The microRNA pathway activation in insect cell model upon Autographa californica multiple nucleopolyhedrovirus infection

Background:The immune system of insects exerts fundamentally different antiviral mechanisms than mammals.MicroRNAs(miRNAs)play vital roles in developing insect antiviral immunity.MiRNAs expression profiles of insects changed significantly during baculovirus infection.Methods:Differential expression profiles of miRNAs in Spodoptera frugiperda were monitored by next-generation sequencing(NGS)and RT-qPCR during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.The transcription levels of genes were detected by RT-qPCR.The 50%tissue culture infective dose(TCID_(50))endpoint dilution assay was used to determine the proliferation of progeny virus.Results:NGS revealed that 49 miRNAs were differentially expressed in Sf9 cells,and 10 of them were significantly up-or down-regulated.Though RT-qPCR analysis,we observed the similar trends for the expression patterns of significantly differentially expressed miRNAs from NGS.Moreover,the transcription levels of core genes,Exportin5,Dicer1,and Argonaute1,in miRNA biogenesis pathways were significantly increased after AcMNPV infection.For five selected miRNAs,miR-34-5p could regulate the proliferation of baculovirus progeny virus and energy metabolism.Conclusion:The miRNAs biogenesis pathway in Sf9 cells plays an important role and may be stimulated to resist AcMNPV infection.This work provides evidence for the molecular mechanism of baculovirus-insect interaction and offers novel ideas and directions for green pest control technology.

Analysis and expression of the polyhedrin gene of Antheraea pernyi nucleopolyhedrovirus (AnpeNPV)

The polyhedrin (polh) gene is often used to analyse evolution of baculovirus. In this report, the polh of Antheraea pernyi nucleopolyhedro-virus (AnpeNPV) was cloned and sequenced. The Open reading frame (ORF) of the AnpeNPV consists of 738 nucleotides encoding 245 amino acids with molecular masses of 29 kDa. The deduced amino acids were significant homol-ogy with other baculoviruses, such as Attacus ricini NPV (ArNPV) and Autographa californica NPV (AcNPV). A strongly hydrophilic region was predicted at positions from 30 to 50 of the An-peNPV Polh protein by bioinformatics analysis. Expression of the polh gene of AnpeNPV in E. coli was examined by SDS–PAGE, Western blot and Mass-spectrum analysis. The result showed that the bacterium expression system was suitable for the virus gene expression. It indi-cated that the products of the polh gene ex-pressed in this system can be easier to use for raising antibodies.

Encyclopedia of Autographa californica Nucleopolyhedrovirus Genes

The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.

基于NPV Scheduler软件的露天转地下矿山开采境界研究

基础的L-G图论法未考虑地下开采可能的成本优势,在露天转地下矿山开采境界圈定时存在一定的局限性。本文通过对价格法与储量盈利比较法计算的经济合理剥采比进行分析,结合NPV Scheduler软件研究了露天转地下开采境界圈定方法,并通过工程实例验证了该方法的合理性。研究成果对露天转地下矿山开采境界的确定具有一定的指导意义。

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?, 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?, 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?, 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?, 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.

Production, Application, and Field Performance of Abietiv^(TM),the Balsam Fir Sawfly Nucleopolyhedrovirus

Beginning in the early 1990s, the balsam fir sawfly (Neodiprion abietis) became a significant defoliating insect of precommercially thinned balsam fir (Abies balsamea (L.) Mill.) stands in western Newfoundland, Canada. In 1997, a nucleopolyhedrovirus (NeabNPV) was isolated from the balsam fir sawfly and, as no control measures were then available, NeabNPV was developed for the biological control of balsam fir sawfly. In order to register NeabNPV for operational use under the Canadian Pest Control Products Act, research was carried out in a number of areas including NeabNPV field efficacy, non-target organism toxicology, balsam fir sawfly ecology and impact on balsam fir trees, and NeabNPV genome sequencing and analysis. As part of the field efficacy trials, approximately 22 500 hectares of balsam fir sawfly-infested forest were aerially treated with NeabNPV between 2000 and 2005. NeabNPV was found to be safe, efficacious, and economical for the suppression of balsam fir sawfly outbreak populations. Conditional registration for the NeabNPV-based product, Abietiv?, was received from the Pest Management Regulatory Agency (Health Canada) in April 2006. In July 2006, Abietiv was applied by spray airplanes to 15 000 ha of balsam fir sawfly-infested forest in western Newfoundland in an operational control program.

