The Parapoynx Stagnalis Nucleopolyhedrovirus (PastNPV), a Divergent Member of the Alphabaculovirus Group I Clade, Encodes a Homolog of Ran GTPase

We report the analysis of the genome of a novel Alphabaculovirus, Parapoynx stagnalis nucleopolyhedrovirus isolate 473 (PastNPV-473), from cadavers of the rice case bearer, Parapoynx stagnalis Zeller (Lepidoptera: Crambidae), collected in rice fields in Kerala, India. High-throughput sequencing of DNA from PastNPV occlusion bodies and assembly of the data yielded a circular genome-length contig of 114,833 bp with 126 annotated opening reading frames (ORFs) and six homologous regions (hrs). Phylogenetic inference based on baculovirus core gene amino acid sequence alignments indicated that PastNPV is a member of the group I clade of viruses in genus Alphabaculovirus, but different phylogenetic methods yielded different results with respect to the placement of PastNPV and four similarly divergent alphabaculoviruses in the group I clade. Branch lengths and Kimura-2-parameter pairwise nucleotide distances indicated that PastNPV-473 cannot be classified in any of the currently listed species in genus Alphabaculovirus. A unique feature of the PastNPV genome was the presence of an ORF encoding a homolog of Ran GTPase, a regulator of nucleocytoplasmic trafficking. PastNPV appears to have acquired a homolog of Ran relatively recently from a lepidopteran host via horizontal gene transfer.


Introduction
Baculoviruses (family Baculoviridae [1]) have two features that continue to inspire their development as biopesticides: their high virulence against insect pests, and their lack of toxicity against non-host organisms and the environment [2]. The latter feature, however, is accompanied by a narrow host range usually consisting of only one or a few related species of pests against which any given baculovirus is optimally active. For this reason, the identification and characterization of new baculoviruses is necessary to identify isolates with insecticidal activity against current pests of interest as well as potential or emerging pests.
Species of Baculoviridae are classified into four genera [3]. Viruses from two of these genera, Alphabaculovirus and Betabaculovirus, infect larvae of Lepidoptera, and some have been formulated for use against lepidopteran pests of agriculture [4,5]. These viruses have large (80-180 kbp) double-stranded DNA genomes that are replicated and packaged into rod-shaped nucleocapsids in the nucleus of infected cells. Initially, these nucleocapsids exit the nucleus and bud from the plasma membrane, forming enveloped virions that spread infection to other cells and tissues within infected larvae. Later during the replication cycle, progeny nucleocapsids are enveloped within the nucleus and assembled into paracrystalline occlusion bodies (OBs). Alphabaculovirus OBs are polyhedral in shape with a typical diameter of 0.5-5 µm, and contain several enveloped virions, often with multiple nucleocapsids bundled within a single virion. Betabaculovirus OBs are smaller (0.12 µm wide × 0.50 µm long), ovocylindrical in shape, and contain a single virion with a single A PastNPV occlusion body (OB) sample was received from Abraham Jacob at the Kerala Agricultural University College of Agriculture in Vellayani, India, on 29 January 1979. The virus was designated isolate 473 and deposited in the USDA-ARS insect virus collection in Beltsville, MD.

DNA Isolation and Sequencing
OBs were pelleted from 500 µL of the PastNPV sample by microcentrifugation at 9168× g for 2 min. The supernatant was removed and the pelleted material was resuspended in 0.1 M Na 2 CO 3 . The suspension was incubated for 30 min at the benchtop, then neutralized by addition of Tris-HCl pH 7.5 solution to a final volume of 0.1 M. Sodium dodecyl sulfate (10%) and proteinase K (Thermo Fisher Scientific, Waltham, MA, USA, catalog #AM2546) were added to final concentrations of 0.25% v/v and 500 µg/mL, respectively, and the suspension was incubated at 55 • C for 1 hr. DNA was extracted by mixing the dissolved OB solution with an equal volume of 25:24:1 phenol: chloroform: isoamyl alcohol saturated with 10 mM Tris-HCl pH 8/1 mM EDTA and separating aqueous and organic phases by centrifugation. The aqueous phase was transferred to a fresh Eppendorf tube and the DNA was precipitated with ethanol at −30 • C overnight. Precipitated DNA was pelleted by centrifugation, re-suspended in deionized distilled H 2 O, and quantified with Quant-iT ™ PicoGreen ™ dsDNA Assay Kit (Thermo Fisher Scientific, #P7589) and a QuantiFluor ™-ST Fluorometer (Promega, Madison, WI, USA). The procedure yielded 197 ng of DNA.
