昆虫质型多角体病毒的研究进展

质型多角体病毒(Cytoplasmic polyhedrosis virus,CPV)隶属呼肠孤病毒科Reoviridae质型多角体病毒属Cypovirus,通常基因组由10个节段双链RNA构成。RNA分子量为3～27u。根据病毒基因组dsRNA片段在聚丙烯酰胺或琼脂糖凝胶中电泳图谱的差异,目前CPV已被分为19个电泳型。不同于呼肠孤病毒科其它成员,CPV为单层衣壳,而不是常见的双层衣壳结构,衣壳蛋白主要由衣壳蛋白、大突起蛋白及塔式突起蛋白组成。大部分质型多角体病毒引起昆虫慢性疾病,造成寄主死亡或适应性降低。随着RNA病毒基因序列测定技术的成熟,质型多角体病毒的序列测定方面取得较大进展,目前GenBank核苷酸序列数据库中已经公布了家蚕Bombyx mori CPV电泳型1两个株系(H株和I株)、舞毒蛾Lymantria dispar CPV电泳型1、舞毒蛾CPV电泳型14及粉纹夜蛾Trichoplusia ni CPV电泳型全基因组序列,为该病毒的进化与起源的研究提供更多的遗传信息。本文从结构功能、侵染特点、基因组特点及应用前景等方面综述了昆虫质型多角体病毒的研究进展。     

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)。     

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DpGPV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%.     

家蚕对马尾松毛虫质型多角体病毒的敏感性

用虫体克隆技术,对马尾松毛虫质型多角体病毒湖南株（DpCPV-HN）进行了分离纯化,鉴定为质型多角体病毒1型。以家蚕春蕾×镇珠杂种F₁代及自交的F₂代4或5日龄幼虫进行毒力测定,以纯化的家蚕质型多角体病毒对F₁代幼虫的毒力测定为对照。结果表明：家蚕品种春蕾×镇珠对家蚕质型多角体病毒敏感,马尾松毛虫质型多角体病毒湖南株能引起其感染发病;马尾松毛虫质型多角体病毒湖南株感染家蚕品种春蕾×镇珠F₁代幼虫和F₂代幼虫28天后的半致死剂量（LD₅₀）分别为885个和18个CPB(质多角体),前者为后者的49倍。马尾松毛虫质型多角体病毒湖南株感染后的家蚕,其结茧率、化蛹率、羽化率、全茧量、茧层量和单蛾产卵数均有所下降,全茧量、茧层量、茧层率和单蛾产卵数与病毒感染剂量之间无显著关联。     

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%。     

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.     

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49．8kDa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1．0mmol／L IPTG诱导2h,分子量约为37.3kDa的融合蛋白在大肠杆菌BL21中得到大量表达。     

棉铃虫单粒包埋型核型多角体病毒极早期基因(ie-1)分析(英文)

克隆了棉铃虫Helicoverpaarmigera单粒包埋型核型多角体病毒 (HaSNPV)C1株基因组DNA ,并通过随机测序的方法测定了经XbaI酶切后的H片段的核苷酸全序列。序列比较和分析发现该片段中ORF1 3与苜蓿丫纹夜蛾Autographacalifornica多粒包埋型核型多角体病毒 (AcMNPV)基因组ORF1 47(ie 1 )同源。ie 1基因编码区全长 1 986bp ,根据推测的氨基酸序列 ,可编码 6 6 1个氨基酸残基组成的多肽 ,预计分子量为 76 .5kD。将所推导的HaSNPVIE 1氨基酸序列与其它已知的杆状病毒IE 1氨基酸序列进行比较 ,结果表明 ,HaSNPV和谷实夜蛾H .zea单粒包埋型核型多角体病毒IE 1氨基酸序列最为相似 ,同源性高达 98%。与AcMNPV、家蚕Bombyxmori核型多角体病毒 (BmNPV)、云杉卷叶蛾Choristoneurafu miferana多粒包埋型核型多角体病毒 (CfMNPV)、舞毒蛾Lymantriadispar多粒包埋型核型多角体病毒(LdMNPV)、黄杉毒蛾Orgyiapseudotsugata多粒包埋型核型多角体病毒 (OpMNPV)、甜菜夜蛾Spodopteraex igua多粒包埋型核型多角体病毒 (SeMNPV)、小菜蛾Plutellaxylostella颗粒体病毒 (PxGV)和Xestiac ni grum颗粒体病毒 (XcGV)的IE 1氨基酸序列同源性较低 ,分别为 2 3 %、2 3 %、2 3 %、2 5 %、2 3 %、1 4%、2 7%和 7%。根据氨基酸序列由GENETYX     

马尾松毛虫质型多角体病毒NS5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,获得一株单一质型多角体病毒。提纯的病毒粒子经SDS－热酚法抽提,在使用低熔点琼脂凝胶电泳分离基因组dsRNA,回收纯化第九片段S9。SgRNA双链经高温变性,使用正反两种引物逆转录合成cDNA双链,使用同样的引物经PCR扩增后,克隆在pMD18-T载体上。利用两种引物组合,获得了大小两个亚克隆片段。序列测定结果表明,较小亚克隆片段长405bp,较大亚克隆片段长677bp,经过序列拼接,得到一个977bp的序列,其中包含一个963bp大的开放阅读框（ORF）。推测DpCPVS9基因编码一个320个氨基酸的多肽,分子质量35.66kDa。和家蚕1型质型多角体病毒的I株（BmCPV-I Istrain)位于第九片段的NS5蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为86．0％和93．4％。     

