Genome structure of DhNV
The circular genome of DhNV is 119,754 bp and contains 106 hypothetical ORFs, with 56 on the positive strand and 50 on the negative strand (Fig. 1). Fifty-nine of the ORFs met our comparative e-value threshold of < 0.001 and were directly comparable to other members of the Nudiviridae. Up to 17% and 37% of the ORFs aligned most closely with genes from HgNV and PmNV, respectively. Two ORFs, DhNV_008 and DhNV_002, scored above 50% similarity to protein sequences on BLASTp. DhNV_008 was 52.33% similar to pif-2 from HgNV (QBB28614) with per os infectivity as the only identified protein domain. DhNV-002 was 50% similar to a hypothetical protein from PmNV (YP_009051845) where the cytoplasmic, non-cytoplasmic, tmhelix, and transmembrane domains were identified. Among the 47 ORFs that provided no similarity to other protein sequences within the threshold, InterProScan assessment identified 20 ORFs with functional domains. A protein signature match to the inhibitor of apoptosis repeat superfamily in DhNV_059 may indicate the presence of a homolog of the Iap nudivirus gene; however, BLASTp annotations did not yield any similarity results to the Iap gene found in other nudiviruses. The remaining 19 ORFs contained proteins with Zinc finger domains (DhNV_045), Tmhelix (DhNV_044), signal peptide (DhNV_084), p-loop containing nucleoside triphosphate hydrolase (DhNV_047), non-cytoplasmic domain (DhNV_025, 028, and 057), disorder predictions (DhNV_011, 020, 024, 072, 077, and 078), predicted to be cytoplasmic (DhNV_019 and 065), or had ‘coil’ feature(s) (DhNV_022, 026, 042, and 086).
Seven genes involved with DNA processing were identified: DNA polymerase, helicase, two copies of helicase 2, integrase, DNA ligase, and fen-1. Five genes involved with RNA transcription were also identified: p47, lef-4, lef-5, lef-8, and lef-9. Eight genes known to be involved in per os infectivity were found: vp91 (pif-8), pif-1, pif-2, pif-3, odv-e28 (pif-4), odv-e56 (pif-5), ac68 (pif-6), and p74 (pif-0), along with the 11 k gene. Nine genes can be grouped into packaging, assembly, and release: two copies of ac92 (p33), two copies of odv-e66, two copies of vlf-1, 38 k, ac81, and 31 K (vp39). The recently identified p6.9 gene in crustacean-infecting nudivirus genomes, a baculovirus core gene associated with encapsulation of the viral genome, could not be found in DhNV despite similarity searches and checks for hypothetical SRSR repeat regions common to the p6.9 protein. A putative DUTPase, cg30-1, p-loop NTPase, guanosine monophosphate kinase, esterase, and p51 were also identified. Definitively, 20 out of the 21 core baculovirus genes conserved among nudivirus were identified in the DhNV genome (Table 1).
Multiple genes were specific to DhNV; however, some were similar to other protein groups from various taxa. One ORF, DhNV_076, revealed 31.98% to 35.97% similarity to proteins from seven different organisms. The LOC108666550-like protein from HgNV is 32.35% similar (85% coverage) to DhNV_076 (Table 1). Uncharacterized proteins from Hyalella azteca (QBB28676, sim. 35.97% cov. 82%), an amphipod native to North America; Penaeus vannamei (XP_027235023, sim. 34.07% cov. 83%), the Whiteleg shrimp; Crassostrea gigas (XP_011433877, sim. 31.98% cov. 85%), the Pacific Oyster; Armadillidium vulgare (RXG54766, sim. 34.15% cov. 83%), the common pill-bug; and Tigriopus californicus (TRY76277, similarity 33.26% coverage 88%), a North American coastal copepod, all met the e-value threshold, as did the actin-binding IPP-like protein from Brachionus plicatilis (RMZ96256 similarity 33.65% coverage 87%). Only the undescribed PANTHER protein family, PTHR38566, was identified as a domain in DhNV_076.
