SeMNPV抑制HaSNPV诱导的Se—UCR细胞凋亡

在采用共感染和共转染的方法构建扩大杀虫范围的重组病毒的研究过程中发现棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)能诱导甜菜夜蛾细胞,Se-UCR发生典型凋亡,但不能诱导另一株甜菜夜蛾细胞Se-301产生凋亡。以5MOI的HaSNPV感染Se-UCR。在12h左右可以观测到少量细胞凋亡。24h能观察到明显的凋亡,凋亡细胞数量随时间不断增加,到72h基本上所有的细胞均发生凋亡,成为凋亡小体,基因组DNA片段化。同时发现HaSNPV诱导的甜菜夜蛾Se-UCR细胞凋亡能够被甜菜夜蛾多核衣壳核多角体病毒(Spodoptera exigua multicapsid nucleoplyhedrovirus,SeMNPV)所抑制,进一步点杂交试验发现SeMNPV和HaSNPV共同感染Se-UCR获得了HaSNPV在该细胞中的复制。     

AcMNPV P35抑制HaSNPV诱导的Tn-Hi5细胞凋亡

苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nuclear polyhedrosis virus,AcMNPV)能够抑制棉铃虫核多角体病毒(Helicoverpa armigera Nucleopoly-hedrovirus,HaSNPV)诱导的Tn-Hi5细胞凋亡,并能辅助HaSNPV在Tn-Hi5细胞中复制,产生具有感染能力的子代病毒.瞬时表达实验证明,在Tn-Hi5细胞中,p35具有明显抑制凋亡的能力,但是不能辅助HaSNPV在Tn-Hi5细胞中的复制;进一步构建超表达p35的重组病毒vHap35,发现vHap35能够抑制Tn-Hi5细胞凋亡,但是不能产生具有感染力的病毒粒子.电镜观察发现感染重组病毒的部分细胞中存在单粒包埋的病毒粒子(ODV).     

AcMNPVP35抑制HaSNPV诱导的Tn-Hi5细胞凋亡

苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nuclear polyhedrosis virus,AcMNPV)能够抑制棉铃虫核多角体病毒(Helicoverpa armigera Nucleopoly hedrovirus,HaSNPV)诱导的Tn Hi5 细胞凋亡,并能辅助HaSNPV在Tn Hi5细胞中复制,产生具有感染能力的子代病毒。瞬时表达实验证明,在Tn Hi5细胞中,p35具有明显抑制凋亡的能力,但是不能辅助HaSNPV在Tn Hi5细胞中的复制;进一步构建超表达p35 的重组病毒:vHap35,发现vHap35能够抑制Tn Hi5细胞凋亡,但是不能产生具有感染力的病毒粒子。电镜观察发现感染重组病毒的部分细胞中存在单粒包埋的病毒粒子(ODV)。     

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)     

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)     

棉铃虫单核衣壳核多角体病毒解螺旋酶基因的序列分析

对棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组中EcoRI-N片段进行序列分析,获得了完整的解螺旋酶基因(hel),其开放阅读框大小为3 762bp,编码一个分子量为146kD的蛋白质.在hel起始密码子ATG上游50位有强晚期启动子转录起始信号ATAAG,在-112位和-189位存在两个TATA box,但未发现早期转录信号CAGT.其在终止密码子下游第12位有一PolyA终止信号AATAAA.在其它真核或原核解螺旋酶中存在的7个保守基元(I、Ia、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ),只有5个(I、Ia、Ⅱ、Ⅲ、Ⅳ)在杆状病毒中保守.同源性比较发现,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒(Spodoptera exigue MNPV,SeMNPV)的解螺旋酶具有最高的同源性(66%),与Xestia c-nigrum颗粒体病毒(XcGV)解螺旋酶的同源性最低(43%).HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因.     

棉铃虫单核衣壳核多角体病毒解螺旋酶基因的序列分析

对棉铃虫单核衣壳核多角体病毒（Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV）基因组中EcoR I-N片段进行序列分析,获得了完整的解螺旋酶基因（hel）,其开放阅读框大小为3762bp,编码一个分子量为146kD的蛋白质。在hel起始密码子ATG上游50位有强晚期启动子转录起始信号ATAAG,在－112位和－189位存在两个TATA box,但未发现早期转录信号CAGT。其在终止密码子下游第12位有一PolyA终止信号AATAAA。在其它真核或原解螺旋酶中存在的7个保守基元（Ⅰ、Ⅰa、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ）,只有5个（Ⅰ、Ⅰa、Ⅱ、Ⅲ、Ⅳ）在杆状病毒中保守。同源性比较发现,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒（Spodoptera exigus MNPV,SeMNPV）的解螺旋酶具有最高的同源性（66％）,与Xestia c-nigrum颗粒体病毒（XcGV）解螺旋酶的同源性最低（43％）。HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因。     

