ABSTRACT
Aim: The present study was undertaken to understand the phylogenetic relationships of NPVs (Nucleopolyhedroviruses) isolated from two pest species, Hyposidra talaca and Biston (=Buzura) suppressaria. The phylogenetic analyses based on the polyhedrin gene were assessed. Methodology: Occlusion bodies (OBs) were isolated separately from NPV infected dead Hyposidra talaca and Buzura suppressaria larvae, and DNA was isolated from OBs. The polyhedrin gene was amplified and sequenced followed by sequence divergence and phylogenetic analyses using MEGA5. Results: The phylogenetic analyses based on the polyhedrin gene revealed that the NPV isolated from Hyposidra talaca (HytaNPV-ITK1) formed a single cluster with the isolates of NPVs infecting Hyposidra specimens in India sharing 99% nucleotide identity, whereas the NPV isolated from Buzura suppressaria (BusuNPV-ITK1) showing 99% nucleotide homology with the NPV isolate of B. suppressaria reported from China formed a different cluster. A nucleotide identity of 85% was found between HytaNPV-ITK1 and BusuNPV-ITK1. Interpretation: Phylogenetic analyses, based on the polyhedrin sequence of 47 baculoviruses, revealed that these two variants of NPVs (HytaNPV-ITK1 and BusuNPV-ITK1) infecting Hyposidra talaca and Buzura suppressaria were comparatively closer to each other than those infecting specimens of other lepidopteran genera.
ABSTRACT
Chrysodeixis includens is an important pest of soybean crop who has gained more visibility in the Brazilian Cerrado due to damage caused in this region. Foliar consumption, feeding period and mortality level of soybean loopers in laboratory, as well as their control in the field conditions, were evaluated after application of the ChinNPV virus in soybean plants. In the laboratory, were tested six concentrations of isolate Chin-IA (I-A) (1 × 1011, 2 × 1011, 4 × 1011, 6 × 1011, 8 × 1011 and 10 × 1011 PIB ha-1), one dose of methomyl chemical insecticide (172 g ai ha-1) and distilled water (control). The field experiment was carried out in the 2016/2017 season using the same cultivar and laboratory treatments, except for the lowest virus concentration. The population density of small and large larvae was evaluated before and at 5, 8 and 12 days after application (DAA) of the treatments in soybean plants. All concentrations of the isolate Chin-IA (I-A) have reduced the soybean loopers consumption and their feeding period, showing 100% of mortality after 3 4 days without differing from treatment with the chemical insecticide. After eight DAA of virus in the field, the population density of small and large larvae was reduced, providing satisfactory levels of control. These results showed the evident potential of ChinNPV in the reduction of defoliation power and maintenance the soybean loopers population under of control level, and thus may be used as complementary method in the integrated management of this pest in soybean crops.(AU)
Chrysodeixis includens é uma importante praga da cultura da soja que tem ganhado maior visibilidade na região do Cerrado brasileiro em razão do dano causado. Consumo foliar, período de alimentação e mortalidade de lagartas falsa-medideira foram avaliados em laboratório, assim como seu controle em condições de campo, após a aplicação do vírus ChinNPV em plantas de soja. Em laboratório, foram testadas seis concentrações do isolado Chin-IA (I-A) (1 × 1011, 2 × 1011, 4 × 1011, 6 × 1011, 8 × 1011 e 10 × 1011 CIP ha-1), uma dose do inseticida químico metomil (172 g i.a ha-1) e água destilada (controle). O ensaio de campo foi realizado na safra 2016/2017 utilizando o mesmo cultivar e tratamentos do ensaio em laboratório, exceto para a menor concentração do vírus. A densidade populacional de lagartas pequenas e grandes foi avaliada antes (pré-contagem) e 5, 8 e 12 dias após a aplicação dos tratamentos nas plantas de soja. Todas as concentrações do isolado Chin-IA (I-A) reduziram o consumo e o período de alimentação das lagartas falsa-medideira, mostrando 100% de mortalidade entre 3 4 dias, sem diferir do tratamento com o inseticida químico. Depois de oito dias após a aplicação do vírus no campo, a densidade populacional de lagartas pequenas e grandes foi reduzida, promovendo um controle satisfatório. Esses resultados mostram o evidente potencial de ChinNPV na redução do poder de desfolha, mantendo a população das lagartas falsa-medideira abaixo do nível de controle, e indicam o seu uso como um método complementar no manejo integrado dessa praga em culturas de soja.(AU)
Subject(s)
Nucleopolyhedroviruses , Lepidoptera , Glycine max , Pest Control, BiologicalABSTRACT
ABSTRACT Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.
ABSTRACT
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 232Tbr 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.
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In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), belonging to group Ⅱ nucleopolyhedrovirus, was studied in mammalian cells.We amplified the SeMNPV ie1 gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn't influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.
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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.
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Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.
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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 vfgftranscripts were detected from 18 to 96 h post-infection (hpi) of Hz-AMI 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-AM 1, 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.