The ORF 113 of Heliocoverpa armigera Single Nucleopolyhedrovirus Encodes a Functional Fibroblast Growth Factor

Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group II NPV. The HearNPV vfgf transcripts were detected from 18 to 96 h post-infection (hpi) of Hz-AM1 cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.

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.

Putative Phosphorylation Sites On WCA Domain of HA2 Is Essential For Helicoverpa armigera Single Nucleopolyhedrovirus Replication

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Thr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.

Phylogenetic Analysis of Orgyia pseudotsugata 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.

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often coevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Ac16 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac16 interacts with baculoviral and cellular proteins. Bm8/Ac16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bm8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group I NPVs.

BmNPV不同地理株之间毒力差异的分子机制探究

家蚕核型多角体病毒(Bombyx mori nucleopolyhe⁃drovirus,BmNPV)是家蚕的主要病原之一,由BmNPV引发的血液型脓病对蚕桑产业的危害极大。对BmNPV3个地理分离株BmNPV⁃Zhenjiang、BmNPV⁃Baoshan和BmNPV⁃Sanya进行全基因组测序分析。在3个地理株基因组序列之间检测到位于编码区的90个单核苷酸多态性(single⁃nucleotide pol⁃ymorphism,SNP)位点和7个插入⁃缺失多态性(insertion⁃de⁃letion,InDel)位点,导致26个杆状病毒核心基因中的21个所编码的氨基酸序列发生了突变。这21个基因所涉及的功能包括:杆状病毒基因组的复制与转录、病毒粒子的组装以及病毒的侵染。此外,检测了3个地理株对菁松×皓月2龄幼蚕的致死中浓度(LC_(50)),并分析了ie⁃1、polh和p143这3个基因在菁松×皓月5龄幼蚕中肠组织中的相对表达水平。BmNPV⁃Zhenjiang与BmNPV⁃Baoshan的LC_(50)无显著差异,而BmNPV⁃Sanya的LC_(50)显著高于BmNPV⁃Zhenjiang和BmNPV⁃Baoshan(P≤0.01)。在幼蚕感染3个地理株48 h、72 h和96 h后,BmNPV⁃Zhenjiang与BmNPV⁃Baoshan中ie⁃1、polh和p143的相对表达水平无显著差异,而BmNPV⁃Sanya中polh、ie⁃1和p143的相对表达水平显著低于BmNPV⁃Zhenjiang和BmNPV⁃Baoshan(P≤0.01)。分析结果表明,BmNPV⁃Zhenjiang与BmNPV⁃Baoshan的毒力不具有较大差异,而BmNPV⁃Sanya的毒力弱于BmNPV⁃Zhenjiang和BmNPV⁃Baoshan。3个地理株之间核心基因的差异可能与其毒力差异存在一定关系,研究结果为阐明造成BmNPV不同地理分离株产生毒力差异的分子机制提供了初步的线索。

采用NPV Scheduler软件对某大型露天矿山进行境界优化

某大型矿床随着开采的进行以及补充勘探的深入,加上市场的变化,使得原来的境界设计条件发生了一些变化,因此需要根据最新的边界条件对境界进行优化设计。采用NPV Scheduler软件用L-G图论法对该铜矿的最终开采境界进行了优化圈定,使得最终境界更符合当前采选成本和市场条件。

NPV与IRR评价互斥方案结论差异的原因分析

NPV和IRR指标用于评价多个互斥项目时,评价结论可能不一致。财务管理、成本会计理论通常认为其原因在于项目净收益的再投资收益率假设不同。证明二者不一致的原因并非再投资收益率假设之不同,而是由于总量指标和比率指标所致。

带有VaR方法的模糊NPV模型及其混合智能算法

基于可信性理论建立一类带有模糊Va R方法的NPV模型,并设计含有逼近方法、神经网络和遗传算法的混合智能算法对提出的模型进行求解。给出一个数值例子表明模型和算法的有效性。通过改变目标函数和可信性约束中的可信性水平对最优解进行灵敏度分析。

