Comprehensive analysis of single molecule sequencing-derived complete genome and whole transcriptome of Hyposidra talaca nuclear polyhedrosis virus

Abstract

We sequenced the Hyposidra talaca NPV (HytaNPV) double stranded circular DNA genome using PacBio single molecule sequencing technology. We found that the HytaNPV genome is 139,089 bp long with a GC content of 39.6%. It encodes 141 open reading frames (ORFs) including the 37 baculovirus core genes, 25 genes conserved among lepidopteran baculoviruses, 72 genes known in baculovirus, and 7 genes unique to the HytaNPV genome. It is a group II alphabaculovirus that codes for the F protein and lacks the gp64 gene found in group I alphabaculovirus viruses. Using RNA-seq, we confirmed the expression of the ORFs identified in the HytaNPV genome. Phylogenetic analysis showed HytaNPV to be closest to BusuNPV, SujuNPV and EcobNPV that infect other tea pests, Buzura suppressaria, Sucra jujuba, and Ectropis oblique, respectively. We identified repeat elements and a conserved non-coding baculovirus element in the genome. Analysis of the putative promoter sequences identified motif consistent with the temporal expression of the genes observed in the RNA-seq data.

Figure 1

(a) H. talaca healthy larvae. (b) H. talaca larvae exhibiting typical NPV infected symptom hanging from tea leaves. (c) Scanning electron micrograph showing the polyhedral inclusion bodies.

Figure 2

Circular diagram of the HytaNPV genome and annotation. The arrows represent position and direction of ORFs. The first ORF (orf1) is the polyhedrin gene. red – core genes, which are conserved among all baculoviruses; blue – genes conserved among lepidopteran baculoviruses; gray – known genes, which are found in baculovirus; green - unique genes, which are only found in HytaNPV.

Figure 3

Molecular phylogenetic analysis by Maximum Likelihood method. The tree was constructed using 37 core genes from 81 baculovirus complete genomes (Supplementary Table S4). Bootstrap value resulted from 1000 replications is shown in each node. Red arrow - HytaNPV.

Figure 4

Comparison of HytaNPV against other known baculoviruses. Heatmap showing amino acid identity (%) resulting from blastp (evalue <= 1) of HytaNPV protein sequences against protein sequences of all complete baculovirus genomes. Rows – 141 HytaNPV genes arranged by 4 groups as shown in Fig. 2. Columns – 81 baculoviruses including HytaNPV in the order determined by phylogenetic analysis (Fig. 3). HytaNPV was indicated by red arrow.

Figure 5

Multiple genome alignment of HytaNPV, BusuNPV, SujuNPV and AcMNPV genomes using progressiveMauve algorithm with default parameters, where HytaNPV was used as the reference. Conserved genomic regions (locally collinear blocks - LCB) are shown as rectangle blocks with unique colors.

Figure 6

Repeat analysis of HytaNPV. Shown are 6 repeat regions identified in HytaNPV genome.

Figure 7

Multiple sequence alignment of Conserved Noncoding Functional Elements (CNEs). (a) CNEs identified in 53 alphabaculovirus genomes were aligned using ClustalW. Nucleotides that are identical at a given position across all CNEs are highlighted in light green while those that are identical in at least 70% of all CNEs are highlighted in light blue. (b) Consensus CNE sequence from multiple sequence alignment using Weblogo.

Figure 8

HytaNPV promoter motif analysis. (a) Number of predicted early and late (or both) genes in HytaNPV based on the presence of known core promoter motifs in sequences 200 bp immediately upstream of each annotated HytaNPV ORF. (b–d) Boxplots that show the distribution of distances (in bp) of key core promoter motifs from the start codon (‘ATG’) (b,d) or TATA box (b).