Identification Of A New Bombyx Mori Nucleopolyhedrovirus And Analysis Of Its Bro Gene Family

Bombyx mori nucleopolyhedrovirus (BmNPV) is a member of the baculoviridae, adiverse family of viruses with circular double-stranded (ds) DNA genomes, which arepathogenic for invertebrates, particularly insects of the order Lepidoptera. Its main host isBombyx mori. Infection of BmNPV has seriously affected the quality and quantity ofcocoon and is very harmful for sericulture. Baculovirus repeated ORFs (bro) gene family isa polygene family in baculovirus of insect. They have different copies in different isolates,and have obvious differences at nucleic acid level. In the past years, the bro genes ofBmNPV were widely studied, for instant, several studies have suggested that bro geneshave function in the process of viral replication in the host. According to the sequencecomparison between BmNPV T3isolate and SC7isolate, we found there were somedivergences in genome sequences of BmNPV isolated from different areas, and thesedifferences mainly located in regions of bro gene family. There are five bro gene copieswhich are named as bro-a, b, c, d, e in BmNPV T3genome isolated from Japan, and theydistribute in three regions of genome. Previous study indicated that BmNPV CQI isolate(isolated from Chongqing, China) lack bro-b, while GD isolate (isolated from Guangdong,China) lack bro-e. Based on Thailand isolate, our studies did some analysis, includingidentification and analysis of BmNPV Thailand isolate, bro gene sequencing andphylogenetic analysis to discuss their function and evolution.1. Purification and identification analysis of BmNPV Thailand strainRecently, we have isolated a new BmNPV strain from the sericulture area ofMahasarakham Province, Thailand and named as BmNPV-Thai. In this study, we clonedand sequenced their conservative genes (p10, p35, polh, egt, vp39) and further comparedtheir sequences to those of BmNPV-T3, confirming that BmNPV-Thai is indeed a newBmNPV strain.2. Sequence analysis of bro genes of BmNPV Thailand strainUsing the genomic DNA of the Thailand strain as template, the fragments of bro-a,bro-c and bro-d were amplified. We analyzed the distribution of bro genes in each genome,bro genes mainly distributed in three regions of the genome. Taking the bro distribution ofT3isolate as standard, the second region of CQI isolate lack bro-b, the third region of GDisolate lack bro-e, and Thailand isolate lack bro-b, bro-e simultaneous, bro-d existed inevery isolate. The obtained sequences were aligned to the related sequences in GenBank. The result shows that insert and deletion have happened in bro genes of Thailand strain,and the changes of amino acid mainly occur at the N terminal of corresponding protein.Our analysis found that the three BRO proteins of BmNPV-Thai coincide with model ofthe conservative sequence reported by Zemskov at their N-terminals, suggesting that BROproteins may interact with nucleic acids. In addition, analysis using ANTHEPRO5.0software also found that all BRO proteins of BmNPV-Thai do not contain transmembraneregion and signal peptide.3. Phylogenetic analysis of bro genes of BmNPV Thailand strainUsing Neighbor-Joining method of Mega4.0software package, we analyzed thephylogenetic tree of BmNPV-Thai’s bro genes, the three bro genes of BmNPV-Thai arepresent in two subgroups. Similar to bro-d of BmNPV-T3, GD and CQ1and Bro-III ofBmNPV SC7, the bro-d gene of BmNPV-Thai is classified into subgroup A.Correspondingly, the bro-a and bro-c genes of BmNPV-Thai are classified into subgroup Btogether with the bro-a and bro-c genes of BmNPV-T3, GD and CQ1and bro-II gene ofBmNPV SC7. However, no bro gene of BmNPV-Thai is classified in subgroup C. Ingeneral, bro-a and bro-c in subgroup B have closer evolutionary relationship with bro-d ofsubgroup A than bro-b and bro-e of subgroup C. The evolutionary relationship of bro genesshows vague relevance to their location. The distribution characteristics of bro genes indifferent isolates of BmNPV is coincidence with the KANG’s theory that the presence ofbro-d and functional complementation between bro-a and bro-c are essential for thesurvival of BmNPV. The most simple form of bro genes in BmNPV is not confirmed andneeds to be further explored.Our study identified a new BmNPV strains according to the sequence analysis ofconservative genes, and analysis the phylogenetic of bro genes of this strain.Phylogenetic analysis of different isolates of BmNPV, have made contribution tofurther research of the mechanism of BmNPV infection.