Transcriptome Analysis Of Porcine PBMCs Of Differentially Virulent Classical Swine Fever Virus Strains And Dissection Of Antiviral Mechanisms Of HES4

Classical swine fever(CSF),caused by classical swine fever virus(CSFV),is a highly contagious,hemorrhagic,and fatal disease of pigs.CSFV is an enveloped virus containing single-stranded and positive RNA genome and that belongs to the Pestivirus genus within the Flaviviridae family.Previous studies have conducted microarray technology to analyze CSFV-infected cells and to classify the differentially transcriptional genes,but the mechanism by which differentially transcriptional genes involved in virus replication has not been elucidated.Therefore,screening the differentially transcriptional host molecules induced by the highly virulent and avirulent strains of CSFV,identifying the effects on CSFV replication and clarifying the mechanisms will provide novel clues for further understanding of the pathogenesis of CSFV and the host's antiviral response.In this study,we constructed the experimental animal models of specific-pathogen-free(SPF)pigs infected with highly virulent and avirulent strains of CSFV to analyze the host response comprehensively.Eleven 8-week-old SPF pigs were randomly divided into three groups and inoculated with the CSFV Shimen strain(a highly virulent strain),C-strain(an avirulent strain),and DMEM(mock control),respectively.Infection with the Shimen strain resulted in fever,clinical signs and histopathological lesions,which were not observed in the C-strain-inoculated pigs,though low viral genome copies were detected in the peripheral blood and tissue samples.The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus.Furthermore,leukopenia,lymphocytopenia,differentiation of T-cells,and the secretion interleukin 2(IL-2),IL-4,IL-6,and IL-10 were induced by CSFV infections,the differences reflected in onset and extent of the regulation.The data revealed that virulence of the CSFV does not affect the pattern of the spread in the host,but influences the onset,intensity,duration and outcome of the disease,and the host responses to infection.Furthermore,RNA-Sequencing was conducted to screen the differentially transcriptional genes induced by differentially virulent CSFV strains.The results showed that there were 3,582 genes with significant changes in transcriptional level at 3 dpi,including 3,205 up-regulated genes and 377 down-regulated genes,and there were 7,456 genes changed significantly in transcriptional level,among which 83 were up-regulated and 7,373 were down-regulated.The first 30 signaling pathways related to immune and viral infection include NF-?B,RIG-I receptor,Toll-like receptor and JAK-STAT signaling pathways.Combining with bioinformatics analysis,we screened 20 candidate molecules for further study.To verify the effects of host factors on the CSFV replication,PK-15 cells were transfected with small interfering RNAs(siRNAs)and infected with CSFV.We demonstrated that knockdown of IL-10,VLDLR and USP18 could significantly inhibit the replication of the CSFV Shimen strain,while the downregulation of HES4(Hairy and enhancer of split 4),Stefin A1,CYSLTR2,LGASL1,IFIT1,ISG15,CASP1,MOV10,USP25,Trim5,DDX58 and ISG20 could significantly promoted the replication of CSFV.HES4 is a transcriptional suppressor that belongs to bHLH(basic-helix-loop-helix)superfamily.We selected HES4 for further study,because it was upregulated in the infection group of the CSFV Shimen strain,while there was no significant change in the infection group of C-strain.In addition,we found that the transcriptional level of HES4 was upregulated in PAM cells infected with CSFV Shimen strain,and in various tissues or PBMCs of CSFV Shimen-infected pigs.We showed that overexpression of HES4 significantly inhibited CSFV growth,whereas knockdown of the endogenous HES4 expression by specific siRNAs remarkably promoted the replication of CSFV.In terms of the molecular mechanism of HES4 against CSFV,we demonstrated that HES4 did not activate the IFN-? or NF-?B signaling pathways,but inhibited the efficiency of protein translation mediated by CSFV internal ribosome entry site(IRES)in a dose-dependent manner.To further verify the results,we conducted RNA pulldown assay with biotin-labeled IRES,and revealed that HES4 could bind to IRES.In addition,we performed the competitive experiments with free IRES and showed that the free IRES can effectively inhibit the interaction,indicating that is the specific interaction between HES4 and IRES.To sum up,we demonstrated that differentially transcriptional host molecule HES4 induced by differentially virulent strains of CSFV inhibits the replication of CSFV via binding to CSFV IRES and inhibiting its activity.