Nai et al. BMC Genomics 2010, 11:116 http://www.biomedcentral.com/1471-2164/11/116 electrophoresis in 1% agarose. LY cell line was cultured in TNM-FH medium [73] at 28°C. The medium con- tained 8% foetal bovine serum (FBS) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin, and 1.25 μg/ml fungizone. Nucleotide sequence determination The LyxyMNPV genome was sequenced to six-fold cov- erage by a shotgun approach. The viral DNA was sheared by hydrodynamic shearing forces into fragments with an average size of 2000 bp (HybroShear; GeneMa- chines). DNA fragments were size fractionated by gel electrophoresis and cloned into the EcoRV site of pBlue- script II SK (-) (Stratagene). The cloned plasmids were transformed into Escherichia coli BH10B (Invitrogen) and the recombinant bacterial colonies were grown on LB agar containing ampicillin, X-gal and IPTG. The DNA templates were prepared using the 96-well plasmid preparation method and the sequencing was performed by using KS/SK primer set (Stratagene) and ABI 3730 DNA analyser (Applied Biosystems), and the data was compiled into contigs using the PHRED/PHRAP soft- ware package [74,75]. The assembled sequences were then edited and completed using the Sun workstation interface [76]. DNA sequence analysis Open reading frames (ORFs) were identified using GeneWorks software (IntelliGenetics, Inc.) and ORF Finder http://www.ncbi.nlm.nih.gov/gorf/gorf.html[77]. The criterion for defining an ORF was a size of at least 150 nt (50 aa) with minimal overlap. Promoter motifs present upstream of the putative ORFs were screened. To screen the early promoter motifs, the conserved pat- tern is a TATA-box motif with a cap-site CAKT of 20- 30 bp located downstream within 180 bp of the initia- tion codon [28,78] and DTAAG within 120 bp of the initiation codon is a conserved motif of the late promo- ter motif [28,29]. In addition, the genome was checked in detail for the presence of any ORFs identified for AcMNPV [22], MaviMNPV [24], LdMNPV [14], SeMNPV [30] and CpGV [31] in GenBank. Homology searches were done through the National Centre for Biotechnology Information (NCBI) website using BLAST [79]. Multiple alignments and percentage identities of all LyxyMNPV ORFs with their homologues in selected genomes were generated using CLUSTAL_X [80]. The Tandem Repeats Finder http://tandem.bu.edu/trf/trf. html[81] was used to locate and analyse the homologous regions (hrs). GeneParityPlot analysis was performed on the LyxyMNPV genome versus the genomes of AcMNPV [22], MaviMNPV [24], LdMNPV [14], SeMNPV [30] and CpGV [31] as described previously Page 12 of 14 [82] and AcMNPV were renumbered manually, starting with the polh gene as number one. Phylogenetic analysis The phylogenetic tree was inferred from a data set of combined amino acid sequences of the 29 baculovirus core genes [22,6] of the 46 baculoviruses that were com- pletely sequenced at the time of analysis (Additional file 1). NJ and MP analyses were performed using MEGA, version 4.0 [83]. Culex nigripalpus NPV (CuniNPV) was selected as the out-group. Bootstrap analyses were per- formed to evaluate the robustness of the phylogenies using 1000 replicates for both NJ and MP analyses. Additional file 1: Characteristics of baculovirus genomes. This file lists the characteristics of baculovirus genomes, including virus name, genomic size, G+C content, coding regsion, number of ORFs, hrs and bro and GeneBank assession number. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 116-S1.DOC ] Additional file 2: ORFs predicted in the genome of LyxyMNPV. This file lists the ORFs predicted in the genome of LyxyMNPV. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 116-S2.DOC ] Additional file 3: LdMNPV and OpMNPV ORFs with no homologue in LyxyMNPV genome. This file lists the LdMNPV and OpMNPV ORFs with no homologue in LyxyMNPV genome. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 116-S3.DOC ] Additional file 4: The difference in the number of amino acids in the LyxyMNPV genome compared with the LdMNPV genome. Bars below zero show smaller ORFs in LyxyMNPV compared to LdMNPV. This file shows the difference in the number of amino acids in the LyxyMNPV genome compared with the LdMNPV genome. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 116-S4.TIFF ] Acknowledgements This work was supported by the Grant National Science Council, Republic of Taiwan and Grant NSC-96-2314-B-212-001 from National Science Council of Taiwan. Author details 1Department of Entomology, National Taiwan University, Taipei, Taiwan. 2Department of Zoology, National Taiwan University, Taipei, Taiwan. 3Department of Molecular Biotechnology, Dayeh University, No 112, Shanjiao Rd, Dacun, Changhua, Taiwan. 4Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Authors’ contributions YSN carried out the molecular cloning, analysed these sequences and drafted the manuscript. MFT, TCW and YRC purified the virus genomic DNA and analysed the sequences, CYW, WHL, CFL and CHW carried out the design and draft of the manuscript. All authors read and approved the final manuscript. Received: 11 August 2009 Accepted: 18 February 2010 Published: 18 February 2010