We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus (PstDNV).

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Insect parvoviruses (densoviruses) belong to the Densovirinae subfamily of the Parvoviridae and are small, isometric, nonenveloped viruses (diameter, ϳ25 nm) that contain a linear single-stranded DNA of 4 to 6 kb (2, 3, 27). These viruses can be subdivided into two large groups, those with ambisense genomes and those with monosense genomes. Like vertebrate parvoviruses, all densoviruses have a genomic DNA with hairpins at both ends, often (but not necessarily for all genera) as inverted terminal repeats (ITRs). All densoviruses with ambisense genomes package both complementary strands in equimolecular ratios as single strands in separate capsids (27). The nonstructural (NS) gene cassette is found in the 5Ј half of one genome strand, and the structural protein (VP) gene cassette is found in the 5Ј half of the complementary strand. By convention, the genome is oriented so that the NS cassette is found in the left half. Expression strategies of densoviruses often involve (alternative) splicing and leaky scanning translation mechanisms (28). So far, the near-atomic structures of three densoviruses, Penaeus stylirostris densovirus (PstDNV), Bombyx mori densovirus 1 (BmDNV-1), and Galleria mellonella densovirus (GmDNV), have been solved (10,11,21). The capsid of densoviruses consists of 60 subunits (Tϭ1) of identical proteins that may contain N-terminal extensions not involved in capsid formation but that confer additional functions to the capsid. One of these functions is a phospholipase A2 (PLA2) activity that is required for genome delivery during infection (34). Densoviruses are usually highly pathogenic for their natural hosts (5).The monosense densoviruses have been classified into 3 uniform genera, i.e., Iteravirus, with a 5.0-kb genome, 0.25-kb ITRs, and a PLA2 motif in VP; Brevidensovirus, with a 4.0-kb genome, no ITRs but terminal hairpins, and no PLA2 motif; and Hepanvirus, with a single member, hepatopancreatic parvovirus, with a 6.3-kb genome also lacking a PLA2 motif and ITRs but with 0.2-kb terminal hairpins (23,27). In contrast, the ambisense densoviruses have been classified into one uniform genus, Densovirus, with a 6-kb genome and 0.55-kb ITRs, and a second genus, Pefudensovirus, with only Periplaneta fuliginosa densovirus (PfDNV) as a member, with a 5.5-kb genome, 0.2-kb ITRs, and a split VP gene cassette (2, 26). Ribosome frameshifts have been proposed to connect its VP open reading frames (ORFs) (33). So far, all ambisense densoviruses have an N-terminal PLA2 motif in their largest VP. Some sequenced ambisense densoviruses, e.g., Myzus persicae densovirus (MpDNV) (32), Blattella germanica densovirus (BgDNV) (18), and Planococcus citri densovirus (PcDNV) (25), are as yet unclassified. The ambisense virus Culex pipiens densovirus (CpDNV) has a different genome organization for both the NS

The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345 (NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promoters, explored using luciferase assays, were functional in insect and human cell lines.