| The Baculoviridae form a large family of rod-shaped, invertebrate infecting viruses with a large double-stranded, covalently closed circular DNA genome. The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. In this study, two complete genomes of Pseudaletia separate Granulovirus (PsGV) and Spodoptera litura Nucleopolyhedrovirus II (SpltNPV II) were sequenced and analyzed.1. Morphological characteristics of PsGV and SpltNPV IINucleopolyhedrovirus (NPV) and Granulovirus (GV), two genera in the family Baculoviridae, are distinguished by their occlusion body (OB) morphology where NPVs occlude many virions within large, polyhedral shaped OBs or polyhedra, and GVs occlude a single virion in a granular OB. Scanning electron microscope (EM) observed that purified occlusion bodies (OBs) of PsGV were of regular shape and size. Its length is 0.5μM and its diameter is 0.3μM. Transmission electron microscope (EM) observed that a single rod-shaped virion, measuring about 310nm in length and 40nm in width, were embedded in each OB with a single nucleocapsid pachaged within the envelope of the virion.Scanning electron microscope (EM) observed that purified occlusion bodies (OBs) of SpltNPV II were of irregular shape and size. Its diameter is about 1.3μM. Transmission electron microscope (EM) observed that multiple rod-shaped virions, measuring about 260nm in length and 50nm in width, were embedded in each OB with multiple nucleocapsid pachaged within the envelope of the virion.2. Genome sequence and organization of PsGVThe genome of Pseudaletia separate granulovirus (PsGV) was completely sequenced. The size of the PsGV genome is 176,677 bases with a G+C content of 39.79%. It contained 183 open reading frames (ORFs) with 150 or more nucleotides that showed minimal overlap, making it the second largest baculovirus genome analyzed to date. Comparison with previodsly sequenced baculovirus including AcMNPV, XecnGV and HearGV indicated that the genomes of PsGV with XecnGV and HearGV exhibited extensive sequence similarity and co-linearity, with three genomes containing the same nine homologous regions (hrs) with conserved structure and locations. 69 of putative ORFs, which collective accounted for 37.7% of genome, are homologs of genes previously identified in AcMNPV. In addition, 166 and 169 of putative ORFs, which collective accounted for 90.7% and 92.3% of genome, are homologs of genes previously identified in XecnGV and HearGV genomes, respectively. These homologs showed on average 79% amino acid sequence identity to those from XecnGV and HearGV and larger than that from AcMNPV (34%). In addition to 7 ORFs which were not found in other baculovirus genomes, 12 bro, 2 helicase and 3 enhancin genes were identified. Especially, two repeated genes (ORF39 and 49) which were reverse and complementary each other were confirmed.12 proteins which constituted in PsGV (OBs) virions have been confirmed including six ODVs own proteins and one ODVs and BVs common protein. The ORF174 is a new viral occlusion body structural protein which was confirmed by mass spectrum.3. Genome sequence and organization of SpltNPV IIThe nucleotide sequence of the DNA genome of Spodoptera litura Nucleopolyhedrovirus II (SpltNPV II) was determined and analysed. The genome is composed of 148,634 bp and has a G+C content of 45%. It contains 149 ORFs of 150 nucleotides or more that show minimal overlap. 141 (94.6%) of these putative genes are homologous to genes previously identified in other baculoviruses. Among them, 97 are homologous to genes of AcMNPV, whereas 96 and 133 are homologous to genes of SpltNPV I and SeMNPV, respectively. These homologues show on average 41.4% overall amino acid sequence identity to those from AcMNPV and 44.6% and 76.5% to those from SpltNPV I and SeMNPV. 8 ORFs which were not found in other baculovirus genomes and 7 hrs were identified. In addition to two bro genes, there were two other repeat ORFs, including p26 and odv-e56 in SpltNPV II genome. Phylogenetic analysis indicated that each member of the paired ORFs was acquired independently. SpltNPV II and SpltNPV I were isolated from same host insects. But comparison of them showed there were noticeable differences in genome size, number of ORFs, G+C content, gene content, gene order and amino acid sequence identity between them. Combining with the former insecticidal efficiency and restriction endonuclease pattern of genome DNA (personal communication with Prof. Zhu of Suzhou University), SpltNPV II is a new baculovirus NPV different from SpltNPV I.4. Phylogenetic analysis of PsGV and SpltNPV II based on 29 core genesUp to date, 45 baculovirus genomes have been sequenced. They share 29 genes, which are considered as the baculovirus core set of genes. These genes generally encode basic functions. A phylogenetic tree based on the concatenated amino acid sequences of all core genes is shown in this study. This tree clearly allows the new taxonomic division which follows the evolution of the host more closely and divides the family baculovirus into the following genera: lepidopteran NPVs; lepidopteran GVs; hymenopteran NPVs and dipteran NPVs. In addition the lepidopteran NPVs split into two groups utilizing different fusion proteins. This tree suggested that PsGV belongs to lepidopteran GVs and is most closely related to XecnGV and HearGV. This accorded with the result of comparision of nucleotide and amino acid sequences in Chapter 4 of this paper. Moreover SpltNPV II blongs to lepidopteran group II NPVs and is most closely related to SeMNPV in this tree. Although both SpltNPV II and SpltNPV I blong to lepidopteran group II NPVs, their phylogenetic relationship is correspondingly far. This result suggested that SpltNPV II is a new baculovirus NPVs different from SpltNPV I again.
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