Combined use of dill essential oil and tannic acid creates stage-specific, and time-dependent antiviral effect against bovine viral pathogens (BoHV-1, BVDV, BPIV-3, BRSV, and BEV).

Bovine viral pathogens, including bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine enterovirus (BEV), cause substantial economic losses in cattle production. This study evaluated the antiviral activities of dill essential oil (DEO) and tannic acid (TA), individually and in combination, and investigated their stage-specific, time-dependent, and direct virucidal effects in MDBK cells. Based on cytotoxicity assessments, 0.0125% (v/v) DEO and 50 µM TA were selected as working concentrations (CC50: 0.0155% and 112.83 µM, respectively), while the DEO/TA combination maintained cell viability above 80% throughout the 144-h observation period. TA showed particularly strong activity against BVDV, whereas DEO was more effective against BoHV-1 and BPIV-3 at selected time points. The DEO/TA combination generally produced enhanced antiviral effects compared with the individual treatments, with single-dose reductions reaching 4.5 log10TCID50 and repeated administration producing reductions of up to 7.0 log10TCID50 in a virus- and time-dependent manner. Stage-specific assays demonstrated distinct patterns of activity: BoHV-1 was particularly affected during adsorption; DEO-containing treatments reduced BVDV and BPIV-3 titers during penetration; and no significant inhibition at the adsorption or penetration stage was detected for BRSV. Virucidal assays further showed significant direct reductions in BoHV-1 and BRSV titers across all exposure periods, significant activity against BVDV after ≥ 1 h of exposure, and significant effects against BPIV-3 at selected exposure times. In contrast, BEV showed no significant time-specific virucidal response. Overall, DEO, TA, and particularly their combination exhibited broad but strongly virus- and stage-dependent antiviral activity. These findings support further investigation of the DEO/TA formulation as a plant-derived antiviral candidate, including dose optimization, mechanistic studies, additional cell-based models, and in vivo studies.