感染HaSNPV棉铃虫幼虫的组织病理和免疫组化研究

本文报道了棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)感染棉铃虫幼虫的病理时相及HaSNPV多角体蛋白在幼虫组织中表达的免疫组化研究。以5×10~3PFU的HaSNPV出芽病毒粒子(BV)注射4龄初的棉铃虫幼虫,石蜡切片的H.E染色表明,在感染后24h,病理变化不明显;48h后脂肪体、气管组织的细胞核开始肿大,细胞开始变形;72h后脂肪体、气管、真皮细胞核肿大十分明显,组织结构松散;但中肠和肌肉组织未见明显病变;96h后,脂肪体、气管、真皮组织结构完全被破坏,肌肉组织变疏松。免疫组化结果表明,感染后24h,未能检测到多角体蛋白在幼虫组织中的表达;感染后48h,多角体蛋白可在部分脂肪体、气管组织、血细胞和真皮组织的细胞核中表达;72h后,被感染的脂肪体、气管组织、和真皮组织的细胞数比48h多;96h后,多角体蛋白可以在脂肪体、气管、真皮组织中大量表达,48h到96h间,被感染的血细胞数目基本不变,中肠组织和肌肉都未检测到多角体蛋白的表达;幼虫死亡后,可在中肠上皮细胞的基底膜间隙中检测到多角体蛋白的表达,肌肉组织中未见表达信号,其它组织全部被感染并且组织结构被破坏,H.E的结果与免疫组化的结果基本相符。     

感染HaSNPV棉铃虫幼虫的组织病理和免疫组化研究

本文报道了棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)感染棉铃虫幼虫的病理时相及HaSNPV多角体蛋白在幼虫组织中表达的免疫组化研究.以5×103PFU的HaSNPV出芽病毒粒子(BV)注射4龄初的棉铃虫幼虫,石蜡切片的H.E染色表明,在感染后24h,病理变化不明显;48h后脂肪体、气管组织的细胞核开始肿大,细胞开始变形;72h后脂肪体、气管、真皮细胞核肿大十分明显,组织结构松散;但中肠和肌肉组织未见明显病变;96h后,脂肪体、气管、真皮组织结构完全被破坏,肌肉组织变疏松.免疫组化结果表明,感染后24h,未能检测到多角体蛋白在幼虫组织中的表达;感染后48h,多角体蛋白可在部分脂肪体、气管组织、血细胞和真皮组织的细胞核中表达;72h后,被感染的脂肪体、气管组织、和真皮组织的细胞数比48h多; 96h后,多角体蛋白可以在脂肪体、气管、真皮组织中大量表达,48h到96h间,被感染的血细胞数目基本不变,中肠组织和肌肉都未检测到多角体蛋白的表达;幼虫死亡后,可在中肠上皮细胞的基底膜间隙中检测到多角体蛋白的表达,肌肉组织中未见表达信号,其它组织全部被感染并且组织结构被破坏, H.E的结果与免疫组化的结果基本相符.     

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2635bp,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（leucine zipper）。     

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.     

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.     

Nucleopolyhedrovirus: scanning electron microscopy technique

A simplified methodology was developed to study the geometric form of multiple Bombyx mori Nucleopolyhedrovirus by scanning electron microscopy. The virus belongs to Baculoviridae family and was isolated from the silkworm Bombyx mori (L.) (Lepidoptera: Bombycidae). The polyhedra of Nucleopolyhedrovirus were obtained from the filtrate, inoculum and hemolymph of the silkworm experimentally infected with nuclear polyhedra. This material was placed on stubs, where a copper tape was previously adhered. After dry at room temperature the virus was covered with carbon and gold. Scanning electron microscopy analysis revealed a well defined morphology for the polyhedra of multiple Bombyx mori Nucleopolyhedrovirus, making possible the mathematical study that identified it as a truncated octahedron. The form of the polyhedron can present taxonomic value, once it is specific for each viral lineage.     

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.     

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.     

Gender-Mediated Differences in Vertical Transmission of a Nucleopolyhedrovirus

With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H♂)×healthy females (H♀); ii) infected males (I♂)×healthy females (H♀); iii) healthy males (H♂)×infected females (I♀) and iv) infected males (I♂)×infected females (I♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.     

Genomic Sequencing and Analysis of Sucra jujuba Nucleopolyhedrovirus

The complete nucleotide sequence of Sucra jujuba nucleopolyhedrovirus (SujuNPV) was determined by 454 pyrosequencing. The SujuNPV genome was 135,952 bp in length with an A+T content of 61.34%. It contained 131 putative open reading frames (ORFs) covering 87.9% of the genome. Among these ORFs, 37 were conserved in all baculovirus genomes that have been completely sequenced, 24 were conserved in lepidopteran baculoviruses, 65 were found in other baculoviruses, and 5 were unique to the SujuNPV genome. Seven homologous regions (hrs) were identified in the SujuNPV genome. SujuNPV contained several genes that were duplicated or copied multiple times: two copies of helicase, DNA binding protein gene (dbp), p26 and cg30, three copies of the inhibitor of the apoptosis gene (iap), and four copies of the baculovirus repeated ORF (bro). Phylogenetic analysis suggested that SujuNPV belongs to a subclade of group II alphabaculovirus, which differs from other baculoviruses in that all nine members of this subclade contain a second copy of dbp.