Genomic Characterization And Detection Of Atypical Porcine Pestivirus

Recently discovered Atypical porcine pestivirus?APPV?,one of the most important pathogens of congenital tremor?CT?in newborn piglets,causes serious harm to our swine herds.However,there is no detection method for APPV so far,which has seriously hampered our country's prevention and treatment for APPV.Therefore,in this study,we analyzed the genome-wide sequence of the GD3 strain APPV.Based on the results of sequence analysis,a real-time fluorescent quantitative RT-PCR assay was established.What's more,it was the first time that E2 recombinant protein antigen had been applied to APPV serum antibody detection,facilitating the establishment of an indirect ELISA assay.The following results were acquired:Sequence Analysis of GD3 Strain APPVThe APPV genome was amplified by RT-PCR using the total RNA of diseased piglets as template.The APPV genome of GD3 strain was obtained by splicing RT-PCR,and the sequence was submitted to GenBank?Accession No.:KY612413?.By using biological software,the sequence analysis of GDV strain APPV showed that the complete genome sequence of GD3 strain APPV was silimar to that of German,American,Dutch and Spanish strains with the homology ranging between 83.1%and 83.4%.Besides,the homologies of strains GD1,GD2 and GD were 99.5%,99.6%and 83.4%,respectively,and the homologies of E2,N^pro and E^rns encoded by GD3 strain to domestic and foreign strains were 83.1%-99.7%,80.2%-99.8%and 81.9%-99.5%,respectively.Establishment of real time RT-PCR detection methodAccording to the sequence of structural protein E2 gene encoded by APPV genome,specific primers were designedand quantitative RT-PCR detection was evaluated by sensitivity,specificity and reproducibility tests.The results showed that the fluorescence quantitative RT-PCR detection method established in this study is highly specific and has the lowest detection concentration of 10 copies/?L.It is simple and time-consuming and has good prospects for veterinary clinical application as well.Prokaryotic expression and antigenicity of APPV E2 proteinThe pMD18-T-APPV plasmid was used as template to design specific primers,amplify the gene fragments containing EcoR I and Xho I cleavage sites,and construct PGEX-6P-1/APPV-E2 prokaryotic expression plasmids.The successfully constructed expression plasmid was then transformed into the expressed bacterium BL21?DE3?.Induced by IPTG and analyzed by SDS-PAGE and Western-Blot,results showed that APPV E2 protein were successfully induced and expressed with good antigenicity.The establishment of indirect ELISA detection methodBy putting the purified E2 protein into the reaction plate at 10 g/mL per hole with the concentration of 100 g/mL for 4 hours,the ELISA detection method could then be evaluated by sensitivity,specificity and reproducibility tests.The results showed that the method can detect antibodies in pig sera of swine infected with atypical swine disease quickly,specifically and sensitively.And its sensitivity to detect serum antibodies is1:1000.In summary,the complete gene sequence of GD3 Strain APPV was successfully obtained and submitted to GenBank?Accession No.KY612413?,and the results of sequence analysis provided basic data for revealing the pandemic situation of APPV in China.Moreover,the real-time RT-PCR detection method based on E2 gene and the indirect ELISA detection method based on E2 protein serum antibody were established for the first time,providing detection method for comprehensive prevention and control of congenital tremor?CT?in newborn piglets.