Iteradensoviruses are 5-kb parvoviruses with typical J-shaped inverted terminal repeats of about 250 nucleotides and terminal hairpins of about 165 nucleotides. The single-stranded DNA genome contains several open reading frames, but their expression strategy is still unknown. Here the transcription maps and expression of the viruses in this genus were explored. As for brevidensoviruses, the two nonstructural (NS) genes were expressed by overlapping promoters with alternate transcription starts at both sides of the NS1 start codon.
Invertebrate densoviruses (DVs) form a separate subfamily (Densovirinae) within the Parvoviridae family (1-4) and have recently been reclassified (1). They share many physicochemical and genome properties with Parvovirinae of vertebrates (5-7), and most viruses of these subfamilies have a phospholipase A2 (PLA2) activity for cell entry (8-10). The left-hand side of the genome contains the nonstructural (NS) genes with the rolling-circle replication endonuclease and the superfamily 3 helicase in NS1 (11,12). Open-reading frames (ORFs) for structural proteins (VPs) are on the right-hand side of the genomes (5, 6).Densovirinae include the Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus, and Iteradensovirus genera, which have distinct genome sizes and organizations and the presence or absence of inverted terminal repeats (ITRs) and PLA2 (1, 2). Iteradensoviruses have a 5-kb genome with ITRs, a monosense genome organization, and PLA2 activity. Four previously known iteradensoviruses are those from Bombyx mori (BmDV) (13), Casphalia extranea (CeDV) (14), Dendrolimus punctatus (DpDV) (15), and Helicoverpa armigera (HaDV2) (16). The reported sequence of HaDV2 is incomplete, differs significantly from the sequences of the others, and remains to be confirmed. Additionally, we isolated iteradensoviruses from Papilio polyxenes (black swallowtail butterfly) (PpDV) (17), Sibine fusca (oil palm pest) (SfDV) (18), and the monarch butterfly (DpIDV) (19) (Fig. 1A). Based on NS1 identities, the taxons of these viruses have been defined as lepidopteran iteradensovirus (LI) 1 with BmDV, the LI 2 species with CeDV, SfDV, and DpIDV, the LI 3 species with DpDV, the LI 4 species with PpDV, and the LI 5 species with HaDV2 (1). NS1 and NS2 proteins of the Chinese isolates (LI 3 and LI 5) had Ͻ40% identity scores with other iteradensoviruses. In contrast, except for HaDV2, the VP proteins of iteradensoviruses all have similar identities of 70 to 80%, independent of the species.Infectious clones were created and sequenced for BmDV (pIN919), CeDV (pSMART-CeDV), PpDV (pCR2.1-PpDV), SfDV (pBlue-SfDV), and DpIDV (pBlue-DpIDV) (14,(17)(18)(19)(20). The completely sequenced genomes all have typical J-shaped ITRs with lengths ranging from 230 to 271 nucleotides (nt) and terminal hairpins of 159 to 175 nt (Fig. 1B and C). The 5=-and 3=-terminal hairpins occurred in two orientations, "flip" and its reverse-complement orientation, "flop" (Fig. 1C). These were identical among the three LI 2 viruses and h...