Study On Various Strains Of BmNPV And The Molecular Mechanism Of Host Range Determination

The family Baculoviridae is a highly selective virus in arthropods, especially in insects of the order Lepidoptera. Until now, genomes of58baculovimses have been sequenced, which have31common conserved genes. These baculoviruses can be classed into four genera, Alphabaculovirus, Betabaculovirus, Gammabaculovirus, Deltabaculovirus, based on linkage evolutionary analysis of31conserved genes. As development of new genome sequencing techniques are driving a boom in sequencing, more and more genomes of baculoviruses have been sequenced, and phylogenetic evolution history of baculoviruses becomes increasingly clear. However, only a few studies focus on various strains of the same species of baculovirus, except studies on Helcoverpa armigera nucleopolyhedrovirus. Taken the BmNPV as the research object, it is the first time to comparatively analyze six various strains from a host at genome level where the blank needs to be filled, and that will enrich our knowledge of evolution of the baculoviruses. Meanwhile, we firstly report a wild nucleopolyhedrovirus from Bombyx mandarina, which can successfully propagate in Bm cell lines, High Five and Sf cell lines. Study on this virus is of great important to host range researches of baculoviruses and application of baculovriuses as biocontrol pesticide. In addition, considering the great threat of BmNPV to silk industry, it is meaningful to study the interaction between BmNPV and its host. Although there were lots of researches on genes’differential transcription of silkworm infected with BmNPV, there is no reports on the whole genomic profile of gene transcription of silkworm infected with BmNPV. Using RNA-Seq technique, we report the transcriptome of Bm5cells infected with BmNPV, and obtain lots of trancription message, which will be definitely useful for studies on interaction between silkworm and BmNPV.The main results are as following:1. Comparatively study on various strains of BmNPVIt is the first time to comparatively analyze various strains from a host at genome level. We found differences in the number of bro gene and composition of homologous region (hr) are two main divergences within these genomes. The total number of palindromes of hrs seems to affect viral DNA replication in Bm5cells, but difference in number of palindromes in the same hr of different viruses seems to be not related to transcript level of its downstream genes. While we compared ORFs from different viruses, we noticed big differences are in the ORFs that are not core genes. This result is consistent with results from evolutionary pressure analysis of ORFs, indicating these ORFs should play important roles in evolution of BmNPV strains. Based on the cascaded DNA of134common ORFs, a phylogenetic tree of these six virus strains was constructed. India strain is closer to Cubic strain but far away from others. Guangxi strain and Zhejiang strain have a close relationship. Boma S1strain and Cubic strain are closely related, even though they are from somewhat different hosts. T3strain locates between Boma S1strain and Guangxi strain (or Zhejiang strain). Bioassays of BVs and OBs were also carried out. BVs of Boma S1strain and T3strain take longer time to kill silkworm larvae than BVs of other four viruses. OBs of India strain are significantly more competitive than OBs of other viruses, and OBs of Boma S1fall behind. There seems no obvious connection between biological characteristics and phylogenic relationship between different strains of BmNPV.2. Study on the molecular mechanism of host range determination of a wild nucleopolyhedrovirus from B. mandarinaThis virus was named "BomaNPV S2", which is isolated from B. mandarina. BomaNPV S2can successfully propagate in B. mori cell lines, Trichoplusia ni cell line (High Five) and Spodoptera frugiperda cell line (Sf9). OBs in nuclei of Bm5cells seem rough and contain less virions than that in High Five cells. Interestingly, BomaNPV S2is SNPV in Bm cells, but MNPV in High Five cells. BomaNPV S2generates much more BVs than BmNPV T3while it infects Bm5cells, indicating a complement to fewer virions in polydedra. Sequence analysis of BomaNPV S2genome has revealed that most sequences of this genome are more identical to DNAs from BmNPV (or BomaNPV S1) and some else are more identical to DNAs from AcMNPV. Compared with the genome of a hybridized virus that is isolated from cells cotransfected with BmNPV and AcMNPV, some genomic DNA regions of BomaNPV S2are thought to be associated with its host range determination. Recombinant viruses containing these sequences were constructed for analyzing their infectivity to Bm cells and High Five cells. Our result showed that gp64is the key gene for BomaNPV S2being able to infect both Bm cells and High Five cells.3. Transcriptome of Bm5infected with BmNPVThe transcriptome of Bm5cells infected with BmNPV was obtained via RNA-Seq. This is a big addition to data library of gene transcription or gene express profile of the silkworm. We found about ninety percent of annotated genes of the silkworm was transcripted in Bm5cells infected with BmNPV. And these transcribed genes distribute different within different chromosome. Classified by GO and KEGG, most genes are related to basic biological functions. However, only a small number of genes undergo alternative splicing, of which exon skipping and intron retention are the two main kinds of alternative splicing events. Noteworthily, some pathways like Ribosome Pathway and some function Brites like Chromosome Brite and Ubiquitin Brite have lots of genes experiencing alternative splicing. Surprisingly, Spliceosome Pathway also has a number of genes undergoing alternative splicing. These results demonstrate the importance and complication of alternative splicing.