Genomic sequencing of Troides aeacus nucleopolyhedrovirus (TraeNPV) from golden birdwing larvae (Troides aeacus formosanus) to reveal defective Autographa californica NPV genomic features

Abstract

Background: The golden birdwing butterfly (Troides aeacus formosanus) is a rarely observed species in Taiwan. Recently, a typical symptom of nuclear polyhedrosis was found in reared T. aeacus larvae. From the previous Kimura-2 parameter (K-2-P) analysis based on the nucleotide sequence of three genes in this isolate, polh, lef-8 and lef-9, the underlying virus did not belong to any known nucleopolyhedrovirus (NPV) species. Therefore, this NPV was provisionally named "TraeNPV". To understand this NPV, the nucleotide sequence of the whole TraeNPV genome was determined using next-generation sequencing (NGS) technology.

Results: The genome of TraeNPV is 125,477 bp in length with 144 putative open reading frames (ORFs) and its GC content is 40.45%. A phylogenetic analysis based on the 37 baculoviral core genes suggested that TraeNPV is a Group I NPV that is closely related to Autographa californica nucleopolyhedrovirus (AcMNPV). A genome-wide analysis showed that TraeNPV has some different features in its genome compared with other NPVs. Two novel ORFs (Ta75 and Ta139), three truncated ORFs (pcna, he65 and bro) and one duplicated ORF (38.7 K) were found in the TraeNPV genome; moreover, there are fewer homologous regions (hrs) than there are in AcMNPV, which shares eight hrs within the TraeNPV genome. TraeNPV shares similar genomic features with AcMNPV, including the gene content, gene arrangement and gene/genome identity, but TraeNPV lacks 15 homologous ORFs from AcMNPV in its genome, such as ctx, host cell-specific factor 1 (hcf-1), PNK/PNL, vp15, and apsup, which are involved in the auxiliary functions of alphabaculoviruses.

Conclusions: Based on these data, TraeNPV would be clarified as a new NPV species with defective AcMNPV genomic features. The precise relationship between TraeNPV and other closely related NPV species were further investigated. This report could provide comprehensive information on TraeNPV for evolutionary insights into butterfly-infected NPV.

Keywords: TraeNPV; Troides aeacus; Troides aeacus nucleopolyhedrovirus.

Fig. 1

Genomic circular map and heat map identity of the TraeNPV. The heat map identity of the species AcMNPV, BmNPV, MaviMNPV, LdMNPV and CpGV compared to the orthologous ORFs of TraeNPV are shown on the inner rings in sequence. The darker the red is, the higher the correlated ORF identity. The positions for these 144 ORFs, which are listed in Additional file 1: Table S2, are presented as arrowheads with the direction of the arrowhead indicating the orientation of each ORF. The locations for the eight homologous repeat regions (hrs) are indicated

Fig. 2

Genomic fragments of TraeNPV and AcMNPV contain the CNE region. (a) The location of the CNE for TraeNPV and AcMNPV is flanked by the ie-2 and pe38 genes. The CEN of AcMNPV is overlapped in the ORF-152. The ClustalX alignment of the CNEs of TraeNPV and AcMNPV. The consensus sequence was determined and described by Kikhno [20]. The clusters of conserved nucleotides are indicated (C1~C7). The lines mark the dyad symmetry elements, each of which is indicated by the abbreviation “DS” in conjunction with the lowercase letters (l, c, and r) that specify the DS position in the CNE (left, central, and right, respectively). The inverted repeats are indicated with arrows, and the abbreviation “IR” in conjunction with the letters l, c, and r, assigns each IR pair to a particular DS. (b) The comparison of the gene locations by using the relative restriction sites in the TraeNPV with those of the corresponding AcMNPV fragment. Arrows denote ORFs and their direction of transcription. Grey boxes represent the CNE region; black boxes represent the homologous repeat regions (hrs). ORF homologues in the corresponding regions are drawn with the same patterns

Fig. 3

Baculovirus phylogeny inferred from a combined dataset of the 37 baculoviral core protein sequences. An unrooted ML tree is shown. CuniNPV was selected as the outgroup. The numbers at the nodes indicate bootstrap scores above 50% for the ML analyses (100 replicates, ML bootstrap)

Fig. 4

Mauve (multiple alignment of conserved genomic sequence with rearrangements) representation of alphabaculoviruses from group I and II and CpGV. The alignment was performed on collinear sequences in which NPV was a reference sequence and the polh gene was considered as a first ORF (Except AcMNPV). Coloured sections (bordered with a curve that indicates the level of nucleotide similarity) represent the homologous fragments of compared genomes. The section that is located beneath the X-axis shows the inversion of this genome fragment in comparison to the reference

Fig. 5

Gene parity plot analysis of TraeNPV in comparison with (a) AcMNPV, (b) BmNPV, (c) MaviNPV, (d) LdMNPV and (e) CpGV, as indicated. Axes: the relative position of each ORF; dots: ORFs

Fig. 6

Amino acid length difference for TraeNPV compared to (a) AcMNPV, (b) BmNPV, (c) MaviNPV, (d) LdMNPV and (e) CpGV, as indicated. X-axis: the relative position of each ORF; Y-axis dots: amino acid differences

Fig. 7

Alignment of IE-2 amino acid sequences. The identical residues occupying > 50% of the aligned positions are shaded in black, and residues similar to the conserved residues or to one another are shaded in grey. The lines above the aligned sequences indicate the locations of different functional motifs. The acidic domain required for transcriptional activation is indicated with a thick line

Fig. 8

Comparison of TraeNPV hr. palindromes with (a) each hr. palindrome, which was identified from the TraeNPV genome; and (b) palindrome consensus sequences from other baculoviruses. The alignment of the consensus hr. palindrome from TraeNPV, AcMNPV, BmNPV, MaviNPV and LdMNPV; and (c) a comparison of the genomic context of the hrs and hr. locations relative to the homologous ORFs between TraeNPV, AcMNPV, BmNPV, MaviNPV and LdMNPV in the linearized genomes. The ORFs flanking the hrs: below the line. Grey rectangles: the major inserts relative to AcMNPV and the ORFs within the inserts are shown above the line. For consistency, all the linearized genomes start with polh, but the hrs and ORF numbers remain the same as in the original papers