昆虫质多角体病毒研究的若干新进展

质多角体病毒隶属呼肠孤病毒科质多角体病毒属,病毒粒子为二十面体球形颗粒,具有3～5种结构蛋白,基因组由10或11个节段双链RNA构成。按病毒基因组RNA片段在聚丙烯酰胺或琼脂糖凝胶中电泳图谱的差异,将质多角体病毒分为15个电泳型。随着RNA病毒序列测定策略的逐步成熟与完善,质多角体病毒的序列测定方面取得一定的进展,家蚕质多角体病毒1的两个毒株（H株和I株）,舞毒蛾质多角体病毒1和14,及粉纹夜蛾质多角体病毒15的基因组全序列得到了测定,但质多角体病毒的进化与起源的研究因缺乏足够的遗传信息仍受到限制。     

新型夹竹桃天蛾质型多角体病毒的分离与初步鉴定

为了开发新型的夹竹桃天蛾的生物杀虫剂,本文从自然死亡的夹竹桃天蛾幼虫分离到病原微生物。通过电镜观察、FLAC(Full Length Amplification of cDNAs)技术以及病原体的基因组S2和S10片段的同源性分析,初步确认该病原体是一种质型多角体病毒(Daphnis nerii Cypovirus,DnCPV))。该病毒基因组电泳分析显示病毒基因组由10条dsRNA组成,大小在89 2bp和(约)4 160bp之间,其条带大小与现有的22类型均不相同。采用FLAC技术克隆了一种该CPV的基因组cDNA,并完成了基因组S2片段和S10片段序列测定工作。测序结果显示,DnCPV基因组S2片段编码RNA聚合酶,S10片段编码多角体蛋白。两者末端保守序列相同,正链5’端和3’端分别存在末端保守序列5’AGUCAAA·AGC3’。基于RNA聚合酶和多角体蛋白的氨基酸序列的系统发育分析显示该质型多角体病毒与19型和5型质型多角体病毒有较近的亲缘关系,但是电泳型上存在明显差异。因此推测该病毒是一种多角体病毒新类型,暂命名为夹竹桃质型多角体病毒南昌株(DnCPV-NC)。     

家蚕质多角体病毒(BmCPV)基因组dsRNA片段V的全序列测定

质多角体病毒属于呼肠孤病毒科质多角体病毒属,代表种为家蚕质多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,BmCPV).质多角体病毒粒子为正二十面体,直径约60nm～80nm,正二十面体的十二个顶点均有管状突起.     

棉铃虫5型质型多角体病毒RNA聚合酶的确定与功能机制研究

棉铃虫5型质型多角体病毒属于呼肠孤病毒科质型多角体病毒属,以重要农业害虫棉铃虫为其天然宿主,对棉铃虫的生物控制具有重要意义.本文对棉铃虫5型质型多角体病毒第3片段编码的蛋白的功能进行了初步研究.首先通过同源性对比,推测其所编码的蛋白可能行使RNA依赖的RNA聚合酶(RdRP)的功能.通过体外活性研究确定了该蛋白的RdRP活性,并确定了其保守活性位点GDD.随后以病毒基因组RNA和3′-OH封闭的病毒基因组RNA为模板,利用Northern blot方法研究该蛋白起始病毒基因组RNA合成的分子机制.结果表明,该病毒的RdRP主要通过引物非依赖的方式起始病毒基因组RNA的合成,并且该RdRP蛋白并不具有末端转移酶活性.最后,对RdRP行使功能的生化条件进行探索,发现RdRP功能的发挥需要二价金属离子Mg2+的存在.     

