Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide

The Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egt locus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigera larvae. The LT(50) of the egt deletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT(50) was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egt locus, giving a reduction in LT(50) of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.     

Helicoverpa armigera nucleopolyhedrovirus ORF80 encodes a late, nonstructural protein

The Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ORF80 (ha80) has 765 bp encoding a protein with approximately 254 amino acids and a predicted molecular weight of 30.8 kDa. Homologues of ha80 are found in most baculovirus sequences, including those from lepidopteran NPVs, lepidopteran granuloviruses (GVs), hymenopteran baculoviruses, and one dipteran baculovirus, yet their functions remain unclear. In this study we characterized ha80, and showed that it was transcribed late in infected host cells (HzAM1). The product of ha80 was a 31 kDa protein that was not a structural protein of budded virus (BV) or occlusion-derived virus (ODV) particles. Ha80 was first detected in the cytoplasm of infected HzAM1 cells at 12 h p.i., and was observed in the nucleus at later stages of infection, suggesting that it may be involved in transporting viral proteins into the host cell nucleus or play its roles in the nucleus.     

Horizontal and vertical transmission of wild-type and recombinant Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus

Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts. Here, we report on the horizontal and vertical transmission dynamics of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV-WT) and a genetically modified variant (HaSNPV-AaIT) with enhanced speed of action through the expression of an insect-selective scorpion toxin (AaIT). In caged field plots, horizontal transmission of both HaSNPV variants was greatest when inoculated 3rd instar larvae were used as infectors, transmission was intermediate with 2nd instar infectors and lowest with 1st instar infectors. Transmission was greater at a higher density of infectors (1 per plant) than at a lower density (1 per 4 plants); however, the transmission coefficient (number of new infections per initial infector) was lower at the higher density of infectors than at the lower density. HaSNPV-AaIT exhibited a significantly lower rate of transmission than HaSNPV-WT in the field cages. This was also the case in open field experiments. In the laboratory, the vertical transmission of HaSNPV-AaIT from infected females to offspring of 16.7+/-2.1% was significantly lower than that of HaSNPV-WT (30.9+/-2.9%). Likewise, in the field, vertical transmission of HaSNPV-AaIT (8.4+/-1.1%) was significantly lower than that of HaSNPV-WT (12.6+/-2.0%). The results indicate that the recombinant virus will be transmitted at lower rates in H. armigera populations than the wild-type virus. This may potentially affect negatively its long-term efficacy as compared to wild-type virus, but contributing positively to its biosafety.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

棉铃虫单核衣壳核多角体病毒几丁质酶基因的克隆与表达

根据棉铃虫单核衣壳核多角体病毒(HaSNPV)几丁质酶基因的序列,设计引物,引入适当的酶切位点,利用PCR扩增出不含N末端信号肽以及C末端内质网定位肽的几丁质酶基因片段.将该基因片断克隆至原核表达载体pProEXHTb,经IPTG诱导,在大肠杆菌DH5α中获得了高效表达,表达产物的大小为60kD,含量占菌体总蛋白量的40%.利用来源于AcMNPV 几丁质酶的抗体对表达蛋白进行检测,获得特异性的显色信号,证实所获原核表达产物与杆状病毒的几丁质酶具有同源性.     

棉铃虫单核衣壳核多角体病毒几丁质酶基因的克隆与表达

根据棉铃虫单核衣壳核多角体病毒 (HaSNPV)几丁质酶基因的序列 ,设计引物 ,引入适当的酶切位点 ,利用PCR扩增出不含N末端信号肽以及C末端内质网定位肽的几丁质酶基因片段。将该基因片断克隆至原核表达载体 pProEXHTb ,经IPTG诱导 ,在大肠杆菌DH5α中获得了高效表达 ,表达产物的大小为 6 0kD ,含量占菌体总蛋白量的 4 0 %。利用来源于AcMNPV几丁质酶的抗体对表达蛋白进行检测 ,获得特异性的显色信号 ,证实所获原核表达产物与杆状病毒的几丁质酶具有同源性     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini Freplicon、一个卡那霉素抗性基因和一个lacZα基因 8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内 ,构建了既能在E .coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid HZ8)。另外将HaSNPV的多角体蛋白基因和P10启动子序列取代 pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列 ,构建插入HaSNPV多角体蛋白基因和P10启动子序列的HapFastBacPhP10供体质粒。利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上 ,随后将含有eGFP基因的重组HaBacmidDNA转染至HZAm1细胞内。转染 5d后 ,细胞核内能形成典型的多角体 ,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光。结果证明我们构建的HaBactoBcac表达系统能有效的表达外源基因     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini-F replicon、一个卡那霉素抗性基因和一个lacZα基因8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内,构建了既能在E.coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid-HZ8).另外将HaSNPV的多角体蛋白基因和P10启动子序列取代pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列,构建插入HaSNPV多角体蛋白基因和 P10启动子序列的HapFastBacPhP10供体质粒.利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上,随后将含有eGFP基因的重组HaBacmid DNA转染至HZAm1细胞内.转染5d后,细胞核内能形成典型的多角体,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光.结果证明我们构建的HaBac to Bcac 表达系统能有效的表达外源基因.     

