根真菌类型,使得这5种虾脊兰均具有较强的生态适应性。     

RBSDV在玉米叶脉细胞内的侵染状态与灰飞虱传毒活力的关系

根据水稻黑条矮缩病毒(RBSDV)侵染玉米(Zea mays L.)的症状发展过程先后取叶脉做超薄切片,在透射电镜下观察病毒在细胞内的侵染状态,并存取样前用灰飞虱无毒若虫进行饲毒和传毒试验。结果显示RBSDV侵入玉米叶细胞后先出现在细咆壁附近,个别粒子似与胞间连丝相连;细胞质内产生病毒基质,病毒粒子先增殖并分布其周边,后向病毒基质内扩展;当病毒粒子布满病毒基质后在细胞质中出现直径约90nm的管状结构,病毒成串排列在该管状结构中;随后管状结构逐渐消失,最终形成晶格状聚集排列。用灰飞虱无毒若虫在细胞内病毒基质出现和病毒增殖期饲毒的,到成虫时分别有2．93％和7．83％个体传毒率;在细胞内病毒成串分布于管状结构和品格状聚集排列期饲毒的,到成虫时均不能传毒。     

引起马蹄莲花叶病的芋花叶病毒

在采自杭州、北京、上海等地的表现花叶和扭曲症状的马蹄莲(Zanledeschia aethiopica)上检测到一种线状病毒。经测定,其平均粒子长度为745nm,病毒粒子最大分布范围为720—750nm。该病毒经免疫电镜观察与芋花叶病毒(DMV)有紧密的血清学关系,且与3个PVY抗血清也有一定的反应。在病叶细胞中观察到紧密聚集。松散聚集和分散的线状病毒粒子和典型的风轮状内含体。作者认为:感染马蹄莲的该种病毒符合芋花叶病毒的特征。本文对该病毒的血清学关系和组织病变情况进行了分析讨论。     

Genome-wide analysis of small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus synergistically infecting Phalaenopsis

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids and causing economic losses worldwide. Mixed infection of CymMV and ORSV could induce intensified symptoms as early at 10 days post-inoculation in inoculated Phalaenopsis amabilis, where CymMV pathogenesis was unilaterally enhanced by ORSV. To reveal the antiviral RNA silencing activity in orchids, we characterized the viral small-interfering RNAs (vsiRNAs) from CymMV and ORSV singly or synergistically infecting P. amabilis. We also temporally classified the inoculated leaf-tip tissues and noninoculated adjacent tissues as late and early stages of infection, respectively. Regardless of early or late stage with single or double infection, CymMV and ORSV vsiRNAs were predominant in 21- and 22-nt sizes, with excess positive polarity and under-represented 5ʹ-guanine. While CymMV vsiRNAs mainly derived from RNA-dependent RNA polymerase-coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3ʹ-untranslated region, with a specific hotspot residing in the 3ʹ-terminal pseudoknot. With double infection, CymMV vsiRNAs increased more than 5-fold in number with increasing virus titres. Most vsiRNA features remained unchanged with double inoculation, but additional ORSV vsiRNA hotspot peaks were prominent. The potential vsiRNA-mediated regulation of the novel targets in double-infected tissues thereby provides a different view of CymMV and ORSV synergism. Hence, temporally profiled vsiRNAs from taxonomically distinct CymMV and ORSV illustrate active antiviral RNA silencing in their natural host, Phalaenopsis, during both early and late stages of infection. Our findings provide insights into offence–defence interactions among CymMV, ORSV and orchids.     

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.     

Genetic Diversities of Cymbidium mosaic virus and Odontoglossum ringspot virus Isolates Based on the Coat Protein Genes from Orchids in Guangdong Province,China

Orchids are some of the most important ornamental flowers. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent and economically important viruses affecting orchids in China. In this study, 20 CymMV and 28 ORSV isolates were selected for genetic diversity analysis. The CymMV isolates shared 84.6–100% and 89.5–100% identities of coat protein (CP) at the nucleotide (nt) and amino acid (aa) levels, respectively. The identities of ORSV isolates were 96.4–100% (nt) and 92.5–99.4% (aa). The CP genes of CymMV were found to have genetic diversity, and the CP genes of ORSV were genetically conservative. These results can aid in designing effective disease‐control strategies.     

Structurally Modified Plant Viruses and Bacteriophages with Helical Structure. Properties and Applications

Biochemistry (Moscow) - Structurally modified virus particles can be obtained from the rod-shaped or filamentous virions of plant viruses and bacteriophages by thermal or chemical treatment. They...     

