Isolation,Identification And Immunogenicity Of Duck Tembusu Virus

Duck tembusu virus has been widespread in China Since it outbroke in 2010,causing a great number of economic loss to the aquaculture industry.In the situation,there is an urgent need of a safe and effective vaccine to control and prevent this epidemic.So this study conducted some preliminary explorations: 1.Isolation and Identification of Duck Tembusu VirusIn this study,virus isolation and identification from the ducks with symptoms of egg production decline in Guangxi were conducted.The ovary,spleen and liver were homogenized,sterilized by filtration,and then inoculated into BHK-21 cells.After 3 passages,CPE was produced on the cells.The isolated strains were purified using a double-layer plaque method and purified virus was obtained by 3 consecutive plaque assays.The virus had no hemagglutination and was identified as DTMUV by RT-PCR,and designated as GX-15.The virus can be cultured in BHK-21 cells with virus titer up to 107.0 TCID50 / 0.1 m L.The virus can adapt to duck embryonated passaging,and the virus titer rises slightly with increasing number of passages.The E gene of the isolate was sequenced and analyzed,the homology with the isolate MM1775 in Malaysia was 89%,and the homology with the domestic isolate HZ-2014 was 96.9%;the homology with other members of the flavivirus genus was 53.7%-75.2%.The phylogenetic analysis of the isolates was found to be in a separate branch with the domestic isolate HZ-2014,both of which are in the same branch as the Malaysian strain MM1775.2.Establishment of indirect ELISAAn indirect ELISA method for detecting DTMUV antibodies was established using serum-free cultured GX-15 isolate as coating antigens.After optimization of the conditions,it was determined that the amount of antigen coated was 5×103.8 TCID50/hole,the serum dilution was 1:200,the dilution ratio of goat anti-duck secondary antibody was 1:4000,the secondary antibody reaction time was 45 min,the color development time was 10 min,and the critical value of negative or positive was 0.463.This method was used to detect positive serum such as NDV,AIV(H9),DHAV-1,DPV,MDRV,and DRV.And the results were below the critical value and showed that this method has good specificity.The sensitivity was 1:1600;the coefficient of variation of the intra-batch and inter-batch repeatability test was less than 10%,indicating that the method is very stable.3.Establishment of infection model of Cherry Valley DuckThe infection model was established with 56 Cherry Valley Ducks who were 70-days-old infected without Tembusu virus.The ducks were randomly divided into 7 groups,each group has 8 ducks,which were infected with different doses of GX-15 isolate,and the control group was injected with the same dose of PBS.Only the 106.0TCID50 infected group and the 105.0TCID50 infected group showed decreased intake and dark green loose stools to varying degrees after the infection.Pathological necropsy and histopathological observations all showed lesions in the spleen.The lesion gradually reduced until disappeared with the decrease in the dose of infection.The isolation rate of virus in serum of 106.0TCID50?105.0TCID50?104.0TCID50?103.5TCID50 infected groups both were 100% on the second day after the infection,and 87.5% and 37.5% in groups of 102.5TCID50 and 101.5TCID50,respectively.On the 4th day after the infection,the virus isolation rate was 25%,37.5%,37.5%,25%,and 12.5% for each group;no virus was detected in each group on the 6th and 8th day after the infection.The final infect dose for the 70-days-old Cherry Valley Duck infection model was 103.5 TCID50.4.Immunogenicity study of GX-15 IsolateIn this study,the inactivation condition of virus cultured with cells and duck embryos was studied respectively.The results showed that the inactivation condition of the cell-derived virus was as follows: the final concentration of formaldehyde was 0.1%,and the inactivation was performed at 37 °C for 20 h.The inactivated conditions of the duck embryo virus was: the final concentration of formaldehyde was 0.1%,andd the inactivation was performed at 37 °C for 24 h.The GX-15 isolate was used to prepare cell-derived virus inactivated vaccine and duck embryo virus inactivated vaccine,respectively,and their immunogenicity was evaluated.After immunizing with Cherry Valley Duck,which was negative for both nucleic acid and antibody,the vaccine was boosted on the 14 th day after the first immunization,and the infection was evaluated on the 28 th day after the second immunization.The results showed that both of the cell-derived virus inactivated vaccine group and duck embryo virus inactivated vaccine group produced high levels of antibodies,which tended to be stable on the 14 th day after the second exemption,and the antibodies reached the highest values on the 21 st day after the second exemption.The protection rate of the two vaccine groups was not less than 70%,and the GX-15 isolates proved to have good immunogenicity.This study laid the foundation for the development of the DTMUV vaccine.