Characterization of the genome structure of Bombyx mori densovirus (China isolate)

The genome of Bombyx mori densovirus (China isolate), termed as BmDNV-3, is composed of two kinds of different single-stranded linear DNA molecules (VD1 and VD2). In this study, the viral DNA molecules were purified and cloned into pUC119 vector, and the complete nucleotide sequence was determined. Sequence analysis showed that VD1 genome consisted of 6,543 nts including inverted terminal repeats (ITRs) of 224 nts, and VD2 genome consisted of 6,022 nts including ITRs of 524 nts. Comparison of the complete genome sequence between BmDNV-3 and BmDNV-2 (Yamanashi isolate) showed an identity of 98.4% in VD1 and 97.7% in VD2, with a total number of 228 bp substitutions, 11 bp deletions and 3 bp insertions found in BmDNV-3. A single nucleotide "A" deletion at nt 1589 in BmDNV-3 caused a frame shift mutation and brought about a premature stop codon, thus dividing VD2 of BmDNV-3 into two ORFs (named VD2 ORF1a and VD2 ORF1b) within that region, while there was only one ORF (named VD2 ORF1) in the corresponding region of BmDNV-2 (Yamanashi isolate). Comparative polymorphisms of ORFs and ITR regions of the two viral genomes showed that highly variable regions were mainly located in VD1 ORF3, VD1 ORF4, VD2 ORF2, and ITRs of BmDNV-3. Northern blots analysis revealed that VD1 had 1.1 kb and 1.5 kb transcripts from the left half of its plus strand, and one transcript about 3.3 kb from the right half of its minus strand. Sequencing of 3' and 5' RACE products showed that the 1.1 kb transcript started at nt 290 and ended at nt 1437, the 1.5 kb transcript started at nt 1423 and ended at nt 2931, and the 3.3 kb transcript started at nt 6287 and ended at nt 2922. These results help us to further understand the variation between different DNV genera and its possible causes, providing clues for studying the evolutionary history of densoviruses.