The Whole Genome Sequencing,Pathologic Damage And Transcriptional Difference Analysis Of Atypical Porcine Pestivirus SC Strain

Atypical porcine pestivirus(APPV)is a member of the genus Flaviviridae and prion,and is one of the main pathogens causing congenital tremor of type AII.It can cause muscle tremors in infected piglets or the hind limbs cannot stand.The death of newborn piglets due to the inability to lick milk has caused huge losses to the pig industry.Because it is a new disease,there is no specific vaccine for this disease,and the mastery of pathogenic pathogenesis is the key to scientifically formulating prevention and control measures,making it possible to cut off its transmission route and control the source of infection..In this study,the full-length sequence of the popular strain CHN-SC was obtained,and its difference with other reference strains at the nucleotide level was analyzed by molecular biology software.And we establish fluorescence quantitative RT-PCR method and make pathological tissue sections to investigate the mechanism of pathological injury in infected piglets;we also explore the pathogenic mechanism of APPV by studying the difference in transcription levels between infected piglets and control groups.1.Whole gene sequencing and analysis of APPV CHN-SC strainThe tissues and serum of suspected infected piglets were collected in a large-scale farm in Suining,Sichuan Province and examed by RT-PCR.After confirming positive infection,12 pairs of primers were designed and synthesized,and obtained through steps of cloning,sequencing and splicing.The full-length sequence of APPN epidemic strain CHN-SC in Sichuan was 11303 bp,of which ORF1 was 10908 bp.The homology between CHN-SC strain and APPV reference strain was further analyzed by Editseq and Megalign software.The results showed that the homology rate was between 81.7% and 90.6% compared with the reference strain.Among them,the homologous rate was the highest with the APPV-GER01 strain derived from Germany,which was 90.6%,which was the same as the two APPV strains in Guangdong,China.The lowest source rate is 81.7%.2.Study on pathological injury of piglets infected with APPVThis study firstly established a qRT-PCR method for the detection of APPV NS2 gene,and used this method to measure the central nervous system and periphery of APPV in sick piglets.The lymphatic organ has the highest load.Moreover,after excluding the interferenceof other CT-related pathogens,pathological observations were made on various tissues and organs,and the results showed that the main and most serious lesions were concentrated in the central nervous system.Compared with the control group,the experimental group can observe obvious neuronal contraction,deep nuclear staining,meningeal edema,nuclear granule necrosis with nuclear debris,cell chromatin lysis,nerve cell necrosis and other serious pathological changes,suggesting that APPV may It mainly replicates in lymphoid organs and mainly infects the central nervous system.3.Differences in transcriptional differences between cerebellar tissues of piglets infected with APPVThe above-mentioned infected piglets were excluded from the CT pathogens,and the cerebellum tissues were collected before.The transcriptome was sequenced on the Illumina Hiseq 4000 platform,and the cerebellum tissues of healthy piglets were used as the control group to construct two cDNA libraries: APPV infected cerebellum tissue library and healthy cerebellum tissue.A total of 381 differentially expressed genes were screened by this sequencing,of which 163 were significantly up-regulated and 218 were significantly down-regulated.The screening criteria were P<=0.05,|log2(FC)|>=0.263.For the differential gene analysis by GO enrichment,the secondary functional classes with significant proportional differences include the negative regulation of amyloid-beta clearance,regulation of signaling,and activin receptors.Activin receptor signaling pathway,hormone secretion,regulation of cell communication,regulation of signal transduction,endosome,intracellular,empty Vacuole,myelin,etc.Using KEGG to annotate differential genes,the results showed that genes differentially expressed in cerebellum were mainly enriched in Glycerolipid metabolism,Neurotrophin signaling pathway,and Long-term depression.Synaptic Vesicle Cycle,Axon guidance,Focal adhesion,Pathways in cancer,Hippo signaling pathway,Kidney Renin secretion,proteoglycans in cancer,and the like.