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Biological and Molecular Characterization of IrfaNPV and OrerNPV

Author: YangLiRong
Tutor: ZhangChuanXi
School: Zhejiang University
Course: Agricultural Entomology and Pest Control
Keywords: Iragoides ragoides NPV Orgyia ericae NPV Nucleopolyhedrovirus Electron microscopy Infection of Tn-5B-4 cells Polyhedrin gene AcORF150 AcORF38 Molecular phylogeny Droplet-feeding bioassay Virulence Fluorescence quantitative PCR.
CLC: S476.13
Type: PhD thesis
Year: 2006
Downloads: 52
Quote: 1
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Abstract


Mophological, molecular and biological characteristics of two baculoviruses isolated from Iragoides fasciata and Orgyia ericae were studied in this study. The morphs of the two nucleopolyhe- droviruses were observed by electron microscopy. Partial genomic sequences including the major structural polyhedrin genes of the two viruses were determined and their phylogenetic relationship to other baculoviruses was determined. Infection of the non-host cell line with Iragoides fasciata NPV (IrfaNPV) was investigated. Lethal dose (LD) response of different instar larvae of Orgyia ericae to Orgyia ericae NPV (OrerNPV) was also determined in this study. The main results were summarized as follows:1. Morphological characteristics and sequence analysis of genome fragments of IrfaNPVA nucleopolyhedrovirus (NPV) newly isolated from the diseased larva of the tea slug moth Iragoides ragoides was characterized. Electron microscopic observations showed that Iragoides fasciata NPV (IrfaNPV) was a single nucleocapsid type virus. An EcoR I/Xho I 11.6kb insertion clone containing the polyhedrin gene was screened out from a plasmid genomic library by PCR, using the primers designed based on the conserved sequence of the NPV polyhedrin. The sequencing result showed the insertion was 11.626 kb in length with a G+C content of 31.47% and contained 13 open reading frames.Using the amino acid sequences of 58 baculovirus polyhedrin genes deposited in the GenBank , we analysed the phylogenetic relationship of IrfaNPV polyhedrin, Ief2, lefll, odv-e56, ORF1629, pk-1 and ptp with other baculoviruses. The clones containing IrfaNPV AcORF47 homologue and vp80 gene were also sequenced. Sequence analysis showed If-Ac47 shared 42%, 42% and 40;% identities with AcMNPV ORF47, BmNPV ORF38 and RaouMNPV ORF44 in amino acid level, respectively. The 222 amino acid residues in the C-terminal of IrfaNPV VP80 had 25%~58% identities with homologous genes from other 17NPVs. A phylogenetic tree based on the VP80/P87 amino acid sequences was also constructed The phylogenetic analyses revealed that IrfaNPV was a member of the Groupl NPVs and IrfaNPV was most closely related to AcMNPV, BmNPV and RaouMNPV.2. Expression of If-Ac38 and If-AclSO in E.coli, preparation of the antisera and detection of Acl50 and Ac38 in AcMNPV infected cellsThe coding region of If-Ac38 and If-Acl50 were subcloned into the expression vectors, pGEX-4T-2 and pET-28-a, respectively, and then were highly expressed in the E. coli strain B121. The expressed proteins were used to raise the rabbits to produce the specific antibodies. The expression time couses of Ac38 and Ac 150 in AcMNPV-infected Tn-5Bl-4 cells were detected by Western blot analysis, using the polyclonal antibodies against If-Ac38 and If-Acl50. The results showed that Ac38 and Acl50 were expressed from 12 through 96 hours post infection (h p.i.), indicating that these two genes were both late genes in the virus infection circle.3. Infection of IrfaNPV to the non-host cell line Tn-5Bl-4The proliferation of IrfaNPV in the non-host cell line Tn-5Bl-4 was investigated, using a fluorescence Quantitative PCR assay. The results showed that IrfaNPV could infect the TN-5B1-4 cells and form BVs, which could normally release to the medium. But the IrfaNPV polyhedra could not be normally formed in the nucleus and the number of BVs in the medium was very low, only as 1/800 high as that of AcMNPV BVs.4. Morphological, phylogenetic, and biological characteristics of a nucleopolyhedrovirus isolated from Orgyia ericae GermarThe electron microscopic observation showed that each nucleocapsid rod of O. ericae NPV was singly encapsulated within the virus lipoprotein envelope. The O. ericae NPV isolate was therefore assigned to the SNPV. The polyhedra of O. ericae NPV were triangular, quadrangular and hexagonal in shape, and their size ranged from 1.2 to 2.5:um (average 1.96 urn) in diameter. The viron size was approximately 260nmX60nm.The restriction endonuclease digestion of OrerNPV using the EcoR I, Xho I. EcoR V, Hind III and PstI enzymes resulted 22, 30, 19, 23 and 26 fragments, repectively. The average sizeof the OrerNPV genome was calculated as 134.6 kb.A 2.6 kb insertion clone containing the polyhedrin gene was screened out among 40 clones by PCR. The sequencing result showed the insertion was 2655 bp in length, containing a 741-nt open reading frame that encodes a 247 aa polyhedrin protein. A conserved baculovirus late gene motif, ATAAG, associated with transcriptional start sites in other ph genes, was identified 48 nt upstream of the start codon ATG. The poly (A ) signal AATAAA was overlapping with the last A of the stop codon (TAA). In the flanking regions, a partial sequence (1540 bp) of ribonucleotide reductase (rrl) was identified 168 nts upstream of the start codon of ph gene.By using the amino acid sequences of 55 baculovirus polyhedrin genes deposited in the GenBank, the phylogenetic position of OrerNPV polyhedrin was analyzed. OrerNPV was positioned within a monophyletic clade together with OpSNV, BusuSNPV and EcobSNPV, and appeared closely related to OpSNPV. The result strongly suggested that OrerNPV were members of NPV Group II.The leaf disc assay showed that the LD5Os of OrerNPV to neonatal, second, third, and fourth instarO. ericae larvae were 10.08, 77.4, 167.21 and 698.45 OBs per larva, respectively. There was no significant difference in the slope of regression in different instars, indicating that the increase in mortality for a given increase in dose was similar in each instar.

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CLC: > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens > Insect viruses
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