| The Ectropis oblique nucleopolyhedrovirus (EcobNPV), Euproctis pseudoconspersa nucleopolyhedrovirus (EupsNPV) and Helicoverpa armigera nucleocapsid nucleopolyhedrovirus (HearNPV) are pathogenic to Ectropis oblique, Euproctis pseudoconspersa Strand and the bollworm Helicoverpa armigera, respectively. These baculoviruses have been extensively used to control the above pests in China. However, their speed of action is slow compared with chemical insecticides. Novel approaches are sought to improve the efficiency of these viruses by genetic engineering. Prior to the development of improved baculovirus insecticides by genetic engineering, further understanding of the structure of these virus genomes is required. In this study, genomes from EcobNPV, EupsNPV and HearNPV C1 strain were fully sequenced and analyzed. Main results in this dissertation were as follows:1. Morphological and biological characteristics of EcobNPVThe EcobNPV was propagated and purified from E. obliqua larvae. Electron microscopy observed that purified occlusion bodies (OBs) of EcobNPV were of irregular shape and ranged size from 0.7 to 1.7μm(1.15±0.27μm: mean±SD) in diameter. Multiple rod-shaped virions, measuring about 250 nm in length and 40 nm in width, were embedded in each OB with a single nucleocapsid packaged within the envelope of the virion. The EcobSNPV was evaluated for its infectivity in second instar larvae of E. obliqua. A bioassay was designed to determine both its lethal concentration and lethal time. The results showed that mortality of E. obliqua larvae increased and the lethal time was shortened with increasing concentration of EcobSNPV. When the second instar larvae were treated with higher concentrations of EcobSNPV, 20,000 and 2,000 PIBs/ml, all died at 10 d and 14 d post-inoculation, respectively. However, only 30.6% of the larvae died at 18 d post-inoculation when they were treated with a concentration of 0.2 PIBs/ml. The time-dose-mortality analysis showed that the value of LC50 was 104.09 PIBs/ml at 7 d post-inoculation whereas which was 100.8 PIBs/ml at 14 d post-inoculation (Table 1). The values of LT50 and LT90 were 8.5 d and 10.7 d respectively at the same concentration of 2,000 PIBs/ml.2. Genome sequence and organization of EcobNPVThe complete nucleotide sequence of the EcobNPV, was determined and analyzed. The double stranded circular genome is composed of 131,204 bp and is 37.6% G+C rich. The analysis predicted 126 putative, minimally overlapping open reading frames (ORFs) with 150 or more nucleotides that together compose 89.8% of the genome. The remaining 10.2% constitute non-coding and three homologous regions. Comparison with previously sequenced baculoviruses indicated that three ORFs were unique to EcobNPV, while the remaining 123 ORFs shared identity with other baculovirus genes. In addition to two bro homologues, three other repeat ORFs, including dbp, p26, and odv-e66, were identified. Phylogenetic analysis indicated that each member of the paired ORFs was acquired independently. Gene parity plot analysis and percent identity of gene homologues suggested that EcobNPV is a Group II NPV, although its genomic organization was highly distinct.3. Genome sequence and organization of HearNPV-C1The complete nucleotide sequence of HearSNPV-C1 was determined and analyzed by comparing with the genome of HearSNPV-G4 isolate. C1 and G4 isolates occurred in the same host species and geographic location but showed different virulence. The HearSNPV-Cl genome consisted of 130,759 bp and 137 putative open reading frames larger than 150 nucleotides were identified. The two genomes shared 98.1% nucleotide sequence identity, with a total number of 555 bp substitutions, 1354 bp deletions, and 710 bp insertions in HearSNPV-Cl. Comparison of ORFs and homologous repeat (hr) regions of the two genomes showed that there were four highly variable regions hr1, hr4, hr5, and bro-b, all in repeat regions. These results suggest that baculovirus strain heterogeneity may be often caused by single nucleotide polymorphisms (SNPs) and changes in the hrs and bro genes.4. Genome sequence and organization of EupsNPVThe entire EupsNPV genome sequence was determined and compared with other baculoviruses. The circular, double stranded DNA genome contained 140,620 bp and had a G+C content of 40.4%. Of 139 potential ORFs predicted from the sequence, 126 had a homology in other baculoviruses; thirteen were unique to EupsNPV. The coding (124,275 bp) and non-coding (14,664 bp) sequence composed 89.5% and 10.5% of the genome, respectively. Four hrs were present in the EupsNPV genome; the repeat sequences were different between these hrs. Three ORFs were identified to contain two homologues in the EupsNPV genome, including bro, p26 and dbp. Each member of the paired ORFs was acquired independently. Gene parity plot analysis and percent identity of gene homologues suggested that EupsNPV is most related to Group II NPV, although its genomic organization was highly distinct.5. Comparison of EupsNPV, EcobNPV and HearNPV-C1 genomes The genome structure and evolution of EupsNPV, EcobNPV and HearNPV-C1 were described. The genome size and G + C content were similar between these viruses. They shared 102 common ORFs, including the 30 baculovirus core genes. The mean amino acid identity of common ORFs between EupsNPV and EcobNPV was 46%, higher than that between EupsNPV and HearNPV-C1 (38%). All of these three genomes lacked a functional homologue of the major budded virus (BV) glycoprotein gene gp64 specific to Group I NPV. The number of unique ORFs between EupsNPV and EcobNPV was less than that between EupsNPV HearNPV-C1 or between EcobNPV and HearNPV-C1. The hr repeat sequences were totally different between these three genomes. Palindromes were found in EcobNPV rather than in EupsNPV and HearNPV-C1, the latter only contained repeat sequences. Gene content, percent identity of gene homologues, gene parity plots and phylogenetic analysis suggested that the common ancestor of EupsNPV and EcobNPV is much closer than that of EupsNPV and HearNPV-C1 during the evolution.
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