Identity And Genomic Sequence Analysis Of Dendrolimus Kikuchii Nucleopolyhedrovirus

Dendrolimus kikuchii (Matsumura)(Lepidoptera: Lasiocampidae), is a serious forestpest for a variety of conifers. In China, D. kikuchii mainly occurs in the south part andchemical pesticides were primely used as the first control method. However, chemicalinsecticides may poison the non-target organisms (e.g., humans, livestock and natural enemy),pollute environment, and induce insecticide-resistant pests. Baculoviruses are ideal tools inintegrated pest management because they are usually highly specific to their host insects anddo not affect other arthropods, including pest predators and parasitoids. They also pose nohazard to the environment, vertebrates, plants, and natural enemies. Over50baculovirusproducts have been used to control agricultural and forestry pests worldwide. Therefore, thescreening of high toxicity and strong stability virus is an imperative work for the biologicalcontrol of D. kikuchii.In present study, a nucleopolyhedrovirus was isolated from the infected larvae of D.kikuchii, which is a serious pest for a variety of conifers in China. The main results are asfollows:1. Morphological characteristics and virulence of D. kikuchii nucleopolyhedrovirus(DekiNPV)A new strain of nucleopolyhedrovirus has been isolated from infected larvae of the D.kikuchii (Matsumura)(Lepidoptera: Lasiocampidae) in China and named as DekiNPV. Thescanning electron micrograph results showed that OBs of D. kikuchii are mostly polyhedralshape,0.79~2.31μm in diameter (n=100), average diameter is (1.64±0.1) μm, with a lots ofsmall holes on their surface,(173.00~254.00)×(55.10~116.00) nm in size. Ultrathin sectionsrevealed that each OB contains many virions,(252.22~359.38)×(70.18~200.00) nm in size.The virion is rod-shaped with the truncated or obtuse ends, consists of multiple nucleocapsids(up to9) within a single viral envelope. The size of nucleocapsid is(24.00~28.60)×(242.00~340.00) nm in size, and average25.80±0.86nm (mean±SE) inwidth and265.00±12.66nm in length (n=50). The results of infection study showed thatDekiNPV could infect and kill the third-larvae of D. kikuchii. LC50was1.72×105PIB/mL. At the tested five concentrations (from2.2×104to2.2×108PIB/mL), LT50were6.89,7.18,8.16,10.31and11.13days, respectively. A large number of D. kikuchii larvae begin to die from theseventh day of infection, and peak mortality from the senventh to tenth days. Our researchdemonstrates that the virus has been propagated4generations in the fourth-instars larvae of D.kikuchii has stable ultrastructural morphological.2. Genomic sequencing and analysis of DekiNPVThe complete genome of DekiNPV from an important pest of pine, D. kikuchiiMatsumura (Lepidoptera: Lasiocampidae), was sequenced and analysied. The DekiNPVgenome was141,454bp in size with C+G content of48.04%. It is predicted to contain146open reading frames (ORFs).133DekiNPV ORFs are homologues of previously reportedbaculovirus genes, and13ORFs are unique to DekiNPV, accounting for12.4%of the genomein total. A phylogenetic tree was constructed based on29core baculovirus genes. TheDekiNPV grouped into the lepidopteran-specific NPV (Group Ⅰ), and had a closerelationship to MaviMNPV, BomaNPV, BmNPV, PlxyMNPV, RoMNPV and AcMNPV. Thisanalysis also suggested that the DekiNPV is more primitive than those6NPVs of the sameclade. The DekiNPV genome contains16conserved structural genes，and absent P6.9andodv-e56. The DekiNPV genome contains apoptosis inhibiting gene, such as iap-1，iap-2andiap-3. The result of the identity-GeneParity analysis of SeMNPV and DekiNPV, LdMNPVand DekiNPV, MacoNPV-B and DekiNPV, ChchNPV and DekiNPV, AcMNPV andDekiNPV, BmNPV and DekiNPV, and XecnGV and DekiNPV, which compares all both theoverall gene arrangement and the homology between genes. It was show that AcMNPV andBmNPV appear to be the most collinear with DekiNPV, but they most genes are inverted withthe genes of DekiNPV. There are11hrs in DekiNPV genome.3. Polymerase chain reaction assay for the detection of D. kikuchii nucleopolyhedrovirusA polymerase chain reaction (PCR) assay was developed with a pair of primers, designedbased on the unique open reading frame (ORF)146of DekiNPV. DekiNPV DNA served astemplate to amplify426bp and655bp segments. Results from the sequenced PCR productswere consistent with the size of selected primer segments. Two primers were applied to assayDekiNPV DNA and detect intact DekiNPV occlusion bodies (OBs), showing a minimumdetection of0.8fg/mL DNA and5OBs/mL, respectively, indicating the existence ofDekiNPV in the infected insects. The assay is characterized with high sensitivity, remarkablespecificity and early assay, demonstrating its potential for applications in the studies ofDekiNPV host range, substitution of DekiNPV hosts, dynamic DekiNPV changes in D.kikuchii population and routine molecular diagnostic procedures.4. Host range and substitution of DekiNPV We examined the infectivity and replication of DekiNPV in11other lepidopteran species,including Dendrolimus houi, Dendrolimus punctatus, Dendrolimus punctatus wenshanensis,Dendrolimus spectabilis, Hyphantria cunea, Lymantria dispar, Ectropis grisescens,Helicoverpa armigera, Spodoptera exigua, Plutella xylostella, Spodoptera litura larvae, sf-9cell, and Clostera anachoreta cell (CAF-clan II) to define the host range of DekiNPV andidentify its suitable alternate hosts. Our study showed that DekiNPV is highly specific to D.kikuchii, but triggers covert infections in H. cunea, D. houi, D. punctatus, D. punctatuswenshanensis, and D. spectabilis larvae into overt symptoms, indicating that DekiNPV issuitable as an ideal biocontrol agent for D. kikuchii, D. houi, D. punctatus, D. punctatuswenshanensis, D. spectabilis, and H. cunea.