Gene synteny among the Epsilonnudivirus, Gammanudivirus and Deltanudivirus genera
A comparison of gene synteny between DhNV and three other nudiviruses determined that DhNV had a different gene synteny to members of the Gammanudivirus and Deltanudivirus genera (Fig. 2). Comparison between ToNV and DhNV revealed a high level of genomic rearrangement, where the 32 genes that showed genetic similarity (e < 0.001) with those ORFs on the DhNV genome were located across the respective genomes, showing little conserved synteny (Fig. 2a). Comparison between DhNV and PmNV/HgNV revealed higher levels of gene synteny (Fig. 2b,c). A comparison using all three viruses identified 12 major regions of genetic novelty in the DhNV genome (Fig. 2d). This included 47 hypothetical ORFs that were unique to DhNV and showed little genetic/protein relatedness to other nudiviruses within the e-value threshold of < 0.001, one of which (DhNV_070) showed highest similarity to a gene from Pyricularia oryzae, a fungal plant pathogen, and another (DhNV_029) with highest similarity to Sucra jujuba nucleopolyhedrovirus (Table 1).
Using the protein similarity data, we determined that there were 11 crustacean-infecting nudivirus genes (DhNV_006, 024, 032, 034, 050, 062, 067, 080, 103, 104 and 106) that show conservation across the crustacean-infecting nudiviruses (Table 1) (i.e. present in PmNV, HgNV and DhNV) but appear absent from other nudiviruses that do not infect crustaceans. Using these genes in addition to the conserved baculovirus core genes across DhNV, HgNV and PmNV 1,2, 35 genes were comparable in a “gene-block” fashion relative to their genomic loci (Fig. 2e). This revealed three major rearrangement events. Reordering of the DhNV_032, DhNV_034, and pif-1 gene block, which is reversed in PmNV and HgNV (Fig. 2, ‘X’). Reordering of the vlf-1 and p74 gene block, which is reversed in PmNV and HgNV (Fig. 2, ‘Y’) and a larger rearrangement of 6 genes (vlf-1, ac68, DhNV_080, ac81, and both copies of helicase 2), which is reversed in PmNV and HgNV and overlaps the ‘Y’ rearrangement event (Fig. 2, ‘Z’).
Morphological and phylogenetic comparison to other Nudiviridae
A concatenated maximum likelihood phylogenetic analysis of eight nudiviruses and one baculovirus (outgroup) using 18 core nudivirus genes (see Sect. 4) supported the positioning of DhNV outside of the two crustacean-infecting nudiviruses with bootstrap values of 100% (Fig. 3). Within this grouping, DhNV is an early branching member of the Gammanudivirus genus and may constitute a different genus altogether. The Betanudivirus genus branches outside of the Gammanudivirus cluster and the Deltanudivirus member ToNV is the earliest branching member of these three genera. The Alphanudivirus group represents the most phylogenetically distinct nudivirus genus represented on our diagram (Fig. 3).
Illustrations of virus morphology provide another dimension of comparison among the Nudiviridae species (Fig. 3). DhNV virions consist of a double membrane surrounding an electron-dense core measuring (n = 30, mean ± SD) 302 ± 13 nm in length and 55 ± 4 nm at its diameter 11. The rod-shaped structure is maintained across all the nudiviruses. DhNV represents one of the larger nudiviruses discovered to date, second to HzNV2, which has a length of 382 ± 30 nm.
A second concatenated maximum likelihood phylogenetic analysis of putative iap and pif-2 genes supported DhNV as an earlier branch of the crustacean-infecting nudiviruses. The addition of Macrobrachium rosenbergii nudivirus CN-SL2011 (MrNV) (NCBI:txid1217568), which only has the aforementioned genes available, branched in the Gammanudivirus genus. ToNV (Deltanudivirus) is the earliest branch of these genera, followed by HzNV2 (Betanudivirus), and the four crustacean-infecting nudiviruses. The Alphanudiviruses represent the most phylogenetically distinct lineage among nudiviruses in this tree (Fig. 4), following the same general phylogenetic theme as the details in Fig. 3.