An adenovirus-vectored strategy expressing IFN-λ3 and IL-22 protects neonatal piglets from porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease in neonatal piglets, often resulting in nearly 100 % mortality. Frequent genetic variation further undermines the effectiveness of existing vaccines and control strategies, highlighting the urgent need for novel antiviral interventions. In this study, we developed a recombinant adenovirus-vectored strategy (rAd5-IFNλ3-IL22) co-expressing interferon lambda 3 (IFN-λ3) and interleukin-22 (IL-22) to concurrently target viral replication and attenuate epithelial injury. In vitro, rAd5-IFNλ3-IL22 expressed and secreted IFN-λ3 and IL-22 in 293A cells, and the culture supernatant significantly upregulated the transcription of antiviral (OASL) and mucosal-protective (Muc1) genes in IPEC-J2 cells. Extending these findings, pretreatment of porcine jejunal enteroids with the same supernatant markedly suppressed replication of PEDV strains (G2b and G2c) and transmissible gastroenteritis virus (TGEV), demonstrating antiviral efficacy against genetically distinct PEDV strains as well as TGEV. In vivo, orally administered rAd5-IFNλ3-IL22 to neonatal piglets delayed diarrhea onset, mitigated villus atrophy, and reduced viral replication and shedding, culminating in significantly enhanced survival. Crucially, under sow-rearing conditions that closely resemble production environments, treated piglets exhibited delayed disease progression, a substantial reduction in viral shedding, and improved weight gain compared to controls. These findings indicate that rAd5-IFNλ3-IL22 confers effective protection against PEDV and alleviates viral burden in neonatal piglets, supporting its potential as a promising antiviral intervention for PEDV control in swine. A caveat is that, without an in vivo empty-vector control, the observed protection likely reflects combined effects of IFN-λ3/IL-22 expression and Ad5 vector-mediated innate immune activation.