Nai et al. BMC Genomics 2010, 11:116 http://www.biomedcentral.com/1471-2164/11/116 (NCBI, GenBank). Baculoviruses are used worldwide as protein expression vectors, biotechnological tools and biological control agents of agricultural and forest pests. The casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), is an herbivore that feeds on casuarina (Casuarina equisetifolia), guava (Psidium guajava L.), longan (Euphoria longana Lam.), lychee (Litchi chinensis Sonn.), acacia (Acacia confusa) forests and more than 60 other species of host plants [9,10]. It is native to Taiwan, Japan, India, and the eastern coast of mainland China [11]. In the last 30 years in Taiwan, many forests have been converted to agricultural land. The moth has expanded into these newly established agricultural areas and simultaneously expanded its host plant range [12]. L. dispar is a closely related species to L. xylina [13]. The L. dispar MNPV (LdMNPV) isolated from its natural host, L. dispar, had been characterised and sequenced in 1999 [14] and used in integrated pest management (I.P.M.) programs to control this pest for many years in America [15]. NPV epizootics occur in populations of L. xylina each year from spring to early summer in Taiwan and main- land China, and the key pathogen was found to be L. xylina multiple nucleopolyhedrovirus (LyxyMNPV) [16]. Following the establishment of an in vitro propagation system for LyxyMNPV in the cell lines IPLB-LD652Y (LD) and NTU-LY (LY) [17,18], LyxyMNPV and host- virus interaction studies were promoted. In previous tis- sue culture infectivity studies of three lymantriid NPVs (LyxyMNPV, LdMNPV, and Orgyia pseudotsugata MNPV), only one cell line, IPLB-LD-652Y, was able to support infection and replication of all three NPVs [17,19,20]. Additionally, Perina nuda NPV (PenuNPV), which was isolated from P. nuda, could also infect both LY and LD cells and L. xylina larvae in our laboratory [18]. Of these Lymantriidae-derived NPV species, Lyx- yMNPV/LdMNPV and PenuNPV/OpMNPV were thought to be closely related [17,21]. However, molecu- lar evidence has supported them as distinct species [12,16]. Therefore, the precise relationship between Lyx- yMNPV and LdMNPV needs to be further clarified. In our previous study, LyxyMNPV and another less preva- lent NPV species were shown to coexsist in the infected larvae in the fields of Taiwan [12]. The less prevalent NPV was isolated and characterised as LdMNPV-like virus [12]. In this study, we reported the complete genomic sequence of LyxyMNPV, which is the most prevalent virus strain in the infected L. xylina larvae and described the whole genomic sequence, gene structure and per- formed a phylogenetic analysis. The genome sequences were compared to the previously published LdMNPV [14] and group I NPV type species, Autographa califor- nica MNPV [22]. Because OpMNPV [23] is another Page 2 of 14 Lymantriidae-derived NPV species that can infect LD cells and Maruca vitrata MNPV (MaviMNPV) [24] can also infect LD cells at low level, these two group I NPVs were also compared to LyxyMNPV. Results and Discussion General characteristics of the LyxyMNPV genome The LyxyMNPV genome size is 156, 344 bp [GenBank: GQ202541] and has a G+C content of 53.4% (see Addi- tional file 1). ORFs were predicted according to the initial criteria for further study. However, three ORFs that had large overlap found in LyxyMNPV were also selected for further study, namely Lyxy37, Lyxy72 and Lyxy139 (arif-1). Lyxy37 (281 aa) overlaps with Lyxy36 (p26) by 192 aa in the same direction, but not in the same frame. A P10 homologue (77 aa) was found located in the C-terminal portion of Lyxy37 (from 205 aa to 281 aa), designated as Lyxy37’. Lyxy72 (homolo- gous to Ld76) is 380 aa long and overlaps with Lyxy71 (bro-g) and Lyxy73 by 182 aa and 119 aa, respectively. Lyxy139 (arif-1), which is 272 aa long, overlaps with Lyxy138 (pif-2) and Lyxy140 by 182 aa and 73 aa, respectively. Therefore, a total of 157 ORFs were identi- fied for further analysis (Fig. 1, Additional file 2), and nucleotides in the LyxyMNPV genome were numbered sequentially, beginning with the A (designated position 1) of the polyhedrin start codon (ATG). The directions of the transcripts are indicated by arrows. The ratio of the ORF orientations was almost equal to 1:1 [clockwise (79/157): anticlockwise (78/157)] for those oriented clockwise with respect to the orientation of the polh gene [25] (ORF1). Most of the 157 LyxyMNPV ORFs have an assigned function or homologues in other bacu- loviruses. As shown in Table 1, four unique ORFs were found in the LyxyMNPV genome. There are 30 con- served genes in all baculovirus genomes, including the dipteran and hymenopteran baculoviruses [6,26,27] and all of these genes were found in the LyxyMNPV gen- ome. A total of 14 baculovirus-repeated ORFs (bro genes, ly-bro-a to ly-bro-n) were also sequenced. Beside these 157 predicted ORFs, other internal spaces were made up of intergenic spaces and homologous regions (hrs). The LyxyMNPV genome has 13 hrs (hr1, 2, 3a, 3b, 3c, 4, 5, 6, 7, 8a, 8b, 8c and hr9) (Fig. 1 and Addi- tional file 2), and the orientations of the hrs were related to those of LdMNPV (Additional file 2). Promoter analysis Promoter motifs present upstream of the 157 putative ORFs were screened. 14 LyxyMNPV ORFs were shown to possess consensus early gene promoter motif (Addi- tional file 2). Three of these ORFs, Lyxy7, Lyxy10 (odv- e56) and Lyxy126 (efp/ld130), were also shown to pos- sess consensus late gene promoter motif, which may