A library for sequencing the DNA sample on an Illumina MiSeq system was constructed from 100 ng of the sample as previously described [10]. Reads were assembled into a contigs and positions with variants were identified using Lasergene NGen v. 16 (DNAStar, Madison, WI, USA).
The first nucleotide of the polyhedrin (polh) open reading frame (ORF) was set as the first nucleotide in the genome sequence, and downstream annotated ORFs were numbered accordingly. The annotated PastNPV-473 genome sequence has been deposited in GenBank with the accession number ON704650.

Genome Annotation
ORFs were initially identified using the NCBI Open Reading Frame Finder (https: //www.ncbi.nlm.nih.gov/orffinder/ (accessed on 3 March 2022)). ORFs were annotated if they encoded amino acid sequences with significant sequence similarity with other baculovirus ORFs or genes from other sources. ORFs of at least 50 codons with no sequence similarity detected by BLASTp were also annotated if (a) they did not occur within an hr; (b) they did not overlap an hr or larger ORF by >75 bp, and (c) they were predicted by both FGENESV (http://linux1.softberry.com/berry.phtml (accessed on 3 March 2022)) and GeneMarkS [11] to be protein-encoding sequences. ORFs with no match in a BLASTp query were used in HMM-HMM queries with HHpred [12].

Phylogeny
Baculovirus core gene amino acid alignments were downloaded from the Baculoviridae chapter of the ICTV Online Report (https://ictv.global/report/chapter/baculoviridae/ baculoviridae/resources (accessed on 7 April 2022)) and re-aligned with core gene amino acid sequences from PastNPV-473 and selected other baculoviruses (Table S1) with MAFFT [16] or MUSCLE [15] as implemented in Lasergene MegAlign v. 17. The core gene alignments, which contained sequences from 97 baculoviruses, were concatenated with BioEdit 7.2.6 [17] and phylogeny was inferred by maximum likelihood (ML) using RAxML [18] from the concatenated core gene alignments using the Le and Gascuel (LG) substitution matrix [19] with variable rates among sites, empirical amino acid frequencies, and 100 rapid bootstrap replicates. Phylogenies were also inferred by minimum evolution (ME) in MEGA XI [20] with distances calculated using the JTT-based matrix method [21] with a gamma shape parameter of 0.79 and 500 bootstrap replicates.
PastNPV-473 ORFs 69 (enhancin) and ORF126 were aligned with other baculovirus homologs (Table S1) by MUSCLE and phylogenies inferred with RAxML as described above for the core gene phylogeny.

Characteristics of the PastNPV-473 Genome
Sequencing of the PastNPV-473 sample yielded 160,135 reads with an average length of 150 nt that assembled into a 114,833 bp circular contig with an average coverage of 211X and a nucleotide distribution of 34.17% GC. One hundred and twenty-six ORFs were annotated, including the 38 ORFs that constitute the core gene set for family Baculoviridae [24,25] ( Figure 1, Table S2).
In addition, six regions with conserved repeat sequences were identified as hrs. These hrs consist of 2-6 59-bp conserved unit sequences containing inverted imperfect repeats, with the consensus sequence 5 -TTGAACTCGCTTTACAAGTTTAAATGTACTCGTAAAGC AAGATCAGTGGATGATGTCA-3 ( Figure 2). In addition, six regions with conserved repeat sequences were identified as hrs. These hrs consist of 2-6 59-bp conserved unit sequences containing inverted imperfect repeats, with the consensus sequence 5′-TTGAACTCGCTTTACAAGTTTAAATGTACTCG-TAAAGCAAGATCAGTGGATGATGTCA-3′ ( Figure 2).
