Genome-wide Analysis Of Transcriptome And DNA Methylation Regulation Of Porcine PBMCs In Differential Antibody Responses Post PRRSV Vaccination

Porcine reproductive and respiratory syndrome(PRRS)is an emerging viral infectious swine disease caused by porcine reproductive and respiratory syndrome virus(PRRSV);typically manifested by reproductive failures in sows and respiratory disorders in growing pigs,including abortions,stillboms and death.PRRSV causes huge economic losses to the swine industry.In the practice of raising pigs,PRRS control stategies by vaccine prevention are widely used to reduce porcine production losses.However,the immune efficiency of PRRSV vaccination in pigs is variable,the protective effect is limited.The main reasons is that the molecular mechanism on variable immune response to vaccines is not clear.In this study,using genome-wide sequencing of transcriptome and methylome of pig peripheral blood mononuclear cells(PBMCs)over two generations in response to PRRSV vaccination,this study explored the changes in gene expression and DNA methylation as well as the role of DNA methylation in regulating immune-related genes.This study aimed to epigenetically disclose the regulatory mechanisms for variable antibody responses,and identify genes or DNA biomarker associated with vaccine responsiveness,thus providing new evidence for PRRSV vaccination and genetic resistance breeding of pigs.Section 1:Identification of candidate genes related to variable antibody response to PRRSV vaccination in pregnant sows using RNA sequencingIn this study,we used RNA-seq to generate the sow transcriptome from PBMCs of 12 pregnant sows with high,median and low antibody level.In total,401 differentially expressed genes(q<0.05)were identified in three comparisons of sow PBMCs.Gene ontology and pathway analysis demonstrated that the 401 differentially expressed genes were enriched in biological processes with regard to inflammatory response,response to wounding,regulation of cytokine production,defense response,regulation of apoptosis,regulation of programmed cell death,cell proliferation,leukocyte chemotaxis and migration,positive regulation of immune system process(P<0.05).Combining analysis of differentially expressed genes,previous reports of quantitative trait loci studies indicated that MEGF9,NDRG1,SLA-8,C2,STX11,C-JUN,FOS,NFKBIA,TNF-a,IL-1? and IL-1? were the most promising candidate genes affecting antibody responses.Section 2:Identification of candidate genes related to variable antibody response to PRRSV vaccination in piglets using RNA sequencingWe used RNA-seq to generate transcriptome from PBMCs of 6 piglets with high and low antibody level.In total,86 differentially expressed genes(q<0.05)between the high and low groups of piglets were revealed.Integrated analysis of bioinformatics results,previously research results of sows and transcriptome studies indicated that C-JUN,FOS,CCL4,TNF-a,NFKBLA,IL7R were the most promising candidate genes affecting piglets antibody responses.This study profiled the piglets DNA methylome with variable antibody responses to PRRSV vaccination,in which with the densities of 10-15 CpGs/kb tending to exhibit a higher methylation level in high antibody group,while the densities of 15-20 CpGs/kb tending to exhibit a higher methylation level in low antibody group.DNA methylation level tending to increase towards gene bodies,and the CpG island in low group showing higher methylated level than that in high group.These negative regressions were found of promoter methylation level with gene expression of 6 genes:PYY,CCR7,IL7R,CD59,RHOB,VASH1.Section 3:Transcriptomic homogeneity in pregnant sows and their offspringThe material of this test is that of test 1 and test 2.We compared differentially expressed genes in two generations of pigs for comparison of HA versus LA.We observed negative regression between expression levels of TNF-a,CCL4 and NFKBIA with PRRS-MLV-specific antibody levels in two generations of pigs.The present study will advance our understanding of transcriptome and epigenetic responses to PRRSV vaccination over two generations with variable antibody responses,and it is expected to constitute a valuable resource for future epigenetic immune responses in pigs and for the analyses of tissue specific DNA methylation pattern.