杨扇舟蛾颗粒体病毒gra基因的分析

本研究利用已知的颗粒体蛋白基因(granulin,gra)设计引物,通过PCR扩增得到ClanGV的gra基因.对PCR结果序列分析表明,ClanGV的gra基因开放阅读框(ORF)全长747bp,共编码248个aa,预计编码的蛋白质大小为29.3kDα.在启始密码子ATG上游-24bp处,有一个杆状病毒晚期启动子序列,ATAAG.有两个TATA框,分别位于ATG上游的26bp和65bp位置.基于gra的同源分析和进化树分析表明,ClanGV和茶小卷叶蛾颗粒体病毒(Adoxophyes orana granulovirus,AoGV)、苹果蠹蛾颗粒体病毒(Cydia pomonella granulovirus,CpGV)、云杉卷叶蛾颗粒体病毒(Choristoneura fumiferana granulovirus,CfGV)、马铃薯块茎蛾颗粒体病毒(Phthorimaeaoperculella granulovirus,PoGV)的亲缘关系较近.利用提取的颗粒体蛋白免疫家兔,制备了抗体进行免疫杂交分析,结果显示ClanGV除了与分月扇舟蛾颗粒体病毒(Clostera anastomosis L.granulovirus,CaLGV)的颗粒体蛋白有较强的杂交信号外,与棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HaNPV)多角体蛋白也有明显的杂交带出现,与甜菜夜蛾核多角体病毒(Spodoptera exigua nucleopolyhedrovirus,SeNPV)只有非常微弱的杂交信号.     

小菜蛾颗粒体病毒晚期表达因子基因的功能分析

小菜蛾颗粒体病毒(Plutella xylostella granulovirus,PlxyGV)基因组含有15个杆状病毒晚期表达因子(Lateexpression factor,lef)基因同源物。PlxyGV的14个lef基因(不包括ie-0)预期编码产物与苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus,AcMNPV)LEF蛋白的序列相似度为13%～53%。其中,LEF-8、LEF-9和P47在两种病毒之间的相似度较高,分别为49%、53%和46%。为了研究不同杆状病毒种间lef基因的功能相关性,验证PlxyGVlef基因的功能,本文利用AcMNPV的一个瞬时表达实验系统测试PlxyGVlef基因在Sf9细胞中激活AcMNPV晚期启动子控制的报告基因表达的能力。实验结果显示,在其它AcMNPVlef基因都存在的情况下,PlxyGVlef-2能够部分替代AcMNPVlef-2的活性。序列对比结果显示Plx-yGV LEF-2的C端末比其它GV和鳞翅目NPV的LEF-2分别多出约100aa和70aa。     

杨扇舟蛾颗粒体病毒gra基因的分析

本研究利用已知的颗粒体蛋白基因(granulin,gra)设计引物,通过PCR扩增得到ClanGV的gra基因。对 PCR结果序列分析表明,ClanGV的gra基因开放阅读框(ORF)全长747bp,共编码248个aa,预计编码的蛋白质 大小为29．3kDe。在启始密码子ATG上游-24bp处,有一个杆状病毒晚期启动子序列,ATAAG。有两个TATA 框,分别位于ATG上游的-26bp和-65bp位置。基于gra的同源分析和进化树分析表明,ClanGV和茶小卷叶蛾颗 粒体病毒(Adoxophyes orana granulovirus,AoGV)、苹果蠹蛾颗粒体病毒(Cydia pomonella granulovirus,CpGV)、 云杉卷叶蛾颗粒体病毒(Choristoneura fumiferana granulovirus,CfGV)、马铃薯块茎蛾颗粒体病毒(Phthorimaea operculella granulovirus,PoGV)的亲缘关系较近。利用提取的颗粒体蛋白免疫家兔,制备了抗体进行免疫杂交分 析,结果显示ClanGV除了与分月扇舟蛾颗粒体病毒(Clostera anastomosis L．granulovirus,CaLGV)的颗粒体蛋 白有较强的杂交信号外,与棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HaNPV)多角体蛋 白也有明显的杂交带出现,与甜菜夜蛾核多角体病毒(Spodoptera exigua nucleopolyhedrovirus,SeNPV)只有非常 微弱的杂交信号。     