Variant positions were documented in the assembly, including 116 single-nucleotide polymorphisms (SNPs) and 16 insertions and deletions (indels) occurring at frequencies >10% (Table S3). Of the 116 SNPs, 30 of the positions occur in hrs or intergenic regions, while the remainder occur in annotated ORFs, resulting in synonymous (40 SNPs) and nonsynonymous (46 SNPs) changes. Of the 16 indels, 12 occur in hrs or intergenic regions. The remaining four indels occurred in annotated ORFs, with three maintaining the reading frame and one indel causing a frame-shift. Variant positions were documented in the assembly, including 116 single-nucleotide polymorphisms (SNPs) and 16 insertions and deletions (indels) occurring at frequencies >10% (Table S3). Of the 116 SNPs, 30 of the positions occur in hrs or intergenic regions, while the remainder occur in annotated ORFs, resulting in synonymous (40 SNPs) and nonsynonymous (46 SNPs) changes. Of the 16 indels, 12 occur in hrs or intergenic regions. The remaining four indels occurred in annotated ORFs, with three maintaining the reading frame and one indel causing a frame-shift. Repeats present in an orientation opposite to that of the hr1 repeats are indicated (reverse), as are the consensus sequence and a sequence logo displaying the relative frequency of nucleotide residues at each position [26]. Arrows denote an imperfect palindrome within the unit repeats.
Phylogenetic inference based on baculovirus concatenated alignments of core gene amino acid sequences placed PastNPV-473 in the group I clade of viruses in genus Alphabaculovirus with strong bootstrap support ( Figure 3). The assignment of PastNPV as a group I alphabaculovirus is further supported by the occurrence of PastNPV-473 ORF11, a homolog for the alphabaculovirus gene gp64 (Table S2). The gp64 gene encodes a budded virus envelope protein (GP64) and is the distinguishing feature of clade I alphabaculoviruses [36]. A previous analysis divided all group I alphabaculoviruses into two sub-clades designated clade I.a and clade I.b [37]. However, in a phylogram produced with the same procedures (ML with the LG substitution matrix inferred from MAFFT-aligned core gene sequences, Figure 3a), PastNPV and four other alphabaculoviruses-CapoNPV, Cyclophragma undans nucleopolyhedrovirus (CyunNPV; [38]), Oxyplax ochracea nucleopolyhedrovirus (OxocNPV; [39]), and LoobMNPV-were not placed in either clade I.a or Figure 2. Alignment of the conserved repeats in PastNPV-473 hrs. The four bases in the sequences are shaded with different colors to help visualize sequence identity. Repeats present in an orientation opposite to that of the hr1 repeats are indicated (reverse), as are the consensus sequence and a sequence logo displaying the relative frequency of nucleotide residues at each position [26]. Arrows denote an imperfect palindrome within the unit repeats.
Phylogenetic inference based on baculovirus concatenated alignments of core gene amino acid sequences placed PastNPV-473 in the group I clade of viruses in genus Alphabaculovirus with strong bootstrap support ( Figure 3). The assignment of PastNPV as a group I Alphabaculovirus is further supported by the occurrence of PastNPV-473 ORF11, a homolog for the Alphabaculovirus gene gp64 (Table S2). The gp64 gene encodes a budded virus envelope protein (GP64) and is the distinguishing feature of clade I alphabaculoviruses [36]. A previous analysis divided all group I alphabaculoviruses into two sub-clades designated clade I.a and clade I.b [37]. However, in a phylogram produced with the same procedures (ML with the LG substitution matrix inferred from MAFFT-aligned core gene sequences, Figure 3a), PastNPV and four other alphabaculoviruses-CapoNPV, Cyclophragma undans nucleopolyhedrovirus (CyunNPV; [38]), Oxyplax ochracea nucleopolyhedrovirus (OxocNPV; [39]), and LoobMNPV-were not placed in either clade I.a or clade I.b. Instead, PastNPV-473 and the other four viruses occurred on branches lying outside of these two subclades, with PastNPV-473 occupying a position that is basal to the rest of the viruses in   Table S1. Clade I.a and I.b are indicated by brackets, and a red bracket and asterisk denotes a group of five alphabaculoviruses with variable placement in the (a,b) group I subtrees. PastNPV-473 is indicated in bold type.  (Table S4). These values were well above the 0.050 substitutions/site demarcation criterion defined for baculovirus species [23,40], further indicating that PastNPV-473 should be classified into a new Alphabaculovirus species.
