Genome Sequence Analysis Of Atypical Porcine Pestivirus And Functional Study Of APPV-N^pro

As the main causative agent for congenital tremor(CT)type A-II in piglets,Atypical porcine pestivirus(APPV)mainly caused the tremor disease of the first litter piglets produced by newly introduced gilts,presenting spasm and contraction of whole body or local muscles with ataxia.So far,it has been reported in 15 countries around the world,since APPV was first discovered from the USA in 2015,threatening the health of global swine herd.In order to study the genetic evolution of APPV,epidemiological investigation of APPV in partial areas of China and the analysis of APPV codon usage pattern were conducted in this study.Pestivirus Npro is the first protein translated in the viral polypeptide,which has important biological functions for the virus.Npro has the activity of cysteine protease,which could cleave itself off from the translated virus polypeptide and further play the role of the virus against the natural immunity of the host.Classical swine fever virus and bovine viral diarrhea virus can inhibit the natural immune response of the host,thus leading to immune escape and persistent infection,where Npro has been shown to play an important role.However,as a newly discovered pestivirus,whether APPV-Npro has self-cleavage activity remains to be determined.At the same time,the pathogenesis and immunosuppressive mechanism of APPV are not clear,the analysis of the role of Npro in natural immunity will help to elaborate this issue1 Isolation and identification,sequences analysis of APPVIn order to further understand the prevalence of APPV and its genetic relationship in China,69 samples of suspected infected tissues collected from partial areas of China were tested for APPV by RT-PCR.The positive rate of APPV was 33%.4 full-length genome sequences and 26 partial genome sequences of APPV were obtained by amplification,sequencing and splicing.A statistical analysis of the geographical distribution of APPV in China has revealed that since China first reported APPV in 2016,12 provinces,municipalities and autonomous regions have reported the virus in succession,indicating that APPV has a national prevalence trend.Phylogenetic analysis showed that the existing APPV strains could be divided into 3 genotypes.Genotype 1 and 2 included APPV strains isolated from China,and genotype 3 could be divided into several subtypes,including strains isolated from Europe,North America and Asia.All the APPV strains amplified in this experiment belong to genotype 3.The results of this study revealed the diversity of Chinese APPV epidemic strains and update the genotyping of global APPV epidemic strains,which will provide important reference information for the detection and prevention of APPV.2 An analysis of APPV codon usage patternCodon preference analysis is often used to understand the evolutionary relationships between different virus strains in order to identify their genetic characteristics.Therefore,in order to further understand the genetic evolution of APPV strains,codon usage pattern analysis of available APPV-ORF sequences was performed using bioinformatics.The results showed that PC A analysis was consistent with the phylogenetic trees constructed based on APPV-ORF sequences.All the APPV-ORF sequences were AU rich,and there is a preference in nucleotide usage.The results of ENC analysis showed that APPV-ORF has a low codon usage bias.The codon usage pattern of APPV-ORF was influenced by dinucleotide abundance,mutation pressure and natural selection,which is the most important factor.These results are helpful to the further analysis of APPV evolution and its adaptability in host.3 Study of APPV-Npro autoprotease activityIn order to understand whether the APPV-Npro protein has self-cleavage activity and the key amino acid residues which affect its protease activity,wild-type recombinant plasmid expressing APPV Npro-C and mutant recombinant plasmid expressing Npro were constructed and then transfected into HEK293T cells,western blot was used to analyze the self-cutting activity of APPV-Npro protein and to identify the key amino acid residues related to its protease activity in this study.The results showed that the APPV-Npro had self-cleavage activity and could cleave itself off between Cys80 and Ser181.The cleavage site Cys 180 could be replaced by amino acids of medium or small molecular weight,but the cleavage activity of Npro protein was decreased.His67,His69,and Cys89 are the key amino acids that affect the cleavage activity of APPV-Npro.The cleavage activity of APPV-Npro is not necessary for the first seven amino acids,but the integrity of other sequences of APPV-Npro is essential for the cleavage activity of APPV-Npro.The results enrich the molecular biological information of APPV4 APPV-Npro suppressed IFN-? expression through IRF3To investigate whether APPV-Npro protein is involved in the immune regulation of APPV to the host,the effect of APPV-Npro on the production and promoter activity of IFN-? was investigated in this study.Then,the key proteins that affect the function of APPV-Npro were explored by stimulating with natural immune genes in type I IFN signal pathway.Finally,Co-IP,Western blot and indirect immunofluorescence were used.All the results demonstrated that Npro could inhibit the IRF3 promoter activity activated by a variety of natural immune genes and inhibit IFN-? production through specific interaction with IRF3.The results suggested that APPV-Npro may play an important role in IFN-production of antagonistic host and the persistence of APPV infection.