Comparative Study of Thermal Remodeling of Viruses with Icosahedral and Helical Symmetry

Study of the possibilities of virions and viral proteins modifications and structural remodeling is an important problem of the modern molecular virology. A technique of heat treatment of rod-shaped tobacco mosaic virus that allowed producing structurally modified spherical particles consisting of the virus coat protein was previously developed in our laboratory. These particles possessed unique adsorption and immunogenic properties and were successfully used to develop a new candidate vaccine against rubella virus. Later, the possibility of thermal remodeling of the filamentous virions of potato virus X was demonstrated. The present work reports a comparative study of thermal remodeling of viruses with different structure belonging to various taxonomic groups. The generation of structurally modified spherical particles by the heat treatment of rod-shaped virions with helical symmetry (dolichos enation mosaic virus and barley stripe mosaic virus) has been demonstrated. The dependence of the size of spherical particles derived from dolichos enation mosaic virus on the initial virus concentration was revealed. The process of thermal remodeling of the filamentous virions and virus-like particles of alternanthera mosaic virus was studied. Heat treatment of plant viruses with icosahedral symmetry was shown to cause no morphological changes.     

Specific incorporation of host cell surface proteins into budding vesicular stomatitis virus particles

The specific incorporation of cell surface proteins into budding Vesicular Stomatitis Virus (VSV) particles was shown by two approaches. In the first, monolayer cultures of Vero or L cells were labeled by lactoperoxidase-catalyzed iodination and the cells were then infected with VSV. Approximately 2% of the cell surface ¹²⁵1 radioactivity was incorporated into particles which co-purify with normal, infectious virions by both velocity and equilibrium gradient centrifugation and which are precipitated by antiserum specific for the VSV glycoprotein. Control experiments establish that these ¹²⁵I-labeled particles are not cell debris or cellular material which aggregate with or adhere to VSV virions. VSV virions contain only a subset of the 10–15 normal ¹²⁵1-labeled cell surface polypeptides resolved by SDS gel electrophoresis; VSV grown in L cells and Vero cells incorporate different host polypeptides. In a second approach, Vero cells were labeled with ³⁵S-methione, then infected with VSV. Two predominant host polypeptides (molecular weights 110,000 and 20,000) were incorporated into VSV virions. These proteins, like VSV G protein, are exposed to the surface of the virion. They co-migrate with the major incorporated ¹²⁵1 host polypeptides. These host proteins are present in approximately 10 and 80 copies, respectively, per virion. Specific incorporation of host polypeptides into VSV virions does not require the presence of viral glycoprotein. This was shown by use of a ts VSV mutant defective in maturation of VSV G protein to the cell surface. Budding from infected cells are noninfectious particles which contain all the viral proteins except for G; these particles contain the same proportion and spectrum of ¹²⁵1-labeled host surface polypeptides as do wild-type virions. These results extend previous conclusions implicating the submembrane viral matrix protein, or the viral nucleocapsid, as being of primary importance in selecting cell surface proteins for incorporation into budding VSV virions.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

Ultrastructural Studies of Barley Mesophyll Cella lnfected with Barley Yellow Mosaic Viruses

lexuous filamentous, rod-shaped particles, and laminated, pinwheel inclusions were observed in the mesophyll cells of the barley plants naturally infected with barley yellow mosaic viruses. These virus particles had a length of 480–920 nm and a width of 10–20 nm. In addition, bundles of filamentous structures which consisted of many particles with more 2000 nm in length were found in the leaves of the infected barley plants. The ultrastructural alterations of the infected mesophyll cells were rather conspicuous. The cytoplasmic matrix was lost seriously, and the chloroplast membrane system was destroyed. The cristae and matrix of the mitochondrium were decreased and some of them became vacuoles. The endoplasmic reticulum (ER) expanded teristic membranous network structures occurred in the cytoplasm of infected cells. The virus particles were often associated at one end with ER and with the membranes of network structures. The nucleus, membrane and wall of ceils also had somewhat variation.     

Odontoglossum ringspot virus host range restriction in Nicotiana sylvestris maps to the replicase gene

The experimental host range of Odontoglossum ringspot virus (ORSV), a member of the tobamoviruses, includes several species of Nicotiana , but not N. sylvestris . However, ORSV was able to replicate in protoplasts from N. sylvestris leaves. By using the green fluorescent protein (GFP) as a marker inserted into ORSV, it was found that a small number of single epidermal cells became infected in mechanically inoculated leaves, but the virus did not move cell to cell. The ORSV movement protein (MP) and coat protein (CP) were examined for their ability to effect movement by substitution into Tobacco mosaic virus (TMV) hybrids. Both proteins and the 3' non-translated region (NTR) of ORSV allowed movement of TMV hybrids in N. sylvestris . These results suggested that the inability of ORSV to move in N. sylvestris was due to the replicase gene or the 5'NTR. One possibility was that the replicase gene could indirectly affect movement by failing to produce subgenomic (sg) RNAs for expression of MP or CP, but this appeared not to be the case as ORSV replicated and produced MP and CP sgRNAs, both of which were translated in N. sylvestris protoplasts. Additionally, genomic RNA was encapsidated into virions in N. sylvestris protoplasts. Because the 5'NTR permitted efficient replication and production of replicase proteins, these findings suggest that the replicase of ORSV is responsible for the defect in cell-to-cell movement of ORSV in N. sylvestris .