In gene-parity plots comparing PastNPV to a clade I.a virus (AcMNPV), a clade I.b virus (OpMNPV), and the four viruses with variable placement in the group I subtree (LoobMNPV, CapoNPV, CyunNPV, and OxocNPV), a small number of ORFs in the region of the polh (polyhedrin) ORF were found to occur in an orientation in PastNPV that is opposite to their orientation in AcMNPV and OpMNPV, but in the same orientation in the other four viruses (Figure 4). This trend has also been reported in prior comparisons between clade I alphabaculoviruses [30,38,39], indicating that ORF orientation relative to polh is a feature that distinguishes the group of alphabaculoviruses including PastNPV, LoobMNPV, CapoNPV, CyunNPV, and OxocNPV from other clade I.a and I.b alphabaculoviruses. In addition, two inversions are evident in the PastNPV-473 genome: one inversion (red box A in Figure 4) encompasses PastNPV ORFs 6 (p26) to 9 (me53), and the second (red box B) encompasses PastNPV ORFs 21 (p24 capsid) to 26 (alk-exo). The ORFs in the A inversion are in the same orientation in LoobMNPV and PastNPV, suggesting that this inversion was present in both PastNPV and LoobMNPV. The branch lengths in all three phylograms suggested that PastNPV represents a new species in genus Alphabaculovirus. The ranges of Kimura-2-parameter pairwise nucleotide distances in alignments of lef-8, lef-9, and polh between PastNPV-473 and alphabaculoviruses were estimated as 0.860-2.092 (lef-8), 0.400-2.330 (lef-9), and 0.468-1.205 (polh) substitutions/site (Table S4). These values were well above the 0.050 substitutions/site demarcation criterion defined for baculovirus species [23,40], further indicating that PastNPV-473 should be classified into a new alphabaculovirus species.
In gene-parity plots comparing PastNPV to a clade I.a virus (AcMNPV), a clade I.b virus (OpMNPV), and the four viruses with variable placement in the group I subtree (LoobMNPV, CapoNPV, CyunNPV, and OxocNPV), a small number of ORFs in the region of the polh (polyhedrin) ORF were found to occur in an orientation in PastNPV that is opposite to their orientation in AcMNPV and OpMNPV, but in the same orientation in the other four viruses (Figure 4). This trend has also been reported in prior comparisons between clade I alphabaculoviruses [30,38,39], indicating that ORF orientation relative to polh is a feature that distinguishes the group of alphabaculoviruses including PastNPV, LoobMNPV, CapoNPV, CyunNPV, and OxocNPV from other clade I.a and I.b alphabaculoviruses. In addition, two inversions are evident in the PastNPV-473 genome: one inversion (red box A in Figure 4) encompasses PastNPV ORFs 6 (p26) to 9 (me53), and the second (red box B) encompasses PastNPV ORFs 21 (p24 capsid) to 26 (alk-exo). The ORFs in the A inversion are in the same orientation in LoobMNPV and PastNPV, suggesting that this inversion was present in both PastNPV and LoobMNPV.  A BLASTn query with the consensus hr repeat sequence of PastNPV-473 resulted in matches to hr sequences from isolates of clade I.a viruses, especially AcMNPV and BmNPV, with e-values ranging from 1 × 10 −4 to 2 × 10 −9 . An alignment of the hr consensus sequences for the exemplar isolates of BmNPV and AcMNPV with the hr consensus sequence of PastNPV-473 and OxocNPV-435 revealed a conserved imperfect palindromic sequence with the EcoR I endonuclease site of the AcMNPV hr repeats at its center [41] ( Figure S2). The hr sequences from different viruses usually share relatively little sequence similarity, but similarity has been observed among hrs of related viruses of some clades [42].

ORF Content of PastNPV-473
In addition to homologs for the 38 core genes of family Baculoviridae, the PastNPV-473 genome contains homologs for the 26 ORFs reported to be conserved among Alphabaculovirus genomes by Garavaglia et al. [24] (Table S2). The PastNPV-473 sequence is missing ORFs for the viral cathepsin and chitinase genes. These ORFs are present in most alphabaculoviruses and several betabaculoviruses, and have been shown to play a role in liquefaction of the host internal anatomy and weakening of the host larval cuticle [43]. While LoobMNPV is also missing these two genes, cathepsin and chitinase ORFs are present in CapoNPV, CyunNPV, and OxocNPV, located between the pkip and dbp ORFs. PastNPV has a homolog of AcMNPV ORF ac57 present at this this location in place of cathepsin and chitinase ORFs (Table S3).
