Genomic Anlyses of LyxyMNPV and the Characterization of an LdMNPV-like Virus

The casuarina moth, Lymantria xylina Swinhoe (Lepidoptera: Lymantriidae), is an important forest pest in Taiwan. Two nucleopolyhedrovirus (NPV) strains were isolated from L. xylina larvae with an epizootic nucleopolyhedrosis. In the field, LyxyMNPV is the most prevalent vius strain, therefore, the nucleotide sequence was determined and analysed. The genome of LyxyMNPV consists of 156,344 bases with a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The LyxyMNPV genome encoded 14 bro genes. 13 homologous regions (hrs) were identified. In a phylogenetic analysis, LyxyMNPV is a member of Group II NPV and closely related to LdMNPV but with highly distinct genomic organization. The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified homologous to those reported in the LdMNPV genome. Two genes were homologous to other baculoviruses and four unique ORFs were identified in LyxyMNPV genome, including a gag-like gene which was not reported in baculoviruses. Two putative odv-e27 homologues were identified in LyxyMNPV, each of which has been acquired independently and not by gene duplication. LyxyMNPV lacks host range factor-1 (hrf-1). However, in vitro host range assay indicated that LyxyMNPV could infect both NTU-LY and IPLB-LD-652Y cell lines with the absence of hrf-1. Therefore, we compared LyxyMNPV gene content to those of LdMNPV and OpMNPV, and found that these three NPVs shared three genes, iap-3, rr2b, and dutpase, that are absent in AcMNPV genome, of these three genes, iap-3 is one of the baculovirus genes that affect viral host range and prevent apoptosis in baculovirus-infected cells.
Two groups of anti-apoptotic genes, p35 and iap (inhibitor of apoptosis) family, have been identified in baculovirus. There are two iap genes, iap-2 (687 bp) and iap-3 (450 bp), in LyxyMNPV genome. The 5’UTR of iap-2 contains a transcriptional initiation site, TAAG, of late and very late gene motif, but 5’UTR of iap-3 contains CTTGT promoter motif plus TCGT in 25 bp spacing context. The mRNA expression profiles of these two genes in permissive cell line LD were evaluated using RT-PCR. The expression levels of iap-2 and iap-3 in the LD cells infected with LyxyMNPV raised from 3 to 72 hours post-infection (p.i.), but declined after 72 to 120 hours. Interestingly, the expression level of iap-2 and iap-3 of the infected SF cells showed significant difference. The mRNA of iap-2 was not detected; in contrary, iap-3 was detectable. During the process of infecting SF cells with LyxyMNPV, iap-3 presented a delayed expression pattern, which was detected at 48 hours p.i. and continued to rise for 120 hours. Functional assay of two iap genes was performed using over-expression method. In SF cells, full length IAP-2, IAP-3 and IAP-2-BIR domain inhibited the apoptosis induced by Drosophila RPR protein (P<0.05), however, IAP-3-BIR domain could not rescue the apoptosis induced by D-RPR.
LdMNPV-like virus was nominaded base on the sequences analyses of polyhedrin, lef-8 and lef-9. This virus was closely related to LdMNPV but far from LyxyMNPV. LdMNPV-like virus showed a few polyhedra (occlusion bodies) CPE in the infected LD cells. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. This is the first FP virus isolated from the field-collected larvae infected with NPV.