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ABSTRACT Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

To evaluate the effects of recombinant porcine interleukin-18 (rpIL-18) on the replication of viruses in host cells and proliferation of lymphocytes, porcine IL-18 ( pIL-18) isolated from a domestic big-white porcine breed found in the Henan province (HN) was cloned using a reverse transcriptase-PCR. The cloned HN pIL-18 contained an ORF of 579 base pairs encoding a 192-amino-acid precursor protein. The amino acid sequence of HN pIL-18 was compared with all the other pIL-18 amino acid sequences and varied by at least one amino acid to the consensus of all the others available. HN pIL-18 mature protein gene was inserted into a prokaryotic vector pGEX-4T-1 and expressed in Escherichia coli BL21. The expression of glutathione- S-transferase–pIL18 fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The rpIL-18 induced in vitro proliferation of concanavalin-A-stimulated porcine splenocytes, as revealed by the MTT assay. We studied the antiviral activities of the rpIL-18 on the replication of porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine parvovirus (PPV) cultured in two homologous cell lines. The results suggested that rpIL-18 can stimulate the proliferation of lymphocytes and inhibit viral pathogens infecting the porcine population. [ABSTRACT FROM AUTHOR]