Of the remaining 62 ORFs, 51 are homologs of ORFs found in AcMNPV. One ORF, ORF22, appears to be a second, divergent copy of core gene ac78. Although a BLASTx query with the ORF22 sequence did not yield any matches, an HHpred query produced UniProt and Pfam matches with AC78 at a probability of >92% with residues 6-59. The remaining 10 ORFs are a mixture of homologs found in other baculoviruses and ORFs with no similarity to previously reported baculovirus sequences.

ORFs with Homologs in Other Alphabaculoviruses
PastNPV ORF54 encodes a putative polypeptide with BLAST matches to ORFs from group I and II alphabaculoviruses that contain the conserved AAA+ ATPase module. The AAA+ (ATPases Associated with a variety of cellular Activities) superfamily of ATPases play a role in a wide range of cellular functions [44]. The top BLAST match for ORF54 is with ORF152 of the group II Alphabaculovirus Leucania separata nucleopolyhedrovirus (LeseNPV; [45]), with 41% sequence identity. However, the PastNPV homolog encodes a 170-amino acid polypeptide, while the other baculovirus AAA+ ATPase homologs range from 307 to 448 amino acids with an average of 374 amino acids. AAA+ ATPase active sites are formed by two conserved motifs, Walker A (G(x)4GKT) and Walker B (hhhhDE, where h denotes hydrophobic amino acids) [46]. While both motifs are present in other Alphabaculovirus AAA+ homologs, the PastNPV ORF54 sequence only possesses the Walker A motif and thus is unlikely to encode a functional AAA+ ATPase.
The top match of a BLASTx query with ORF69 was with an enhancin encoded by Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV [47]; GenBank accession no. NP_848341). Enhancins are zinc metalloproteases encoded by some alpha-and betabaculoviruses as well as bacteria [48]. The enhancins have been shown to promote primary infection of the host midgut epithelium by degrading proteins of the peritrophic matrix, which acts as a protective barrier [49][50][51], though some results suggest an additional or alternative mechanism for its effects [52,53]. Phylogenetic inference of the relationships among ORF69 and other enhancins grouped the ORF69 sequence with the enhancins from group I Choristoneura spp. alphabaculoviruses, including CfMNPV, Choristoneura occidentalis nucleopolyhedrovirus BC_1 (ChocNPV-BC_1) and Choristoneura rosaceana nucleopolyhedrovirus NB_1 (ChroNPV-NB_1) [54] (Figure 5). ORF69 and the Choristoneura spp. Alphabaculovirus enhancins grouped with a larger set of enhancins from alphabaculoviruses of hosts from subfamily Noctuinae. Other group I Alphabaculovirus enhancin genes were tional or alternative mechanism for its effects [52,53]. Phylogenetic inference of the relationships among ORF69 and other enhancins grouped the ORF69 sequence with the enhancins from group I Choristoneura spp. alphabaculoviruses, including CfMNPV, Choristoneura occidentalis nucleopolyhedrovirus BC_1 (ChocNPV-BC_1) and Choristoneura rosaceana nucleopolyhedrovirus NB_1 (ChroNPV-NB_1) [54] (Figure 5). ORF69 and the Choristoneura spp. alphabaculovirus enhancins grouped with a larger set of enhancins from alphabaculoviruses of hosts from subfamily Noctuinae. Other group I alphabaculovirus enhancin genes were distributed among group II alphabaculovirus sequences in a separate clade that included the vef-1 and vef-2 enhancin genes from viruses of Lymantria spp. hosts.  Table S1. PastNPV-473 ORF69 is indicated in bold type, and group I alphabaculovirus sequences are indicated with red asterisks. Branches for enhancins from betabaculoviruses and from alphabaculoviruses of Lymantria spp. and Mamestra spp. hosts are collapsed.  Table S1. PastNPV-473 ORF69 is indicated in bold type, and group I Alphabaculovirus sequences are indicated with red asterisks. Branches for enhancins from betabaculoviruses and from alphabaculoviruses of Lymantria spp. and Mamestra spp. hosts are collapsed.
