Studies On Hyphantria Cunea Nucleopolyhedrovirus (HcNPV) And Determining Process For Infested Hosts

Studies on the Hyphantria cunea ucleopolyhedrovirus (HcNPV) were conducted forseveral aspects: Morphological characteristics of HcNPV; pathological process of theHyphantria cunea larvae infected by HcNPV; susceptibility of the H. cunea larvae at differentinstar to HcNPV. The main results were summarized as follows:1. Electron microscopic observations showed that polyhedron diameters were about 2～3μm and envelop size were about 0.4～0.6μm×0.57～1.11μm.2. We demonstrated that second-instar Hyphantria cunea larvae were 14 times moresusceptible than third-instar larve and 105 times more susceptible than fourth-instar larvae,3500 times than fifth-instar larvae. We also determined on a weight basis (miligrams) thatsecond-instar larvae were 3.3 times, 4.5 times, and 71 times more susceptible than third-,fourth- and fifth-instar larvae. Initally lethal time prolonged along with the reduced dosage oflarvae fed HcNPV on the same stadium and the increased stadium of larvae fed HcNPV on thesame dosage. Among all assary larvae fed 5.3×10⁶ PIBs/mL (10uL), LT₅₀ values of third-instarlarvae were 5.4 days and fourth-instar larvae were 6.2 days more than that of second-instarlarvae, respectively.3. Pathological variety occured in cells of different tissue after larvae fed on the HcNPV."Ring zone" appeared in midgut cells 48h after treatment. Three or four -stratum structure inepidermal cells under integument 168h after treatment was also observed. It is the particularsymptom of Hyphantria cunea infected HcNPV. Most tissues disorganized when larvae cometo died 216h after treatment. The mature polyhedrons were full of body cavity at the same time.4. According to the polyhedron gene sequences of Hyphantria cunea NPV available inGeneBank, a pair of primers was designed for establishing SYBR Greenâ… quantitativereal—time fluorescence quantitative PCR method. Standard curve was established basing on Ctvs initial input amount of Log(copy number): y=-3.450x+36.154, and the correlationcoefficient was 0.998. The sensitivity arrived at 10¹-10²/uL. The results demonstrated thatmethod developed in this study can quickly detect polyhedron gene in broad range with highefficiency, and high specificity. The experiment provided the foundment for polyhedrin gene ofHcNPV as a control gene in quantitative analysis of early infection.5. To investigate the proliferation of Hyphantria cunea nucleopolyhedrovirus (HcNPV) in its host, the third- nstar larvae were orally administered with determinate dose of HcNPV. Afteroralingestion, midgut, haemolymph tissues and larvae were collected at different time toextract DNA. Fluorescencequantitative PCR amplification of HcNPV Polyhedrin gene wasused to detect copy number of HcNPV. The results showed that Copies/milligram in midgut12～108h after treatment fluctuated in 10⁴-10⁵/μL. Copies in haemolymph 3-108h aftertreatment showed increaed trend but beyond 10⁴ copies/μL. Log (Copies/milligram) in larvaewere linearly related to hours after treatment. Regression equation: y=3.901x±1.094(r=0.966).6. Indirect ELISA detection for hyphantriacunea virus was developed through usingantiserum prepared by purified hyphantriacunea virus particles as antibody. Results showedthat protein content of hyphantriacunea virus immunogen and its rabbit-anti hyperimmuneserum was 17.17mg/mL and 9.728mg/mL, respectively. The optimal determinationconcentration of rabbit-anti hyphantriacunea virus hyperimmune serum was 1:320, namely itsprotein content was 53.65μg/mL. The optimal dilution of hyphantriacunea virus solution was1:100, namely its content was PIBs 1:10⁷. The optimal incubation conditions were as following:0.5% BSA+2% sucrose was incubated in water bath at 37℃for 2h and then maintained for 24hunder 4℃. Under above conditions, the minimum detection limit of hyphantriacunea virusantigen protein was 0.304μg/mL.7. Multiplication pattern of Polyhedrin(HcNPV) in larvae were studied using indirectELISA method. A linear relation was found between log (concentration value) andabsorbance when the concentration range was at 3.12×10⁵～1.0×10⁷ PIBs/mL. Themultiplication of HcNPV entered a stationary phase two days before died.8. According to status of pest management to hyphantria cunea at present in China,several pesticides (including HcNPV) were selected for experiment by imitating field spraymethod in order to determine security to the important natural enemy of Hyphantria cuneDuruy—Chouioia cunea Yang. Effects on the productivity of Chouioia cunea exposured toHcNPV were also studied. Results indicated that bionic pesticide, biopesticide are safe whilechemical pesticide is more toxic to Chouioia cunea adults. HcNPV is not harmful for theproductivity of Chouioia cunea. The findings also suggest that the technology of utilizingHcNPV in larval stages combined with Chouioia cunea in pupal stage for controllinghyphantria cunea is practicable.