Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva

BMC Genomics Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva Shan-Ying Zhu 2 Jian-Ping Yi 1 Wei-De Shen 0 Li-Qun Wang 2 Hua-Gang He 3 Yong Wang 3 Bing Li 0 Wen-Bing Wang 2 0 School of Life Sciences, Soochow University , Suzhou 215123 , PR China 1 Shanghai Entry-Exit Inspection and Quarantine Bureau , Shanghai 200135 , PR China 2 Institute of Life Sciences, Jiangsu University , Zhenjiang 212013 , PR China 3 School of Food and Biological Engineering, Jiangsu University , Zhenjiang 212013 , PR China Background: Baculoviruses are well known for their potential as biological agents for controlling agricultural and forest pests. They are also widely used as expression vectors in molecular cloning studies. The genome sequences of 48 baculoviruses are currently available in NCBI databases. As the number of sequenced viral genomes increases, it is important for the authors to present sufficiently detailed analyses and annotations to advance understanding of them. In this study, the complete genome of Clanis bilineata nucleopolyhedrovirus (ClbiNPV) has been sequenced and analyzed in order to understand this virus better. Results: The genome of ClbiNPV contains 135,454 base pairs (bp) with a G+C content of 37%, and 139 putative open reading frames (ORFs) of at least 150 nucleotides. One hundred and twentysix of these ORFs have homologues with other baculovirus genes while the other 13 are unique to ClbiNPV. The 30 baculovirus core genes are all present in ClbiNPV. Phylogenetic analysis based on the combined pif-2 and lef-8 sequences places ClbiNPV in the Group II Alphabaculoviruses. This result is consistent with the absence of gp64 from the ClbiNPV genome and the presence instead of a fusion protein gene, characteristic of Group II. Blast searches revealed that ClbiNPV encodes a photolyase-like gene sequence, which has a 1-bp deletion when compared with photolyases of other baculoviruses. This deletion disrupts the sequence into two small photolyase ORFs, designated Clbiphr-1 and Clbiphr-2, which correspond to the CPD-DNA photolyase and FADbinding domains of photolyases, respectively. Conclusion: ClbiNPV belongs to the Group II Alphabaculoviruses and is most closely related to OrleNPV, LdMNPV, TnSNPV, EcobNPV and ChchNPV. It contains a variant DNA photolyase gene, which only exists in ChchNPV, TnSNPV and SpltGV among the baculoviruses. - Background Baculoviruses are a large group of rod-shaped, enveloped viruses with circular, covalently closed, double-stranded DNA genomes. These viruses are pathogenic to arthropods, mainly insects within the orders Lepidoptera, Diptera and Hymenoptera [1,2]. According to morphology of the virus occlusion bodies (OBs), the family Baculoviridae comprises two genera: the Nucleopolyhedroviruses (NPVs) and Granuloviruses (GVs). The lepidopteron NPVs can be further divided into two sub-groups on the basis of their envelope fusion proteins, which are essential for the spread of infection in the insect and are required for efficient virus budding. Group I NPVs possess proteins related to GP64, whereas no GP64 homologues have been identified in Group II NPVs [3,4]. Instead, members of Group II encode homologues of LD130 proteins, also known as Fusion (F) proteins [5]. The taxonomy of the Baculoviridae genera has recently been changed on the basis of the hosts. There are now four genera: the Alphabaculoviruses (lepidopteron-specific NPV), Betabaculoviruses (lepidopteron-specific GV), Gammabaculoviruses (hymenopteron-specific NPV), and Deltabaculoviruses (dipteron-specific baculovirus) [6]. In recent years, much research has focused on baculoviruses owing to their potential as agents for biological control of pests in agriculture and forestry [7]. Furthermore, they can be used as efficient expression vectors of foreign genes [8,9]. Forty-eight completely-sequenced baculovirus genomes, including 34 Alphabaculoviruses, 10 Betabaculoviruses, 3 Gammabaculoviruses and 1 Deltabaculovirus (see Additional file 1), with sizes ranging from 81,755 base pairs (bp) in Neodiprion lecontei NPV (Nele NPV) [10] to 178,733 bp in Xestia c-nigrum GV (XecnGV) [11], have been made available in GenBank since the Autographa californica NPV (AcMNPV) genome sequence was reported [12]. Clanis bilineata (Walker), belonging to Lepidoptera Sphingidae, is a major agricultural pest causing considerable damage to soybean production in China. No baculovirus able to infect C. bilineata larvae was reported until 2006 [13], when a novel baculovirus named Clanis bilineata nucleopolyhedrovirus (ClbiNPV) was isolated and purified from the larvae of the sphingid C. bilineata infected with NPV. Transmission electron micrographs showed that this virus occludes single-enveloped nucleocapsids and hence is an SNPV [14]. The ClbiNPV genome comprises 135,454 bp and codes for 139 putative open reading frames (ORFs) with a minimum size of 150 nucleotides. In this report, we present the complete sequence and organization of the ClbiNPV genome and compare them to other baculoviruses through genomic and phylogenetic analyses. Results and discussion Nucleotide sequence analysis of the ClbiNPV genome The genome of ClbiNPV has a size of 135,454 bp [GenBank: DQ504428], slightly smaller than that of Spodoptera exigua NPV (SeMNPV, 135,611 bp) [15]. ClbiNPV has a highly AT rich genome. Its overall G+C content is 37%, similar to that recorded for Agrotis segetum GV (AgseGV) and Ecotropis obliqua NPV (EcobNPV) [16], and higher only than those of Adoxophyes honmai NPV (AdhoNPV, 35%) [17] and Adoxophyes orana NPV (AdorNPV, 34%) among the Alphabaculovirus (see Additional file 1). According to convention [18], the adenine residue at the translational ATG start codon of the polyhedrin gene (polh) was considered to be nucleotide number 1 of the genome, and successive nucleotides were numbered in the direction of the polh gene (see Additional file 2). Analysis of the ClbiNPV genome sequence led to the identification of 139 putative ORFs with 50 or more amino acids and minimal overlapping of adjacent ORFs. There are 60 ORFs with the same orientation as the polyhedrin gene, and 79 with the reverse orientation. Within 150 bp upstream of the ATG start codon, 34 ClbiNPV ORFs have baculovirus early promoter motifs (CAGT), 51 have late promoter motifs (TAAG), and 29 carry both these motifs. Of the 139 ClbiNPV ORFs identified, 126 are homologous to at least one other baculovirus, and the 30 core genes that are probably shared by all baculoviruses are conserved in the ClbiNPV genome [19]. Thirteen ORFs (Clbi5, Clbi6, Clbi18, Clbi31, Clbi35, Clbi42, Clbi47, Clbi49, Clbi56, Clbi57, Clbi70, Clbi75 and Clbi129) are unique to ClbiNPV; they account for 9% of the whole genome. Three baculovirus-repeated ORFs (bro genes) were identified in ClbiNPV (ORF55, 115 and 131) and were designated bro-a, bro-b and bro-c, respectively, based on their order in the geno (...truncated)