Metalloproteases are characterized by a zinc-binding motif, HEXXH, where X is any amino acid. The histidines in this motif coordinate a zinc ion that is essential for proteolytic activity, while the glutamate residue catalyzes the hydrolysis of the substrate peptide bond [55]. An alignment of ORF69 with 89 additional baculovirus enhancin sequences yielded a consensus zinc-binding motif sequence of HEIGH. However, the sequence of this motif in PastNPV ORF69 was found to be REIGH, with an arginine replacing the first histidine in the motif. Some of the Alphabaculovirus and betabaculovirus enhancin sequences deviate from the consensus zinc-binding motif to an even greater extent [48,56], and it is not clear if these ORFs or ORF69 encode an active metalloprotease.
PastNPV ORF75 shared the highest degree of sequence identity with LoobMNPV ORF78. Homologs of this ORF were also identified in a mixture of alphabaculoviruses and betabaculoviruses by BLASTx. An HHpred query with a multiple sequence alignment of 24 ORF75 homologs only yielded one reasonably convincing match with the nonstructural protein NS3 of Diatraea saccharalis densovirus (GenBank accession no. NP_046812.1), with a probability of 90.07%. The NS3 gene of the related Junonia coenia densovirus was shown to be required for densovirus DNA replication [57].
The predicted amino acid sequence of ORF126 shares 51.9% sequence identity with ORF141 of Antheraea pernyi nucleopolyhedrovirus (AnpeNPV, strain Liaoning; [58]). Homologs of this ORF are also found in other isolates of Antherea sp. NPVs and related NPVs from other saturniid hosts, as well as isolates of Choristoneura NPVs, Anticarsia gemmalis multiple nucleopolyhedrovirus (AgMNPV; [59]), Neophasia sp. nucleopolyhedrovirus (NespNPV, GenBank accession no. MK293724), and Epiphyas postvittana nucleopolyhedrovirus (EppoNPV; [60]). No homologs of this ORF were detected in group II Alphabaculovirus or betabaculovirus genomes, and no convincing matches were obtained from an HHpred query with an alignment of 19 ORF126 homologs. In a phylogeny inferred from an alignment of ORF126 and related sequences, ORF126 was placed in a position basal to the other homologs of this ORF ( Figure S3).

ORFs Not Found in Other Baculovirus Genomes
Six ORFs annotated for PastNPV-473 were not found to have homologs in any other baculovirus sequence available in GenBank (Table S2). Promoter motifs associated with both early and late baculovirus gene expression were identified for all six ORFs (Table 1). Queries with four of these ORFs (15,27,92, and 104) using BLASTx and HHpred did not return any matches with significant sequence similarity. A BLASTx query with ORF10 returned a top match with a hypothetical protein of the bacterium Chitinophaga caeni (GenBank accession no. WP_198405806) with 54.5% sequence identity and an e-value of 2.6 × 10 −20 . The predicted amino acid sequence of ORF10 contained eight complete copies and one partial copy of the sequence MAYVTDLS, which aligned with a similar repeat sequence in the C. caeni polypeptide.  Queries with the ORF40 amino acid sequence indicated that it was a homolog of the Ran GTPase, a member of the Ras superfamily [61]. Ran regulates nucleocytoplasmic trafficking of macromolecules across the nuclear envelope and is involved in steps of the eukaryotic cell cycle, including mitotic spindle assembly and nuclear envelope formation [62]. The ORF40 polypeptide was found to share 84.0-88.3% sequence identity with Ran homologs from insects, with the highest BLASTx match being with the Ran GTPase of the navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae; GenBank accession no. XP_013194495). An examination of the alignment of the ORF40 amino acid sequence with other Ran sequences suggested that ORF40 encodes a full-length, functional Ran GTPase, with a conserved phosphate-binding loop (P-loop; GDGGTGKT) ( Figure 6). The Ran Switch I and Switch II motifs, which undergo conformational changes in Ran when GDP is exchanged for GTP, were also present in ORF40, as was a conserved C-terminal acidic tail which interacts with a conserved basic sequence to stabilize the GDP-bound form of Ran [63,64].