棉铃虫多核型多角体病毒v-cath同源基因的克隆及序列分析

为获得棉铃虫多核衣壳型多角体病毒(Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus)基因组序列,采用随机克隆方法,建立HearMNPV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,获得编码组织蛋白酶基因v-cath。该基因阅读框为1026bp,共编码341个氨基酸。核苷酸和氨基酸同源性比较结果表明:HearMNPV的v-cath基因与蓓带夜蛾核型多角体病毒B(Mamestra configurata NPV-B)的同源性最高,而与苹果皮小卷蛾颗粒体病毒(Cydiapomonella GV CpGV)同源性最低,由此认为,杆状病毒科的v-cath基因在进化上存在2种进化方式:一类以点突变为主,基因长度变化不明显;另一类突变以小片段的碱基增减为特征。     

Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures

The serial passaging of baculoviruses in cell lines numerous times can result in a variety of mutations or defective viral populations becoming predominant in the cultures. The generation of these mutants during cell culture passage, also known as "the passage effect," can seriously hinder the use of in vitro methods for large-scale production of baculoviruses for use as biopesticides. In an effort to develop a large-scale in vitro method of producing Helicoverpa armigera singly enveloped nucleopolyhedrovirus (HaSNPV), it was essential to determine whether or not the passage effect was evident when this virus is serially passaged in cell cultures. An isolate of HaSNPV was serially passaged in Helicoverpa zea cell cultures up to 10 times. The production of occlusion bodies decreased with increasing passage number and there was evidence of defective viruses becoming predominant in cultures after 5 passages. The number of virions present within cross sections of passage 3 occlusion bodies was 1.5 times higher than those from passage 10 occlusion bodies when quantified using electron microscopy. A laboratory bioassay showed that potencies of passage 3 isolates against H. armigera larvae were 8 times higher than potencies of passage 10 isolates. This study indicated that changes typical of the passage effect were evident when HaSNPV was serially passaged in H. zea cell cultures up to 10 times.     

Genomic sequencing and analyses of HearMNPV-a new Multinucleocapsid nucleopolyhedrovirus isolated from Helicoverpa armigera

ABSTRACT: BACKGROUND: HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed. METHODS: The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the "partial filling-in" method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses. RESULTS: Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome. CONCLUSIONS: HearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.     

In vivo enhancement of nucleopolyhedrovirus of oriental Armyworm, Mythimna separata using spindles from Helicoverpa armigera entomopoxvirus

When the third instar larvae of M. separata were exposed to eight varying concentrations of polyhedral occlusion bodies (POB's) of nucleopolyhedrovirus of M. separata (MsNPV) ranging from 2.6 x 10(-1) to 2.6 x 10(8) POB's/ml, the percent mortality and incubation period ranged from 16-100% and 14 to 9 days respectively. On the other hand when the same third instar larvae of M. separata were exposed to only five varying concentration of POB's of MsNPV ranging from 2.6 x 10(2) to 2.6 x 10(6), POB's/ml along with a constant dose of entomopox viral spindles from Helicoverpa armigera, the per cent mortality ranged from 63 to 100% with reduction in incubation period from 7 to 4 days respectively. The enhancement index (log10) of the virus was 2.76 or reduction of more than 500 times in LC50. The ability and the mechanism of the spindles from H. armigera entomopoxvirus to enhance the infectivity of MsNPV has been discussed.     

Construction and characterization of a Helicoverpa armigera nucleopolyhedrovirus bacterial artificial chromosome with deletion of ecdysteroid UDP-glucosyltransferase gene

Genetically modified baculoviruses offer a promising alternative to chemical insecticides in the control of agricultural and forest insect pests. A novel bacmid, HaBacYH6, was constructed in which the ecdysteroid UDP-glucosyltransferase gene (egt) was replaced with a bacterial replication cassette containing a mini-F replicon, a kanamycin resistance gene, and the attTn7 site. Insertion of the enhanced green fluorescence protein gene (egfp) into HaBacYH6 showed that the bacmid can express an active exogenous protein. Bioassays showed that median lethal time (LT50) of HaBacYH6 was 89.23 h in third instar Helicoverpa armigera larvae, 15.81 h earlier than that of wild-type HearNPV-G4, though there was no significant difference in median lethal dose (LD50). The data indicate that HaBacYH6 can be used as a new Bac-to-Bac system, and can also provide a technology platform for generating more effective biological insecticides.     

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology,structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.