斜纹夜蛾核型多角体病毒Ⅱ型DNA甲基化研究

目的:检测斜纹夜蛾核型多角体病毒Ⅱ型是否存在DNA甲基化。方法:选取SpltNPVⅡ的DNA聚合酶序列以及全序中其它三段序列,利用亚硫酸盐修饰直接测序法进行甲基化检测实验。结果:DNA聚合酶中共有246处CG,14处发生甲基化,全序中选取的三段序列只有一段发生3处甲基化。结论:实验表明,SpltNPVⅡ中存在DNA甲基化,其DNA聚合酶甲基化程度高于其它三段序列,且不同序列的甲基化程度不一样,这为以后该病毒的表观遗传学进一步研究奠定了基础。     

柞蚕核型多角体病毒基因表达载体系统的构建与基因表达

柞蚕是一种野外饲养的经济昆虫,主要分布在我国东北部山区。柞蚕以蛹滞育越冬,其蚕蛹个大、容易固定、保存时间长、无须饲养、容易运输等优点。利用柞蚕蛹作为生物反应器生产外来蛋白质,可以大规模机械化生产,减少操作上的烦琐和劳动力。本文利用柞蚕核型多角体病毒(AnpeNPV)作为基因表达载体,在柞蚕培养细胞(AnPe细胞)和柞蚕蛹中成功地表达了β-半乳糖苷酶基因（LacZ）,并利用AnPe细胞筛选、纯化获得了AnpeLacZ重组病毒。该重组病毒的β-半乳糖苷酶产量,在TC-100(含10%FBS)培养的AnPe细胞中最高酶活性为感染后第12天的40.9 units/ml,在SF-900Ⅱ培养的AnPe细胞中最高酶活性为感染后第18天的59.9 units/ml,后者表达量稍高,但时间滞后。AnpeLacZ在5℃保存了7个月的柞蚕蛹中,感染后第15天酶活性达到最高,雌蛹14.3 units/g,雄蛹11.7 units/g,雌蛹比雄蛹略高。结果显示,柞蚕核型多角体病毒和柞蚕蛹可以作为一个可以机械化大规模生产的新型杆状病毒基因表达载体系统开发和利用。     

零背景转座技术高效构建重组家蚕杆状病毒表达系统

摘要：【目的】获得零转座背景的基于家蚕核型多角体病毒(Bombyx mori Nucleopolyhedrovirus, BmNPV) Bac-to-Bac 系统,为高效经济构建重组BmNPV在家蚕体内表达目标蛋白提供新系统。【方法】利用R6Kγ作为复制子构建新的条件复制型杆状病毒转移载体pRADM,同时封闭BmNPV-Bacmid（BmBacmid）宿主菌（Escherichia coli BmDH10Bac）的Tn7转座受体位点attTn7,获得新的封闭型宿主菌E.coli BmDH10Bac△Tn7。【结果】由于pRADM无法在宿主菌E.coli BmDH10Bac中复制,封闭了attTn7位点的宿主菌也不能再和BmBacmid竞争与转移载体的重组,显著提高了转座效率。封闭宿主菌的attTn7位点,能使转座效率提高近4倍,使用条件复制型转座载体pRADM时,转座效率提高近10倍。而用pRADM转座E.coli BmDH10Bac△Tn7时,转座阳性率为100％。避免了获得重组病毒DNA的鉴定程序,缩短了获得重组蛋白所需时间。用携带红色荧光蛋白基因DsRed的重组质粒pRADM-Red转座E.coli BmDH10Bac△Tn7,获得重组BmBacmid转染BmN细胞,红色荧光蛋白在细胞中得到高效表达。【结论】结果表明pRADM和E.coli BmDH10Bac△Tn7是一种零背景高效构建重组BmNPV的新系统。     

Phylogenetic analysis of <Emphasis Type="Italic">Orgyia pseudotsugata</Emphasis> single-nucleocapsid nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses. Foundation item: Supported by a scholarship from the European Union (Functional Biodiversity and Crop Protection), contract n^o HPMT-CT-2000-00199.     

