Establishment Of Real-time Fluorescence Quantitative RT-PCR Detection Method For DWV And Evaluation For Egg Yolk Antibody Against Virus

Deformed wing virus(DWV)could infect the different developmental stages of honeybee,which led to deformed and crippled wings,discoloration and paralysis and body color abnormality of honeybee,so it was regard as one of the most pathogenic honey bee viruses.DWV had not only directly caused the loss of bee colonies and decrease of wild pollinating insects in the worldwide,but also indirectly bring about huge economic losses to agricultural production.In this study,we have investigated the rapid diagnosis method of DWV and developed the biologics drug against DWV,which is the basis of prevention and treatment of this disease scientifily.In order to diagnose DWV rapidly and accurately,the conservative 3C-Rd RP gene of DWV was selected as a target gene to design the pair of specific primers,and the real-time fluorescence q RT-PCR detection method for DWV was established based on SYBR Green I technology.Then,the reaction system have optimized,and the sensitivity test,the specificity test and the repeatability tests were conducted.The results show that the optimal primer concentration and annealing temperature of this detection method was 500 nmol/L and 59°C,respectively,and the minimum copy number of detectable standard plasmid is 10 copies,and this method could effectively distinguish CSBV,IAPV,CBPV and DWV.Furthermore,36 clinical samples from different regions of Liaoning,Anhui and Jiangsu province were detected by the real-time fluorescence q RT-PCR and traditional RT-PCR detectio,and the coincidence rate with traditional RT-PCR detection methods reached to 91.6%by the real-time q RT-PCR,so this method could be produced for the rapid diagnosis and molecular epidemiological investigation of DWV.For evaluating the immunogenicity to the three structural protein of DWV,the structural protein genes VP1,VP2 and VP3 of the DWV-LN strain(MF770715)were obtained by RT-PCR,then the amino acid sequences were derived by nucleotide sequences,and the result show that the protein of VP1,VP2 and VP3 were composed of 416,225 and 246 aa,respectively.Compared with the sequences of the reference strains,the homology of the three structural proteins was96.9%-99.3%,96.5%-98.1%and 95.9%-99.2%by the analysis of DNAStar,respectively.Then,based on E.coli-preferred codons without changing the amino acid sequence of the corresponding proteins,the expression plasmids p ET28-VP1-OPTI,p ET28-VP2-OPTI and p ET28-VP3-OPTI were constructed after codon optimization,then transformed into BL21(DE3)to induce expression,and the analysis of SDS-PAGE and Western blot show that the target protein(r VP1:50.5 k Da;r VP2:29.1 k Da;r VP3:32.6 k Da)was obtain accord with the theoretical value.Moreover,the recombinant proteins r VP1,r VP2 and r VP3 were immunized to BALB/c mice three times,and the level of Ig G were detected with ELISA after immunization,and anti-ro VP-antisera(r VP1 or r VP2or r VP3)from the immunized mice was incubated with DWV for injecting healthy white-eyed pupae for the viral challenge test.The result indicated that Ig G level of the r VP3 group was highest in the three groups,and anti-ro VP3-antisera was strongest against DWV,respectively,which showed that r VP3 protein could stimulate the immuned mice to produce the humoral immunity and possessed the excellent immunogenicity.In order to further study IgY prepared by r VP3 to neutralize DWV,Hylan brown laying chicken were injected with different doses by leg and pectoral muscles,with 4 times,once every 2weeks.At 8 weeks after immunization,Ig G level in the serum and IgY titer against DWV in the eggs was detected by ELISA method.The results showed changes law of IgY were basically consistent with Ig G.To verify the ability of anti-r VP3 IgY to neutralize viruses,the healthy bee pupae were injected with mixture after 2~1-2~5 titer IgYand 1×10~4copies/?L DWV had been incubated together.Then,DWV virus copy number was relatively quantified by the real-time q RT-PCR after 72 h incubation.The results indicated that IgY with a titer of 2~5 possessed excellent ability to neutralize the virus.The treatment test against DWV was carried out using anti-r VP3 IgY.The larvae of honey bee were orally fed on the different titer of anti-DWV IgY 24 h after and before inoculation with DWV.Then,DWV virus copies was quantified by q RT-PCR after72 h.The results showed that IgY titer?2~3was effective against DWV,and 2~5 IgY titer had the best effect of inhibiting virus replication.With 2~5IgY as the clinical therapeutic dose,20 honeybee colonies infected DWV in two bee farms from two districts in Liaoning were selected to carry out clinical treatment trials,and IgY was fed on 3 times at 1d intervals.After 12 days of treatment,the virus copy number was detected to evaluate the clinical treatment effect.The clinical treatment indicated that after feeding the IgY with a titer of 25 for the DWV trial colony 3 times,the virus copies were significantly reduced with feeding IgY after 12 days of treatment.In summary,we have established a real-time fluorescence quantitative RT-PCR detection method of DWV and developed the antiviral IgY to inhibit virus replication,which have provided an important material basis for the rapid diagnosis developing the therapeutic drug against DWV.