The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal antibody (mAb) against pig CD69 was produced by peptide immunization and hybridoma technique. One mAb (5F12) showed good reactivity with pig CD69 that was expressed in transfected-HEK-293T cells and on mitogen-activated porcine peripheral blood mononuclear cells (PBMCs) by indirect immunofluorescence assay and flow cytometry. This mAb did not cross-react with activated lymphocytes from mouse, bovine, and chicken. Epitope mapping showed that the epitope recognized by this mAb was located at amino acid residues 147–161 of pig CD69. By conjugating with fluorochrome, this mAb was used to detect the early activation of lymphocytes in PRRSV- and ASFV-infected pigs by flow cytometry. The results showed that PRRSV infection induced the dominant activation of CD4 T cells in mediastinal lymph nodes and CD8 T cells in the spleen at 14 days post-infection, in terms of CD69 expression. In an experiment on ASFV infection, we found that ASFV infection resulted in the early activation of NK cells, B cells, and distinct T cell subsets with variable magnitude in PBMCs, spleen, and submandibular lymph nodes. Our study revealed an early event of lymphocyte and T cell activation after PRRSV and ASFV infections and provides an important immunological tool for the in-depth analysis of cellular immune response in pigs after infection or vaccination.

African swine fever (ASF) is a devastating infectious disease in domestic pigs caused by African swine fever virus (ASFV) with a mortality rate of about 100%. However, the understanding of the interaction between ASFV and host is still not clear. In this study, the expression differences and functional analysis of microRNA (miRNA) in porcine peripheral blood lymphocytes of ASFV infected pigs and healthy pigs were compared based on Illumina high-throughput sequencing, then the GO and KEGG signal pathways were analyzed. The miRNA related to immunity and inflammation were screened, and the regulatory network of miRNA-mRNA was drawn. A total of 70 differentially expressed miRNAs were found (p ≤ 0.05). Of these, 45 were upregulated and 25 were downregulated in ASFV-infected pigs vs. healthy pigs. A total of 8179 mRNA genes targeted by these 70 differentially expressed miRNA were predicted, of which 1447 mRNA genes were targeted by ssc-miR-2320-5p. Five differentially expressed miRNA were validated by RT-qPCR, which were consistent with the RNA-Seq results. The GO analysis revealed that a total of 30 gene functions were significantly enriched, including 7 molecular functions (MF), 13 cellular components (CC), and 10 biological processes (BP). The KEGG enrichment analysis revealed that the differentially expressed genes were significantly enriched in pathways related to immunity, inflammation, and various metabolic processes, in which a total of two downregulated miRNAs after infection and eight upregulated miRNAs related to immunity and inflammation were screened in ASFV-infected pigs vs. healthy pigs. The network of miRNA-mRNA showed that the mRNA target genes were strongly regulated by ssc-miR-214, ssc-miR-199b-3p, and ssc-miR-199a-3p. The mRNA target genes were enriched into the MAPK signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway by using a KEGG enrichment analysis. Therefore, ASFV could regulate immunity and metabolism-related pathways in infected pigs by inducing differential expression of miRNAs. These results provided a new basis for further elucidating the interactions between ASFV and the host as well as the immunity regulation mechanisms of ASFV, which will be conducive to better controlling ASF.