新型抗菌肽Z-d14CFR通过激活3D4/21细胞中RIG-I信号通路抑制猪流行性腹泻病毒OA

Porcine epidemic diarrhea virus(PEDV)poses a serious threat to the swine industry,but effective antiviral strategies remain limited.In this study,in vitro infection models were established in three different cell types,and Western blotting(WB),indirect immunofluorescence assay(IFA),real-time fluorescent quantitative polymerase chain reaction(qPCR),and confocal laser scanning microscopy(CLSM)were used to evaluate the anti-PEDV activity and mechanism of the β-hairpin antimicrobial peptide Z-d14CFR and its structural analogue Z(WK)2.The results showed that,compared with the 0 μg/mL group,Z-d14CFR and Z(WK)2 at 0.5-64 μg/mL had no significant effects on the viability of porcine alveolar macrophage cells(3D4/21),African green monkey kidney cells(Vero),or porcine intestinal epithelial cells(IPEC-J2)(P>0.05).In the post-infection treatment model,compared with the PEDV group,Z-d14CFR at 4,8,and 40 μg/mL and Z(WK)2 at 4 and 32 μg/mL significantly reduced the relative expression level of PEDV N protein in 3D4/21 cells(P<0.05 or P<0.01),whereas no significant effects were observed in Vero or IPEC-J2 cells(P>0.05).In the pretreatment model,compared with the PEDV group,Z-d14CFR at 10-30 μg/mL significantly reduced the relative expression level of PEDV N protein(P<0.01),whereas Z(WK)2 had no significant effect(P>0.05).The antiviral activity of 20 μg/mL Z-d14CFR gradually increased after 16-24 h of pretreatment.In the direct virus neutralization assay,Z-d14CFR at 25-100 μg/mL significantly reduced the relative expression level of PEDV N protein(P<0.01),whereas Z(WK)2 was effective only at 100 μg/mL(P<0.05).WB and CLSM results showed that Z-d14CFR pretreatment enhanced the phosphorylation of interferon regulatory factor 3(IRF3)and altered the temporal expression patterns of retinoic acid-inducible gene I(RIG-I)and mitochondrial antiviral signaling(MAVS)protein.In the absence of viral infection,Z-d14CFR promoted the mRNA expression of interferon alpha(IFN-α)and interferon beta(IFN-β).These results demonstrate that Z-d14CFR exerts anti-PEDV activity through dual mechanisms involving direct virus neutralization and activation of the RIG-I/IRF3 signaling pathway.This study provides a theoretical basis for the development of novel peptide-based antiviral agents and highlights the potential application of immunomodulatory antimicrobial peptides in the prevention and control of veterinary coronavirus infections.