ficking of macromolecules across the nuclear envelope and is involved in steps of the eukaryotic cell cycle, including mitotic spindle assembly and nuclear envelope formation [62]. The ORF40 polypeptide was found to share 84.0-88.3% sequence identity with Ran homologs from insects, with the highest BLASTx match being with the Ran GTPase of the navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae; GenBank accession no. XP_013194495). An examination of the alignment of the ORF40 amino acid sequence with other Ran sequences suggested that ORF40 encodes a full-length, functional Ran GTPase, with a conserved phosphate-binding loop (P-loop; GDGGTGKT) ( Figure 6). The Ran Switch I and Switch II motifs, which undergo conformational changes in Ran when GDP is exchanged for GTP, were also present in ORF40, as was a conserved C-terminal acidic tail which interacts with a conserved basic sequence to stabilize the GDP-bound form of Ran [63,64].

Discussion
PastNPV is one of five group I alphabaculoviruses including LoobMNPV, CapoNPV, CyunNPV, and OxocNPV that can be distinguished from other group I alphabaculoviruses on the basis of branch lengths in phylograms and ORF synteny (Figures 3 and 4). These viruses originate from a disparate group of host families including Crambidae (PastNPV), Pieridae (CapoNPV), Lasiocampidae (CyunNPV), Saturniidae (LoobMNPV), and Limacodidae (OxocNPV). A previous study failed to detect infectivity of PastNPV against larvae of seven other crambid species, three species of family Noctuidae, and one species of family Autostichidae [9]. We similarly were unable to detect infectivity of our sample of PastNPV when fed to neonate larvae of Plutella xylostella (diamondback moth, family Plutellidae) or Trichoplusia ni (cabbage looper, family Noctuidae), although we cannot exclude the possibility that our sample had lost infectivity during storage. Hence, the

Discussion
PastNPV is one of five group I alphabaculoviruses including LoobMNPV, CapoNPV, CyunNPV, and OxocNPV that can be distinguished from other group I alphabaculoviruses on the basis of branch lengths in phylograms and ORF synteny (Figures 3 and 4). These viruses originate from a disparate group of host families including Crambidae (PastNPV), Pieridae (CapoNPV), Lasiocampidae (CyunNPV), Saturniidae (LoobMNPV), and Limacodidae (OxocNPV). A previous study failed to detect infectivity of PastNPV against larvae of seven other crambid species, three species of family Noctuidae, and one species of family Autostichidae [9]. We similarly were unable to detect infectivity of our sample of PastNPV when fed to neonate larvae of Plutella xylostella (diamondback moth, family Plutellidae) or Trichoplusia ni (cabbage looper, family Noctuidae), although we cannot exclude the possibility that our sample had lost infectivity during storage. Hence, the biological control potential of PastNPV may be limited to addressing outbreaks of P. stagnalis in rice.
The remaining 17 group I alphabaculoviruses of Figure 3 occur in two well-supported subclades, clade I.a and clade I.b. Clade I.a includes AcMNPV and other closely related alphabaculoviruses, and clade I.b consists of a more divergent group of alphabaculoviruses mostly from tree and forest pests, such as OpMNPV [65]). While Kimura-2-parameter nucleotide distances clearly indicate that PastNPV cannot be classified into any currently existing Alphabaculovirus species [40], the relationships of PastNPV and the four other more disparate viruses to the clade I.a and I.b alphabaculoviruses are unclear. The incongruence of the group I trees in Figure 3 could be due to the divergent nature of sequences from PastNPV and the other four viruses resulting in relatively long branch lengths, and relationships of these viruses with each other and the other group I alphabaculoviruses will likely require additional sequences from similar viruses to resolve.
The absence of a Ran GTPase homolog in other baculovirus genomes and the high degree of sequence identity between insect Ran GTPases and PastNPV ORF40 suggests that a virus of the PastNPV lineage had acquired a homolog of a cellular Ran sequence by horizontal gene transfer relatively recently. The preservation of motifs involved in Ran's function in the ORF40 sequence further suggests that ORF40 encodes a functional Ran GTPase. Expression of the PastNPV homolog may affect egress of progeny PastNPV nucleocapsids from the host nucleus, or alter the nuclear or cellular mileau in a way that impacts DNA replication or virion assembly. The impact of expression of a Ran GTPase on baculovirus replication will require additional research.

Conflicts of Interest:
The authors declare no conflict of interest. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.