Entry into midgut epithelial cells is a key step in the selection of genotypes in a nucleopolyhedrovirus

An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes (e.g., SfNIC-C), that lack pif1 and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype (SfNIC-B) in coinfected cells. To determine whether selection for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum and budded virus (BV) samples taken from larvae inoculated with OBs comprising 10% SfNIC-B + 90% SfNIC-C indicating that a marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures of 50% SfNIC-B + 50% SfNIC-C or those comprising 10% SfNIC-B + 90% SfNIC-C as most of the OBs in these mixtures originated from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely modulated by changes in MOI during the infection period.     

Nucleopolyhedrovirus introduction in Australia

Nucleopolyhedrovirus (NPV) has become an integral part of integrated pest management (IPM) in many Australian agricultural and horticultural crops. This is the culmination of years of work conducted by researchers at the Queensland Department of Primary Industries and Fisheries (QDPI&F) and Ag Biotech Australia Pty Ltd. In the early 1970's researchers at QDPI&F identified and isolated a virus in Helicoverpa armigera populations in the field. This NPV was extensively studied and shown to be highly specific to Helicoverpa and Heliothis species. Further work showed that when used appropriately the virus could be used effectively to manage these insects in crops such as sorghum, cotton, chickpea and sweet corn. A similar virus was first commercially produced in the USA in the 1970's. This product, Elcar(R), was introduced into Australia in the late 1970's by Shell Chemicals with limited success. A major factor contributing to the poor adoption of Elcar was the concurrent enormous success of the synthetic pyrethroids. The importance of integrated pest management was probably also not widely accepted at that time. Gradual development of insect resistance to synthetic pyrethroids and other synthetic insecticides in Australia and the increased awareness of the importance of IPM meant that researchers once again turned their attentions to environmentally friendly pest management tools such NPV and beneficial insects. In the 1990's a company called Rhone-Poulenc registered an NPV for use in Australian sorghum, chickpea and cotton. This product, Gemstar(R), was imported from the USA. In 2000 Ag Biotech Australia established an in-vivo production facility in Australia to produce commercial volumes of a product similar to the imported product. This product was branded, ViVUS(R), and was first registered and sold commercially in Australia in 2003. The initial production of ViVUS used a virus identical to the American product but replicating it in an Australian Helicoverpa species, H. armigera. Subsequent research collaboration between QDPI&F and Ag Biotech reinvigorated interest in the local virus strain. This was purified and the production system adapted to produce it on a commercial scale. This new version of ViVUS, which was branded ViVUS Gold(R), was first registered and sold commercially in 2004. Widespread insect resistance to insecticides and a greater understanding of integrated pest management is leading to increased adoption of technologies such NPV in Australian agriculture.     

The Polyhedral Membrane does not Protect Polyhedra of AcMNPV Against Inactivation on Greenhouse Chrysanthemum

Polyhedral inactivation of wild-type AcMNPV and an AcMNPV mutant lacking the gene for the polyhedral membrane protein (AcMNPV-Delta pp34) was studied on greenhouse chrysanthemum. It was hypothesized that polyhedra without a polyhedral membrane might be more susceptible to inactivation on plants. The density of infectious polyhedra of both viruses on the leaf surface decreased in time in a near-exponential fashion. The inactivation curves suggested the presence of two distinct fractions of polyhedra with differences in persistence. One fraction of polyhedra is not inactivated at all, whereas the other fraction is inactivated in an exponential fashion. Relative inactivation rates of the inactivated polyhedra fraction for wild-type AcMNPV and AcMNPV-Delta pp34 were 0.16 and 0.13 per day, respectively, which is not significantly different. After 28 days on leaves in a greenhouse, both viruses still showed residual infectivity. The fraction of residual infectious polyhedra were not significantly different and amounted to approximately 20% of the original density for both wild-type AcMNPV and AcMNPV-Delta pp34. Therefore, the polyhedral membrane does not protect polyhedra against inactivation on greenhouse chrysanthemum.     

核型多角体病毒侵染及其与宿主免疫系统互作的研究进展

核型多角体病毒(Nucleopolyhedrovirus,NPV)应用广泛,已被开发成微生物杀虫剂和用于重组蛋白表达等.NPV具有两种病毒颗粒:包埋型病毒粒子(occlusion-derived virus,ODV)和芽生型病毒粒子(budded virus,BV),两者的构成和组装存在差异.病毒包涵体在肠道中溶解后释...