马尾松毛虫质型多角体病毒NS5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-热酚法抽提,在使用低熔点琼脂糖凝胶电泳分离基因组dsRNA,回收纯化第九片段S9.SgRNA双链经高温变性,使用正反两种引物逆转录合成cDNA双链,使用同样的引物经PCR扩增后,克隆在pMD18-T载体上.利用两种引物组合,获得了大小两个亚克隆片段.序列测定结果表明,较小亚克隆片段长405bp,较大亚克隆片段长677bp,经过序列拼接,得到一个977bp的序列,其中包含一个963bp大的开放阅读框(ORF).推测DpCPVS9基因编码一个320个氨基酸的多肽,分子质量35.66kDa.和家蚕1型质型多角体病毒的I株(BmCPV-II strain)位于第九片段的NS5蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为86.0%和93.4%.     

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     

A comparative study of three closely related cytoplasmic polyhedrosis viruses

A cytoplasmic polyhedrosis virus (CPV) from the pine caterpillar, Dendrolimus spectabilis, was compared with Japanese isolates of closely related viruses from the silkworm, Bombyx mori, and gypsy moth, Lymantria dispar. The sizes of the viral RNA genome segments were almost identical, although the CPVs from D. spectabilis and L. dispar could be distinguished from the silkworm virus by a small size difference (0.03 × 10⁶ daltons) in one segment. The same viruses were also distinguishable by RNA homology differences of 25–50% measured by the reannealing of ³H-labeled single-stranded viral messenger RNA (synthesized in vitro) to heat-denatured viral double-stranded RNA. Antigenic differences were also detected by gel immunodiffusion tests. CPVs of D. spectabilis and L. dispar were indistinguishable by these criteria.     

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.     

Biological and molecular studies of a cypovirus from the black fly Simulium ubiquitum (Diptera: Simuliidae)

A cypovirus from the black fly Simulium ubiquitum (SuCPV) was isolated and examined using biological and molecular techniques. SuCPV produces small (typically 0.25mum), polyhedral shaped inclusion bodies (polyhedra), in which the virus particles become multiply embedded. SuCPV is the third cypovirus isolated from Diptera, but the first from Simuliidae that has been characterized using molecular analyses. SuCPV has a genome composed of 10 segments of dsRNA, with an electrophoretic migration pattern that is different from those of recent UsCPV-17 and CrCPV-17 isolates from the mosquitoes Uranotaenia sapphirina and Culex restuans, respectively. The SuCPV electropherotype appears to show significant differences from those of the previously characterized lepidopteran cypoviruses. Sequence analysis of SuCPV segment 10 shows that it is unrelated to either of the two CPV isolates from Diptera or to the CPV species for which Seg-10 has been previously characterized from Lepidoptera. A comparison of the terminal regions of SuCPV genome segments to those of CPV-1, 2, 4, 5 14, 15, 16, 17, 18, and 19 also revealed only low levels of conservation. We therefore, propose that SuCPV is classified within a new Cypovirus species, which we have tentatively identified as Cypovirus-20. We have therefore referred to this virus isolate as S. ubiquitum CPV-20 (SuCPV-20).     

我国蚊虫分离的一种新型胞质多形体病毒

0507BS3是从中国新疆喀什地区采集的库蚊和按蚊混合蚊标本分离的病毒株,对C6/36细胞致病变而对Ve-ro和BHK-21细胞不致病变。电镜观察显示病毒颗粒呈圆球形,直径约60nm(n=20),无包膜,单层衣壳,衣壳内有中央核。基因组核酸电泳显示基因组包括10条双链RNA(double stranded RNA,dsRNA)片段。病毒第10基因片段核酸序列测定显示该片段全长964bp(GenBankID:FJ150869),具有单一开放读码框,编码长度为275个氨基酸的蛋白,分子量约30.8kD。病毒第10基因片段核酸序列比对未发现相似的病毒核酸序列,氨基酸序列与胞质多形体病毒(Cytoplasmic polyhedrosis virus,CPV)第10基因片段编码的多形体蛋白部分区段匹配。病毒第10基因片段和已知各型CPV第10基因片段核酸序列共同进行系统进化分析显示该病毒位于独立的进化分枝,提示0507BS3病毒可能是一种新型CPV病毒。     

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.     

Homologous Terminal Sequences in the Double-Stranded RNA Genome Segments of Cytoplasmic Polyhedrosis Virus of the Silkworm Bombyx mori

The 3′-terminal regions (20 to 32 residues) of the genome double-stranded RNA (dsRNA) segments of cytoplasmic polyhedrosis virus were sequenced. The dsRNAs, which were labeled at their 3′ termini by incubation with [5′-³²P]pCp and T4 RNA ligase, were denatured and resolved into the plus and minus strands by agarose-urea gel electrophoresis. Ten single-stranded RNAs thus obtained from the five dsRNA segments IV, V, VIII, IX, and X were sequenced by postlabeling methods. Common 3′-terminal sequences, -GUUAGCC and -UUACU, were found in the plus and minus strands, respectively, of all five dsRNA segments. However, adjacent sequences diverged and were considerably variable. The homologous sequences found in the 3′ end may be important recognition signals for viral RNA polymerases and for assembly of the genome segments.