A Novel Alphabaculovirus from the Soybean Looper, Chrysodeixis includens, that Produces Tetrahedral Occlusion Bodies and Encodes Two Copies of he65

Abstract

Isolates of the alphabaculovirus species, Chrysodeixis includens nucleopolyhedrovirus, have been identified that produce polyhedral occlusion bodies and infect larvae of the soybean looper, Chrysodeixis includens. In this study, we report the discovery and characterization of a novel C. includens-infecting alphabaculovirus, Chrysodeixis includens nucleopolyhedrovirus #1 (ChinNPV#1), that produces tetrahedral occlusion bodies. In bioassays against C. includens larvae, ChinNPV #1 exhibited a degree of pathogenicity that was similar to that of other ChinNPV isolates, but killed larvae more slowly. The host range of ChinNPV#1 was found to be very narrow, with no indication of infection occurring in larvae of Trichoplusia ni and six other noctuid species. The ChinNPV#1 genome sequence was determined to be 130,540 bp, with 126 open reading frames (ORFs) annotated but containing no homologous repeat (hr) regions. Phylogenetic analysis placed ChinNPV#1 in a clade with other Group II alphabaculoviruses from hosts of lepidopteran subfamily Plusiinae, including Chrysodeixis chalcites nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus. A unique feature of the ChinNPV#1 genome was the presence of two full-length copies of the he65 ORF. The results indicate that ChinNPV#1 is related to, but distinct from, other ChinNPV isolates.

Keywords: Chrysodeixis includens; DNA ligase 3; baculovirus; he65; lef-12; occlusion body; polyhedrin; soybean looper.

Figure 1

Scanning and transmission electron micrographs of ChinNPV#1 occlusion bodies (OBs): (A) A group of ChinNPV#1 OBs; (B) high-magnification view of a single OB; (C) section through a single OB, showing the lattice lines of the paracrystalline polyhedrin matrix surrounding the occluded virions; (D) section through a larger OB. Scale bars: (A) 2 µm, (B) 500 nm, (C) and (D) 200 nm.

Figure 2

Open reading frame (ORF) map of the ChinNPV#1 genome. ORFs are represented by arrows, with the position and direction of the arrow indicating ORF position and orientation. Each ORF is color-coded to indicate whether it corresponds to a baculovirus core gene (yellow), an ORF reported to be conserved among all alphabaculoviruses (green) [36], an ORF with homologs in a subset of other baculoviruses (black), or an ORF not previously identified in any other baculovirus genome (red). ORFs are designated by either a specific name, the designation of their AcMNPV homolog (acXX), or a number corresponding to its annotation in the ChinNPV#1 genome.

Figure 3

Gene parity plots comparing the ORF content and order of ChinNPV#1 (y-axis) with that of related alphabaculoviruses from noctuid hosts of subfamilies Plusiinae (PsinSNPV-IE, ChchNPV, and TnSNPV) and Noctuinae (AgipMNPV). Each point in a plot represents an ORF, and points corresponding to ORFs present in only one of the compared genomes appear on the axis line for the virus in which they are present.

Figure 4

Relationships among ChinNPV#1, representative isolates of other baculovirus species, and other unclassified baculovirus isolates inferred from the predicted amino acid sequences of baculovirus core genes. A phylogram was constructed from the concatenated alignments of 38 baculovirus core gene amino acid sequences using maximum likelihood (ML) and minimum evolution (ME) methods. Shown is the ML tree with bootstrap values >50% for branches in trees produced by both ME and ML methods (displayed as ME/ML). Stars indicate bootstrap values of 100%. Branches for the viruses of genera Betabaculovirus, Gammabaculovirus, and Deltabaculovirus are collapsed, and the numbers of taxa in those nodes are shown in parentheses. Group I and Group II alphabaculoviruses are indicated with colored bars. ChinNPV#1 is indicated in bold red type, and its position denoted with a red arrow. The taxa and sequences used in the analysis are as listed in Table S1.

Figure 5

Alignment of Group II plusiine alphabaculovirus polyhedrin amino acid sequences. Portions of a MUSCLE alignment of these sequences where differences among the sequences exist are shown, encompassing residues 1–50, 101–150, and 151–200, along with the consensus sequence in those regions. Amino acid identities that differ from the consensus identity at a given position are highlighted in a color corresponding to the biochemical class of the residue. Residues that are conserved among alphabaculoviruses with tetrahedral OBs and that differ in the three of the four sequences of alphabaculoviruses not reported to produce tetrahedral OBs are outlined in red. A star indicates a residue (#43) previously reported to determine tetrahedral OB morphology [47]. TnSNPV (AAFC) corresponds to the exemplar TnSNPV isolate whose genome was reported in reference [38]; the other viruses are as described in the text.

Figure 6

Phylogenetic inference of relationships among encoded baculovirus LEF-12 proteins. An ML phylogram shown with branch support for ME and ML phylogenies as described in the legend for Figure 4. The position of the ChinNPV#1 sequence is indicated in bold red type with an arrow. Branches for the 21 Group I alphabaculovirus sequences are collapsed.

Figure 7

Phylogenetic inference of relationships among encoded baculovirus HE65 amino acid sequences. An ML phylogram inferred with a bacteriophage T4 RNA ligase included as an outgroup is shown with branch support for ME and ML phylogenies as described in the legend for Figure 4. The position of the two ChinNPV#1 HE65 sequences are indicated in bold red type with arrows. Genus and group classifications of the different HE65 taxa are indicated with colored boxes.

Figure 8

Mauve alignment of the genomic region of ChinNPV#1 and three other plusiine alphabaculoviruses extending from gp37 to vp39, indicating the positions of homologous ORFs in these viruses. Outlines of the same color correspond to Locally Collinear Blocks (LCBs) of sequence that are conserved among the viruses. The height of the profile within each LCB corresponds to the average level of sequence conservation among the isolates in that region of the genome sequence. ORFs annotated for each virus are indicated below the LCBs.

Figure 9

Phylogenetic inference of relationships among insect and alphabaculovirus DNA ligase 3 (LIG3) amino acid sequences. An ML phylogram inferred with a betabaculovirus DNA ligase 1 deployed as an outgroup is shown with branch support for ME and ML phylogenies as described in the legend for Figure 4. Insect order classifications are indicated with colored bars for different clades and taxa of LIG3 sequences, as are the positions of the alphabaculovirus LIG3 sequences. The position of the ChinNPV#1 LIG3 sequence is indicated in bold red type with an arrow.