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Antheraea pernyi nucleopolyhedrovirus (ApNPV) PstI-B and C fragments were cloned and sequenced. ApNPV PstI-B was 7406 bp long, contained seven open reading frames (orfs)/genes, including p87, he65, pnk/pnl, odv-ec43 and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) orf107,orf108 homologue on either strands of genomic DNA. ApNPV PstI-C was 6663 bp long, contained eleven orfs/genes, including pk-1, orf1629, polh, lef-2, ptp-2, ctl-1, ptp-1 and OpMNPV orf5, orf7, orf8, orf1 1 homologue on either strands of genomic DNA. Among the eighteen baculovirus genes identification, he65 and orf1629 were two diverse genes, while polh and lef-2 were two conserved genes. ApNPV was the third baculovirus found to contain pnk/pnl gene, the fourth baculovirus found to contain both ptp-1 and ptp-2 gene.
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There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.
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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.
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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.
ABSTRACT
Antheraea pernyi nucleopolyhedrovirus (ApNPV) PstI-B and C fragments were cloned and sequenced. ApNPV PstI-B was 7406 bp long, contained seven open reading frames (orfs)/genes, including p87, he65, pnk/pnl, odv-ec43 and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) orf107,orf108 homologue on either strands of genomic DNA. ApNPV PstI-C was 6663 bp long, contained eleven orfs/genes, including pk-1, orf1629, polh, lef-2, ptp-2, ctl-1, ptp-1 and OpMNPV orf5, orf7, orf8, orf1 1 homologue on either strands of genomic DNA. Among the eighteen baculovirus genes identification, he65 and orf1629 were two diverse genes, while polh and lef-2 were two conserved genes. ApNPV was the third baculovirus found to contain pnk/pnl gene, the fourth baculovirus found to contain both ptp-1 and ptp-2 gene.
ABSTRACT
Antheraea pernyi nucleopolyhedrovirus (ApNPV) PstI-B and C fragments were cloned and sequenced. ApNPV PstI-B was 7406 bp long, contained seven open reading frames (orfs)/genes, including p87, he65, pnk/pnl, odv-ec43 and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) orf107,orf108 homologue on either strands of genomic DNA. ApNPV PstI-C was 6663 bp long, contained eleven orfs/genes, including pk-1, orf1629, polh, lef-2, ptp-2, ctl-1, ptp-1 and OpMNPV orf5, orf7, orf8, orf1 1 homologue on either strands of genomic DNA. Among the eighteen baculovirus genes identification, he65 and orf1629 were two diverse genes, while polh and lef-2 were two conserved genes. ApNPV was the third baculovirus found to contain pnk/pnl gene, the fourth baculovirus found to contain both ptp-1 and ptp-2 gene.
ABSTRACT
To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.
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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.
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Os efeitos da estrutura etária e da densidade do hospedeiro na transmissão horizontal de um baculovírus foram investigados em laboratório, utilizando-se populações de lagarta do repolho, Mamestra brassicae L. (Lepidoptera: Noctuidae). Os insetos foram mantidos a três combinações de ínstares e três densidades populacionais em recipientes fechados contendo larvas infectadas. As larvas foram observadas diariamente e o número de mortes e o tempo letal foram registrados. Os níveis de mortalidade viral foram marginalmente superiores em recipientes contendo hospedeiros sob maior densidade populacional. As larvas apresentaram um risco de infecção aparentemente maior quando combinações de instares mais avançados foram usadas. Os níveis de mortalidade de larvas mais velhas foram maiores que as de larvas mais jovens. As médias de tempo letal das populaç ões larvais foram maiores para larvas mais jovens, observando-se um declínio mais acentuado na curva de sobrevivência das larvas em estágio mais adiantado de desenvolvimento.
The effects of stage structure and host density on baculovirus horizontal transmission were examined in the laboratory using larvae of the cabbage moth, Mamestra brassicae L. (Lepidoptera: Noctuidae). Insects were reared at three instar combinations and three host densities in closed containers with infected larvae. Insects were observed daily and the number of deaths and time to death were recorded. Levels of virus mortality were marginally higher in the containers where a higher density of hosts was introduced. Larvae appeared to have a greater risk of infection when late instar combinations were used. Final levels of mortality of older larvae were significantly higher than those of younger larvae. Mean times to death of larval populations were longer for larvae at earlier instar combinations, with a faster decrease in survivorship of older larvae over time.
ABSTRACT
Objective To understand the functions of ORF27 protein of the Thai isolated Helicoverpa armigera nucleopolyhedrovirus(HaNPV-Th) and explain the possible molecular mechanism of HaNPV-Th in passage effect.Methods ORF27 gene was cloned and amplified by polymerase chain reaction(PCR) from HaNPV-Th genome.The physical-chemical properties,subcellular localization,signal peptide,transmembrane structure and functional motifs were analyzed by the software of ProtParam,TMpred,SignalP and ScanProsite.The truncated ORF27 gene which lacked parts of the amino-terminal was expressed by bacterial expression system.Results The full length sequence of ORF27 was 768bp long and encoded 255aa with a molecular weight of 29.5ku.Several functional motifs on ORF27 were predicted by bioinformatics software.The truncated ORF27 recombinant expressed 35ku target protein mainly in the form of inclusion body.Conclusion ORF27 protein of HaNPV-Th is presumed to possess extensive biological activities.It may regulate various cellular signaling pathways,apoptosis and cell cycle,which are involved in passage effect.