Epidemiological Investigation Of PRRSV And Replication Research Of PRRSV And PEDV In Marc 145 Cell Line Deleted With IFNAR1

Porcine reproductive and respiratory syndrome(PRRS)is a serious viral infection caused by Porcine reproductive and respiratory syndrome virus(PRRSV).The disease is characterized by severe reproductive disorders,respiratory diseases,growth delays and increased mortality.Porcine epidemic diarrhea(PED)is a highly infectious intestinal disease due to the infection of Porcine epidemic diarrhea virus(PEDV),which causes acute diarrhea,dehydration,vomiting and other symptoms in the host.In recent years,the outbreak of PRRSV and PEDV in many countries has brought great harm to the pig industry worldwide.According to relevant literature,PRRSV and PEDV in vitro cell culture can both induce the expression of IFN in the later stage of cell infection.Interferon is a cytokine produced by the natural immune response of the host in order to resist virus invasion.Can induce a strong antiviral state in the host.When interferon is combined with interferon receptor on the cell membrane,it will activate protein molecules on the cell jak-stat signaling pathway to regulate cell growth and differentiation,and exert antiviral effect and immune response and other effects.In this study,In order to explore the epidemiology of PRRSV in guangdong province of China,suitable screene PRRSV vaccine,119 samples from guangdong province of China were tested by rt-pcr,and 42 samples were found to be positive.The positive rate was 35.3%.The ORF5 and NSP2 genes of the positive samples were amplified and sequenced by rt-pcr,and 18 strains of ORF5 and 12 strains of NSP2 were obtained.The amino acid sequence and nucleotide sequence homology analysis of the obtained ORF5 sequences of 18 strains showed that the ORF5 nucleotide sequence homology of 18 strains was 83.3%-100%,the amino acid sequence homology was 83.5%-96.3%,the Nsp2 nucleotide sequence homology was 49.7%-96.6%,and the amino acid sequence homology was 57.9%-99.7%.The nucleotide amino acid sequence of the detected strain was homologous with the highly pathogenic strain and the nadc30-like strain.Genetic genetic and evolutionary analysis was performed on the ORF5 genes of the acquired strains.Eight strains belonged to the Lineage8.7(highly pathogenic strain JAX1)branch,five were in the Lineage1(NADC30-like)branch,and five were in the Lineage3 branch.Genetic evolution analysis of Nsp2 gene showed that 7 strains belonged to the Lineage8.7(highly pathogenic strain JAX1)branch,4 strains were in the Lineage1(NADC30-like)branch,and 1 strain belonged to the Lineage3 branch.In this study,JAX1-like and NADC30-like strains were found to be the most prevalent strains of PRRSV in south China.Amino acid sequence comparison of Nsp2 high mutation region showed that several strains had the characteristics of NADC30 and high pathogenicity in the high mutation region of Nsp2.IFNAR1 is interferon mediated by Ⅰ type JAK-STAT signaling pathways of upstream receptors.It is assumed that by knocking out IFNAR1 of marc-145 cells(embryonic kidney cells of African green monkey)and blocking the conduction of the JAK-STAT signaling pathway,the proliferation and replication of virus on cells can be promoted and the titrations of PRRSV and PEDV in vitro can be improved.IFNAR1 on marc-145 cells was knocked out by CRISPR/Cas9 technology,and 5 homozygous ifnar1-deficient marc-145 cell lines were finally obtained.KO cells were identified at the gene level,and the IFNAR1 sequence of 5 cell lines had different base number deletions.Westernblot analysis of STAT1 and p STAT1 expression showed that the IFN-β mediated JAK-STAT pathway was blocked in the knockout cell lines.To investigate the effect of IFNAR receptor knockout on PRRSV and PEDV proliferation in Marc-145 cells.The PRRSV HN strain and PEDV GDgh strain isolated and preserved in our laboratory were subcultured on Marc-145 cells silenced by IFNAR1,and the viral loads of the two cells were determined by q PCR.The results showed that the N gene expression level of PRRSV in several KO cell lines at 12 h,24h and 48 h was lower than that of WT cell lines.PEDV also had a low expression of N gene in KO cell line at 24 h,48h and 72 h.The two cell lines were infected with PRRSV and PEDV48 h respectively.IFA results showed that the amount of fluorescence of PRRSVN and PEDVN protein in KO cell lines was significantly less than that in WT cell lines.PEDV was significantly inhibited.Westernblot assay was performed on two cell lines at 48 h after inoculating PEDV.The results showed that the expression of PEDV N protein in WTMarc-145 cells was higher than that in KOMarc-145 cells.PEDV was further confirmed to be inhibited in KO cell lines.Through the epidemiological investigation of PRRSV in guangdong province of China,it was found that the prevalent strains in guangdong province of China were complex and varied,but the variation trend of the strains was from highly pathogenic strains to NADC30-like strains and QYYZ-like strains.To provide scientific and theoretical basis for selecting suitable vaccine for the disease.IFNAR1 of Marc-145 cells was knocked out by CRISPR/Cas9,and it was found that PRRSV and PEDV were inhibited in the replication of marc-145 cells knocked out by IFNAR1.