草鱼呼肠孤病毒的三维重构与衣壳蛋白特性

草鱼呼肠孤病毒(grass carp reovirus, GCRV)为呼肠孤病毒科水生呼肠孤病毒属一新成员. 最新的基因组序列分析发现, GCRV与哺乳动物呼肠孤病毒(mammalian reovirus, MRV)具有高度的同源性. 为了解GCRV致病机理, 进行了分辨率达到17 Å的三维重构与衣壳蛋白特性研究. 结果表明: GCRV颗粒呈多层排列, 包括RNA核心与内壳层、中间层及外壳层. 由200个按T = 13对称排列的三聚体组成外衣壳, 其典型特征是在5次轴上出现三聚体缺失凹陷区, 暴露出中间层三聚体亚单位. 内壳层由120个单体组成, 按T = 1排列, 结构特点与呼肠孤病毒科成员内衣壳特征相一致. 衣壳蛋白电泳显示, GCRV颗粒含有7种蛋白(VP1-VP7)组分, 与MRV衣壳蛋白特性相近, 两者在衣壳结构组成上的相似性与基因组序列的高度同源性相吻合. 此结果对进一步研究GCRV与宿主细胞相互作用机理具有指导意义.     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'－磷酸,3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端,经逆转录,退火,补齐形成全长双链cDNA。使用单一的互补引物2进行PCR 增,扩增产物克隆在pMD18-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长330bp,S'端具有CPV－1型末端保守序列AGTAAA'端具有保守序列GTTAGCC。起始密码子从ATG位于38－40残基,终止密码子TAA位于1208－1210残基。推测S8片段编码390年氨基酸多肽,分子量为44kDa。与舞毒蛾质多角体病（LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒（BmCPV）第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。     

中华绒螯蟹二株呼肠孤病毒的初步研究

在研究中华绒螯蟹病害的过程中,分离到两株呼肠孤病毒(分别命名为EsRV816和Es RV905).EsRV816从江苏某养殖场分离,EsRV905从武汉某养殖场分离.EsR V816和EsRV905 的病毒粒子为球状对称结构,大小分别为65nm和55nm.病毒粒子基因组分别为10和12个 节段的双链RNA.根据它们的宿主范围、基因组节段数及电泳型,这两种病毒很可能属于呼肠孤病毒科的两个新属.     

昆虫质型多角体病毒同义密码子使用模式分析

昆虫质型多角体病毒(cypovirus,CPV)是害虫种群重要调节因子,可用作生物防治剂。本研究采用多元统计分析方法对7种CPV进行密码子使用模式分析,结果表明:CPV密码子使用偏好性较弱,多数基因密码子使用模式受碱基组成影响,少数基因密码子使用模式除碱基组成外还有其它影响因素;中性绘图分析表明碱基组成主要受选择压力影响,受突变影响较小。同一电泳型CPV之间比同一宿主CPV之间共有的偏好性密码子多。CPV基因组内10个基因组片段之间密码子偏好性存在差异。CPV密码子偏好性与宿主昆虫密码子偏好性存在差异,所有CPV与其宿主昆虫共有的偏好性密码子均较少。对应分析进一步证明碱基组成是影响密码子使用的主要因素,不同电泳型CPV具有不同的密码子使用模式。聚类分析表明同一电泳型CPV密码子使用模式相似,同一宿主CPV密码子使用模式差异较大。     

马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

马尾松毛虫质多角体病毒(湖南株)基因组S7节段(AY180908)cDNA克隆及序列分析结果表明S7由1501个碱基组成,编码由448个氨基酸组成的分子量为49.8 kDa的多肽P50.5′末端和3′末端具有5′-AGTAA-3′和5′-GTTAGCC-3′末端保守序列.该基因组与舞毒蛾质多角体病毒1型和家蚕质多角体病毒1型S7节段有很高的同源性,核苷酸序列同源性分别为97.2%和87.0%,氨基酸序列同源性分别为98.7%和92.8%.P50多肽与人型支原体的 DnaK样蛋白在C-末端有相似性.本文报道了编码P50 C259的cDNA序列的克隆并作了原核表达,当用1.0 mmol/L IPTG 诱导2h,分子量约为37.3 kDa的融合蛋白在大肠杆菌BL21中得到大量表达.     