利用BmNPV多角体包埋外源蛋白的观察

家蚕核型多角体病毒在感染晚期大量表达产生多角体蛋白,并形成结构致密的超大分子蛋白质晶体,以有效保护内部包埋的病毒粒子,保证子代病毒的传播。借鉴多角体包埋病毒粒子的原理,观察到多角体也能将外源蛋白质固定入多角体内部。利用能形成多角体的BmNPV Polh+Bac-to-Bac表达系统构建的含有绿色荧光蛋白(EGFP)的重组病毒能在表达、产生正常多角体的同时也表达EGFP蛋白。共聚焦分析表明,该重组病毒感染后形成的多角体能够发射绿色荧光。被包埋的EGFP蛋白放置1个月后仍能检测到绿色荧光,干燥处理30min后仍能检测到荧光。由此说明将多角体与外源基因同时表达,多角体中会包埋外源蛋白,且能较长时间保存外源蛋白的生物活性。这一现象为解决蛋白质长期保存提供了新思路,对于开发含有毒素蛋白的新型生物杀虫剂也有很好的参考意义。

苏云金杆菌与EoNPV混用的增效作用

采用生物测定方法测定茶尺蠖核型多角体病毒（Ectropis obliqua nucleopolyhedrovirus，EoNPV）和苏云金杆菌（Bacillus thuringiensis，Bt）的联合作用。5组不同含量EoNPV制剂（1．50×10^7 PIB／ml、1．25×10^7PIB／ml、1．00×10^7PIB／ml、7.50×10^6PIB／ml和5.00×10^6PIB／m1）中混合2000IU／mg Bt制剂后共毒系数在106．02至128．03之间，表明Bt与EoNPV混用具有增效作用。其中以1．00×10^7PIB／ml EoNPV制剂与2000IU／mgBt制剂混用增效作用最明显，LC50为817．03μg/ml。对2龄茶尺蠖的杀虫速度，EoNPV＋Bt制剂（1．00×10^7 PIB／ml EoNPV＋2000IU／mg Bt）的LT50较单独使用EoNPV（2．00×10^7 PIB／ml）缩短了2d，且EoNPV＋Bt制剂对茶尺蠖具有拒食作用，茶尺蠖对EoNPV＋Bt制剂处理茶树的食叶量比单独使用EoNPV制剂的食叶量减少65．9％。EoNPV＋Bt制剂对茶刺蛾、茶银尺蠖等茶树鳞翅目害虫的兼治作用分别达85．8％和88．7％。多点田间药效试验结果表明，EoNPV＋Bt制剂药后10d对茶尺蠖防效达81．80％～93．24％。

灰茶尺蠖核型多角体病毒(EgNPV)安全性试验

用20亿PIB/mL灰茶尺蠖病毒(EgNPV)对成年SD大鼠进行急性经口、经皮毒性试验,对白色家兔进行眼刺激和皮肤刺激试验,对豚鼠进行皮肤致敏试验以及进行小鼠急性致病性试验。结果表明20亿PIB/mL灰茶尺蠖病毒(EgNPV)水剂对SD大鼠的急性经口毒性(其LD50雌雄大鼠均>5 000 mg/kg)属于低毒;对SD大鼠的急性经皮毒性(其LD50雌雄大鼠均>2 000 mg/kg)属于低毒;对家兔的皮肤无刺激性;对家兔的眼睛也无刺激性:在染毒后24 h内不洗眼情况下,眼刺激积分最高为0;对豚鼠的皮肤致敏率为0,其致敏性分级为Ⅰ级,属弱致敏物;病理组织学检查结果表明,各实验组小鼠内脏组织均未见有病理改变,无急性致病性。

SpltNPVp49基因的克隆和序列分析

根据新发现的杆状病毒凋亡抑制基因p4 9基因的序列设计了一对引物 ,以SpltNPV基因组DNA为模板 ,通过PCR扩增获得了预期大小的约 1 3kb的DNA片段 ,将此片段克隆到pGEM T载体上并直接测序 .序列分析表明 ,该片段为SpltNPV完整的p4 9基因开放读码框 .与已知的杆状病毒p4 9基因的序列同源性为 87% ,与之对应的氨基酸序列的同源性高达 93% .它是首次从SpltNPV中克隆到的抑制细胞凋亡的基因 .

WHS_3菌株产几丁质酶对棉铃虫HaSNPV的增效作用

WHS3 (Serratiamarcescens)菌是一株几丁质酶的高产菌株 ,在诱导培养基中几丁质酶的产量可达84 .4 μg/mL。通过对中国棉铃虫进行的生物测定表明 :WHS3 菌所产的几丁质酶 (A3 )能有效地提高HaSNPV的毒力 2 0 %～ 70 % ,LT50 、LT90 比对照组缩短天数最高可达 1.1d、1.3d。