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Genomic Analysis of a Newly Identified Multinucleocapsid Nucleopolyhedrovirus Isolated from Helicoverpa armigera

Author: TangPing
Tutor: ZhangZhiFang
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: baculovirus Helicoverpa armigera infection genomic library genomic Analysis
CLC: S482.3
Type: PhD thesis
Year: 2011
Downloads: 27
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Abstract


The baculoviruses is a family of invertebrate viruses with large, circular, and double-stranded DNAgenomes. They are pathogenic to arthropods, mainly insects of the orders Lepidoptera, Hymenoptera,and Diptera. The baculoviruses is the most commonly investigated with regard to its development as amicrobial insecticide due to its favorable characteristics such as safety to the environment, humans, othervertebrates, plants, and natural enemies of pests. Consequently, the baculoviruses are ideal controlagents to be used in integrated pest management (IPM) programs in agriculture, forests.In this study, anew nucleopolyhedrovirus isolated from H.armigera was observed by electron microscope (EM),suggesting it was multinucleocapsid NPV.Experimental infection of insect larvae indicated that hostrange of HearMNPV was different from that of HearSNPV and that the cytopathological effect ofHearMNPV differed from that of HearSNPV. This report describes the sequence and organization of theHearMNPV genome and compares it with sequence data from other baculoviruses, such as HearSNPVand MacoNPV-B.The purified occlusion bodies (OBs) of NPV origin from the infected cotton bollworm have irregularshape by scanning electron microscope. Multiple rod-shaped virions were embedded in each OB withmultiple nucleocapsid packaged within the envelope of the virion by transmission electron microscope.These results indicated that it belong to typical multiple NPV.Therefore the isolate is denominated asHelicoverpa armigera multinucleocapsid nucleopolyhedrovirus(HearMNPV).The quantity of HearMNPV viral DNA, relative to host actin gene copy number, in infected larvae,included decreasing phase, latent phase, exponential phase and stationary phase, the results suggestedthat the cotton bollworm was infected by HearMNPV well. Experimental infection of insect larvaeshowed that HearMNPV can infect the eastern armyworm (Pseudaletia separate) but cannot infect eitherthe cotton leaf worm (Spodoptera litura) or the beet army worm (Spodoptera exigua). In contrast,HearSNPV cannot infect Pseudaletia separate. These results indicated that the host range of HearMNPVdiffered from that of HearSNPV. Moreover, HearMNPV-infected Hz-AM1cells produced no polyhedraand showed no typical cytopathic effects (CPE). However, HearMNPV-infected QB-Ha-E-5cellsproduced polyhedra. It was previously reported that Hz-AM1cells were permissive to HearSNPV, theQB-Ha-E-5cells were infected by HaBacmid-egfp which were constructed from HearSNPV, greenfluorescence were observed by fluorescence microscopy. These results indicated that the host range andcells infected with HearMNPV differed from those infected with HearSNPV.To better understand HearMNPV, the genomic DNA of HearMNPV was partially digested bySau3AⅠ, the plasmid vector pUC19was fully digested by SalⅠ, and subsequently filled in by theKlenow fragment respectively. After ligation and transformation, a genomic library of HearMNPV withthe representation of more than99.9%was constructed by “partial filling-in” method.The HearMNPV genome consists of154,196base pairs, with a G+C content of40.07%. A total of162putative ORFs were detected in the HearMNPV genome which represented90.16%of the genome.The remaining9.84%constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of MacoNPV-B,but distinguished from HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-Bsuggested that HearMNPV has a deletion of a5.4-kb fragment which contained5ORFs. In addition,HearMNPV orf66、 bros、 hrs were different with the corresponding parts in MacoNPV-Bgenome.HearMNPV can replicate in vivo and in vitro of Helicoverpa armigera, which is a new isolateNPV distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but with adistinct genomic structure, content, and organization.

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