Porcine Cytomegalovirus Infection Of Pig Multi-organ MicroRNA Expression Profile

Porcine cytomegalovirus(PCMV)is a member of the genus Cytomegalovirus,subfamily Betaherpesvirinae,family Herpesviridae.It is an icosahedral virus with a double-stranded linear DNA genome.Although Done originally isolated the herpesvirus-like particles of PCMV in England,PCMV is now widely distributed around the world.China,Britain,the United States,Germany,and Japan have all confirmed the prevalence of the virus.PCMV is an immunosuppressive virus that mainly inhibits the immune function of T lymphocytes and macrophages,which has caused great distress to the farming industry.Moreover,the molecular immunosuppressive mechanisms of PCMV,along with the host antiviral mechanisms,are still not well characterized.To understand the potential impact of PCMV on the function of immune organs,we examined the transcriptome of PCMV-infected thymuses by microarray analysis.We identified 5,582 genes that were differentially expressed as a result of PCMV infection.Of these,2,161 were upregulated and 3,421 were downregulated compared with the uninfected group.We confirmed the expression of 13 differentially expressed immune-related genes using quantitative real-time RT-PCR,and further confirmed the expression of six of those cytokines by western blot.Gene ontology,gene interaction networks,and KEGG pathway analysis of our results indicated that PCMV regulates multiple functional pathways,including the immune system,cellular and metabolic processes,networks of cytokine-cytokine receptor interactions,the TGF-b signaling pathway,the lymphocyte receptor signaling pathway,and the TNF-a signaling pathway.microRNAs(miRNAs)are small(18-24 nt),noncoding RNAs that exist widely in eukaryotic organisms.The mature miRNAs play an important regulatory role in organism growth,hematopoiesis,organogenesis,cell differentiation,apoptosis,proliferation,fat metabolism,cancer,plant hormone level,and signal transduction.In order to study the regulatory function of miRNAs during PCMV infection,we obtained the miRNA expression profiles of PCMV-infected porcine lung,liver,thymus,kidney and spleen via high-throughput sequencing.The comprehensive analysis of miRNA profiles showed that 601 miRBase annotated and 295 novel pig-encoded miRNAs were detected.The 12 differentially-expressed pig-encoded miRNAs were further determined by stem-loop RT-PCR,and the results were consistent with the high-throughput sequencing.The results of the Gene Ontology(GO)annotation reflected the regulatory roles that the host-encoded miRNAs play in host physiological processes,including the regulation of cellular components,molecular functions,and biological processes.The target genes of the differentially-expressed miRNAs were mainly related to immune system process,cellular process,metabolic process and signal transduction.Binding site prediction results showed that miR-24-3p?miR-10b?miR-30a?miR-180a target immune-related genes transcripts.To verify that miR-24-3p?miR-10b?miR-30a?miR-180a post-transcriptionally regulate the expression of immune-related genes through binding to the target sites,a recombinant lentiviral vector Lv-mir-24-3p,Lv-mir-10b,Lv-mir-30a,Lv-mir-180awere constructed and used to infect porcine macrophages.Verification results showed that the expression IL-la,IL-15,IL-12 in recombinant lentivirus infected porcine macrophages was significantly inhibited by the over-expression of miR-24-3p,miR-lOb,miR-30a compared with the expression in lentiviral vector-control-infected and normal porcine macrophages,separately.But the expression of IL-12 did not effected by the over-exprssion of miR-180a.This suggests that IL-la,IL-15,IL-12 are the novel target of miR-24-3p,miR-l0b,miR-30a,separately.Therefore,we conclude that PCMV infection interferes with the cytokine-related signaling pathways by effecting the expression of miRNAs,thus affecting the immune function.