灰斑古毒蛾核型多角体病毒三个晚期表达因子基因

将灰斑古毒蛾(Orgyia ericae)单粒包埋型核型多角体病毒(OrerSNPV)基因组DNA的各EcoRI酶切片段进行克隆和测序.序列分析结果表明EcoRI-M(3.0kb)与EcoRI-P片段(2.2kb)相连处含有编码晚期表达因子(LEF-2)的开放阅读框(ORF).同时在EcoRI-E(约10kb)片段两端分别含有lef-3和lef-11基因的部分序列.lef-2基因编码区由648bp组成,可编码216个氨基酸残基的多肽,预计蛋白质分子量为23.7kDa.将OrerSNPVLEF-2氨基酸序列与其它已知的27种杆状病毒的LEF-2序列比较,发现OrerSNPV与其它鳞翅目NPV LEF-2氨基酸有35%～49%同源性,其中与BusuSNPV、MaMNPV-A、HezeSNPV、HearSNPV和LdMNPV同源性最高,和GV有26%～31%同源性,与膜翅目松柏锯角叶蜂NPV的同源性为32%,与库蚊杆状病毒的同源性只有23%.根据氨基酸序列绘制的分子进化树表明28种杆状病毒lef-2基因可以分为鳞翅目NPV、GV、膜翅目NeseNPV与双翅目Culex nigripalpus Baculovirus四个分支.OrerSNPVlef-2与BusuSNPV在进化树上关系最为接近,而OrerSNPVlf-3和LdMNPV的最接近,OrerSNPVlef-11则和SeMNPV的关系最接近.     

灰斑古毒蛾核型多角体病毒三个晚期表达因子基因

将灰斑古毒蛾(Orgyiaericae)单粒包埋型核型多角体病毒(OrerSNPV)基因组DNA的各EcoRI酶切片段进行克隆和测序。序列分析结果表明EcoRI-M(3．0kb)与EcoRI-P片段(2．2kb)相连处含有编码晚期表达因子(LEF-2)的开放阅读框(ORF)。同时在EcoRI-E(约10kb)片段两端分别含有ref-3和lef-11基因的部分序列。lef-2基因编码区由648bp组成,可编码216个氨基酸残基的多肽,预计蛋白质分子量为23．7kDa。将OrerSNPVLEF-2氨基酸序列与其它已知的27种杆状病毒的LEF．2序列比较,发现OrerSNPV与其它鳞翅目NPVLEF．2氨基酸有35％-49％同源性,其中与BusuSNPV,MaMNPV-A,HezeSNPV、Hear3NPV和LdMNPV同源性最高,和GV有26％-31％同源性,与膜翅目松柏锯角叶蜂NPV的同源性为32％,与库蚊杆状病毒的同源性只有23％。根据氨基酸序列绘制的分子进化树表明28种杆状病毒lef-2基因可以分为鳞翅目NPV、GV、膜翅目NeseNPV与双翅目Culex nigripalpus Baculovirus四个分支。oret SNPV lef-2与BUSUsnpv在进化树上关系最为接近,而orerSNPV lef-3和LdMNPV的最接近,OrerSNPV lef-11则和SeMNPV的关系最接近。     

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind Ⅲ-L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组的HindⅢ-L片段的全序列.该片段全长2 635bp,包括5个有意义的开放阅读框HaSNPV ORF227,晚期表达因子10基因(lef10),vp1054基因,Ac55(AcMNPV ORF55的同源基因),Ac56(AcMNPV ORF56的同源基因).与其它6种杆状病毒的氨基酸序列比较表明,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒(SeMNPV)的同源性最高,为64%,与冷杉毒蛾核型多角体病毒(OpMNPV)的同源性最低,为43%;HaSNPV的vp1054基因与SeMNPV的同源性最高,为65%,与OpMNPV的同源性最低,为49%.序列比较表明,HaSNPV的LEF10与VP1054蛋白与其它6种杆状病毒具有相同的保守区和亮氨酸拉链(1eucine zipper)     

中国棉铃虫核多角体病毒基因组XbaI—I片段的序列分析

报道了棉铃虫单核衣壳核多角体病毒 (HaSNPV)XbaI I片段的序列结构。该片段在基因组上定位于 18.4～2 2 .8m .u .,包括 8个完整的开放阅读框架和 p47的 5′端部分序列 :其中ubiquitin ,39K/pp31,lef - 11,p47,Ac34 ,Ac38和Lsel2 5homologue 7个基因是杆状病毒的同源基因 ,另外两个orf5 0 7和orf10 80是HaSNPV的特有基因 ,且具有典型的早期表达基因启动子的特征 ,经软件分析发现这两个基因推导的产物具有典型的跨膜压结构。序列比较分析表明 ,ubiquitin基因与其它杆状病毒的同源基因有相同的保守区 ,39K/pp31具有和其它杆状病毒同源基因不同的启动子结构     

东方粘虫颗粒体病毒超氧化物歧化酶基因的克隆与分析

为获得东方粘虫颗粒体病毒(Pseudelatia separata granulovirus,PsGV)基因组序列,采用随机克隆方法,建立PsGV的质粒基因文库,并通过对插入片段进行克隆鉴定和序列分析,获得编码超氧化物歧化酶蛋白的基因(PsGV-sod)。该基因阅读框为462bp,共编码153个氨基酸。核苷酸和氨基酸同源性比较结果表明该基因与其他颗粒体病毒同源性较高,通过保守基序分析,认为其为铜锌超氧化物歧化酶。     

