Stable expression of Interleukin-18 in a live-attenuated PRRSV backbone confers safe and potent immune protection in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen responsible for substantial economic losses, and current live-attenuated vaccines provide suboptimal cross-protection. Interleukin-18 (IL-18), a multifunctional pro-inflammatory cytokine, has potent immunostimulatory effects that can enhance antiviral defense. In this study, we employed a reverse genetics system to insert the swine IL-18 gene between open reading frame 1b (ORF1b) and open reading frame 2a (ORF2a) of the full-length cDNA clone of the attenuated PRRSV strain pHuN4-F112, generating the recombinant virus rPRRSV-IL18. The genetic stability and sustained expression of IL-18 were confirmed over 25 serial passages using RT-PCR, Western blotting, and immunofluorescence assay (IFA). Comparative growth kinetics revealed that rPRRSV-IL18 exhibited similar plaque morphology, the replication efficiency in the later stage of infection, and peak titers to the parental strain. Recombinant IL-18 protein was expressed in E. coli and used to prepare specific polyclonal antibodies, validated through IFA and immunoblotting. In vivo evaluation in pigs demonstrated that rPRRSV-IL18 was well tolerated, caused no significant lesions compared to controls, and maintained normal growth performance. Immunized animals developed robust humoral and cell-mediated immune responses and were effectively protected against challenge with a highly pathogenic (HP)-PRRSV strain. These results indicate that stable incorporation of IL-18 into a live-attenuated PRRSV backbone yields a safe and immunogenic candidate vaccine. This molecularly engineered platform integrates cytokine-based immunopotentiation with live viral vector technology, offering a promising strategy for developing next-generation immune-enhanced vaccines to improve PRRSV prevention and control in the swine industry.