Construction Of Recombinant Adenoviral Vector Vaccine Expressing Atypical Porcine Pestivirus E2 Protein

Atypical（APPV）is a novel pestivirus that was first reported in the United States in 2015.Subsequently,some researchers confirmed that APPV is the causative agent of type A-II congenital tremor（CT）.So far,there have been many cases of APPV infection in commercial swine herds in more than ten countries around the world.The APPV strains isolated from different regions have great variability.At present,there are several cases of CT-affected pigs caused by APPV infection in China,which have caused considerable economic loss to the swine farms.As a novel pestivirus,the related research of APPV is still in the initial stage,and comprehensive epidemiological investigation and prophylactic measures are of great significance.In this study,116 samples of diseased piglets was collected from 12 pig farms in Guangdong,Guangxi,Anhui,Guizhou,Jiangsu and Hunan provinces from 2017 to 2018.The PCR analysis showed that 84.5%（98/116）of the tissue samples were identified as APPV-positive,in which all piglets with congenital tremors were APPV-positive,and one of the two piglets with spread leg symptoms（1/2）was APPV-positive.The occurrence of CT in affected farms was repeated in the season or a certain period of time.The cases are almost all piglets of the primiparous sows.The rate of affected nests（more than one piglets in the nest）would reach more than 80% in seriously susceptible production line,and more than one or the whole litter of piglets showed clinical typical signs.In addition to tremor,the affected piglets also have symptoms of spreaded hind legs.Above all,there are many CT cases caused by APPV in many farms in China.In order to figure out the diversity of APPV strains in different regions,4 whole genomes and 18 E2 genes which obtained from piglets with typical symptom were amplified,cloned and sequenced,respectively.Phylogenetic analysis based on the amino acid sequences of E2 gene indicated that these strains formed a new branch,except for one strain from Jiangsu Province.The results of whole genome similarity analysis showed that comparing with one strain from Jiangsu Province,three strains from Guangdong Province showed 82.2% to 84.4% similarity,and the other strains from Guangdong Province showed 98.1% similarity.And three strains from Guangdong Province showed 94% to 97% similarity with each other,and showed 79.6% to 85.9% similarity with other reference APPV strains.In addition,phylogenetic analysis based on the whole genomes showed that the Jiangsu strain was in the same branch with some strains from Guangdong Province;and the other three strains from Guangdong Province formed a novel branch.In summary,the domestic APPV strains have great variability and emerged a novel genotype.In this study,the recombinant baculovirus expression vector was constructed by optimizing the immunogenic gene E2 of APPV GD-DH01-2018-China strain.The recombinant bacmid DNA was transfected into Sf9 cells,and recombinant baculovirus was harvested from the supernatant.The recombinant E2 protein with molecular weight of roughly 40kDa was successfully expressed and purified in a baculovirus expression system.The results of Western Blot showed that the recombinant E2 protein could be probed by both positive APPV polyclonal antibody and monoclonal antibody to strep-tag.It also showed that the recombinant E2 protein has good antigenicity.And we developed indirect ELISA with good sensitivity and stability for detecting E2-specific antibody by using the purified recombinant E2 protein as coating antigen.In this study,the E2 gene were amplified from the APPV GD-DH01-2018-China strain and subcloned into recombinant adenovirus shuttle plasmid,and the recombinant adenoviral skeleton plasmid was obtained by homologously recombined the recombinant shuttle plasmid with the adenoviral skeleton plasmid.The recombinant adenoviral skeleton plasmid was linearized and transfected into 293 cells,and recombinant adenovirus was harvested from the supernatant.After several passages,the recombinant adenovirus titer was high and stable.The results of Western Blot and IFA showed that the APPV E2 gene was expressed in recombinant adenovirus.We investigated the immunological characteristics of the recombinant adenovirus in mice.The results indicated that,2 weeks after the vaccination,serum E2-specific antibody titer could be detected,and reached the highest level in the sixth week.In conclusion,the E2 recombinant adenovirus could induce high levels of E2-specific antibody in mice.In conclusion,this study provides a rich reference for the epidemiology survey of APPV in China.The recombinant adenovirus vector of APPV E2 gene produced an effective specific antibody level in mice and is expected to become a alive vector vaccine with developmental value.