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind III-L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒 (Helicoverpaarmigerasingle nucleocapsidnucleopolyhedrovirus,HaSNPV)基因组的HindIII L片段的全序列。该片段全长 2 6 35bp ,包括 5个有意义的开放阅读框 :HaSNPVORF2 2 7,晚期表达因子 10基因 (lef10 ) ,vp10 5 4基因 ,Ac5 5 (AcMNPVORF5 5的同源基因 ) ,Ac5 6 (AcMNPVORF5 6的同源基因 )。与其它 6种杆状病毒的氨基酸序列比较表明 ,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒 (SeMNPV)的同源性最高 ,为6 4 % ,与冷杉毒蛾核型多角体病毒 (OpMNPV)的同源性最低 ,为 4 3% ;HaSNPV的vp10 5 4基因与SeMNPV的同源性最高 ,为 6 5 % ,与OpMNPV的同源性最低 ,为 4 9%。序列比较表明 ,HaSNPV的LEF10与VP10 5 4蛋白与其它 6种杆状病毒具有相同的保守区和亮氨酸拉链 (leucinezipper)     

扁刺蛾核型多角体病毒新分离株的基因组测序与分析

从自然死亡的扁刺蛾幼虫分离到一株核型多角体病毒。扁刺蛾核型多角体病毒新分离株(Oxyplax ochracea nucleopolyhedrovirus-Thosea sinensis,OxocNPV-Ts)具有典型的杆状病毒特征，呈单粒包埋型病毒粒子，病毒颗粒(Occlusion-derived virus,ODV)呈不规则的多边形，直径为0.63μm～1.49μm。OxocNPV-Ts能有效杀灭扁刺蛾幼虫，它对扁刺蛾幼虫的半致死浓度LC₅₀为4×10⁶ PIB/mL，是一种理想的生物防治剂。本实验对OxocNPV-Ts基因组进行了测序和鉴定。OxocNPV-Ts基因组全长114781 bp，包含124个开放阅读框。它与斜纹刺蛾核型多角体病毒(OxocNPV)关系最为密切，序列相似性高达99%。系统进化分析表明，OxocNPV-Ts属于杆状病毒科alpha杆状病毒属(Alphabaculovirus)，根据国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)的种属划分标...     

一株稻纵卷叶螟颗粒体病毒的系统发育分析和流行病学调查

稻纵卷叶螟颗粒体病毒（Cnaphalocrocis medinalis granulovirus,CnmeGV）于1979年首次从我国水稻主要害虫稻纵卷叶螟（Cnaphalocrocis medinalis,Guenée）幼虫中分离,能够在宿主种群内传播,具备发展为生物杀虫制剂的潜力。本研究利用从田间采集的稻纵卷叶螟幼虫,在实验室成功增殖了CnmeGV。编码杆状病毒RNA聚合酶两个亚基的基因lef-8、lef-9和包涵体基因granulin在杆状病毒系统演化过程中非常保守。利用PCR扩增得到CnmeGV的上述三基因,并将其核苷酸序列融合。通过该融合序列与GenBank中相应病毒序列的系统发育分析,发现CnmeGV是未被GenBank收录的病毒。流行病学初步调查结果表明,仅在采集自广东省江门市恩平县的稻纵卷叶螟体内发现了CnmeGV,而其他采集地点,包括广州市岑村、惠州市万芦村、惠州市新岗村和湖北省潜江市均未发现。江门市恩平县是CnmeGV的首次发现地,时隔三十多年,仍然存在因感染CnmeGV而死亡的稻纵卷叶螟幼虫,表明该病毒具有田间控制目标害虫的持效性能。     

粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.     

Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.     

Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno

Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.     

Identification, characterization and phylogenic analysis of conserved genes within the odvp-6e/odv-e56 gene region of Choristoneura fumiferana granulovirus

The genes that are located within the odvp-6e/odv-e56 region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing the 11 kb BamHI restriction fragment on the ChfuGV genome. The global GC content that was calculated from the data obtained from this genomic region was 34.96%. The open-reading frames (ORFs), located within the odvp-6e/odv-e56 region, are presented and compared to the equivalent ORFs that are located at the same region in other GVs. This region is composed of 14 ORFs, including three ORFs that are unique to ChfuGV with no obvious homologues in other baculoviruses as well as eleven ORFs with homologues to granuloviral ORFs, such as granulin, CfORF2, pk-1, ie-1, odv-e18, p49, and odvp-6e/odv-e56. In this study, the conceptual products of seven major conserved ORFs (granulin, CfORF2, IE-1, ODV-E18, p49 and ODVP-6E/ODV-E56) were used in order to construct phylogenetic trees. Our results show that granuloviruses can be grouped in 2 distinct groups as follows: Group I; Choristoneura fumiferana granulovirus (ChfuGV), Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), and Adoxophyes orana granulovirus (AoGV). Group II; Xestia c-nigrum granulovirus (XcGV), Plutella xylostella granulovirus (PxGV), and Trichoplusia ni granulovirus (TnGV). The ChfuGV conserved proteins are most closely related to those of CpGV, PhopGV, and AoGV. Comparative studies, performed on gene arrangements within this region of genomes, demonstrated that three GVs from group I maintain similar gene arrangements.