中华绒螯蟹二株呼肠孤病毒的初步研究

在研究中华绒螯蟹病害的过程中 ,分离到两株呼肠孤病毒 (分别命名为EsRV816和EsRV90 5 )。EsRV816从江苏某养殖场分离 ,EsRV90 5从武汉某养殖场分离。EsRV816和EsRV90 5的病毒粒子为球状对称结构 ,大小分别为 6 5nm和 5 5nm。病毒粒子基因组分别为 10和 12个节段的双链RNA。根据它们的宿主范围、基因组节段数及电泳型 ,这两种病毒很可能属于呼肠孤病毒科的两个新属     

Characterization of the complete genome segments from BmCPV-SZ, a novel Bombyx mori cypovirus 1 isolate

A novel Bombyx mori cypovirus 1 isolated from infected silkworm larvae and tentatively assigned as Bombyx mori cypovirus 1 isolate Suzhou (BmCPV-SZ). The complete nucleotide sequences of genomic segments S1-S10 from BmCPV-SZ were determined. All segments possessed a single open reading frame; however, bioinformatic evidence suggested a short overlapping coding sequence in S1. Each BmCPV-SZ segment possessed the conserved terminal sequences AGUAA and GUUAGCC at the 5' and 3' ends, respectively. The conserved A/G at the -3 position in relation to the AUG codon could be found in the BmCPV-SZ genome, and it was postulated that this conserved A/G may be the most important nucleotide for efficient translation initiation in cypoviruses (CPVs). Examination of the putative amino acid sequences encoded by BmCPV-SZ revealed some characteristic motifs. Homology searches showed that viral structural proteins VP1, VP3, and VP4 had localized homologies with proteins of Rice ragged stunt virus , a member of the genus Oryzavirus within the family Reoviridae. A phylogenetic tree based on RNA-dependent RNA polymerase sequences demonstrated that CPV is more closely related to Rice ragged stunt virus and Aedes pseudoscutellaris reovirus than to other members of Reoviridae, suggesting that they may have originated from common ancestors.     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经SDS－酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10。S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长763bp,起始密码AUG位于3－5残基,终止密码UGA位于747－749残基。推测DpCPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD。和家蚕质型多角体病毒（BmCPV）多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%。     

松毛虫CPV不同分离株的基因组电泳图谱

综述了松毛虫CPV不同分离株基因组电泳图谱的研究状况。松毛虫CPV是松毛虫Dedrolimusspp .的重要病原微生物 ,在松毛虫灾害治理中起着重要作用。病毒基因组电泳图谱是CPV重要的分类依据和研究基础。到目前为止 ,全世界范围共分离报道了 1 0株不同的松毛虫CPV ,基因组电泳图谱研究表明CPV -1型是松毛虫CPV的主要类型。PAGE分析显示 ,松毛虫CPV不同分离株中至少存在有 3种不同CPV -1的型内变异 ,它们有时以纯一型或混合型的形式出现。特别需要指出的是中国马尾松毛虫CPV分离株 ,其存在有 2种不同形态的病毒多角体 ,通过蔗糖密度梯度离心可以分为上下 2条带 ,上带多角体为锥形 ,基因组dsRNA电泳图谱显示 1 3条带 ,不属于目前已确定的 1 4种电泳型中的任何一种 ;下带多角体为六边形 ,基因组为纯合的CPV -1型 ;另外在不同时期不同地方感染马尾松毛虫也分离得到了纯合型的马尾松毛虫CPV -型     

Novel replication complex architecture in rubella replicon-transfected cells

Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo-lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double-stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB-infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross-sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno-EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two-dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin-O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.     

Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.     

Current status of <Emphasis Type="Italic">Deltabaculoviruses,Cypoviruses</Emphasis> and <Emphasis Type="Italic">Chloriridoviruses</Emphasis> pathogenic for mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.      

Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.     

Molecular characterization of a rotaviruslike virus isolated from striped bass (Morone saxatilis).

The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison of SBR virus and SA11 virus by cross-immunoprecipitation and cross-immunofluorescence tests did not show any relationship. These results suggest that SBR virus could represent a new genus within the family Reoviridae.