Influenza D virus (IDV), belonging to the Orthomyxoviridae family, was first discovered in 2011 in pigs. Surveys in humans and animals have been carried out to decipher IDV ecology. In this seroepidemiological study, we investigated the circulation of IDV lineages across Italy in livestock and wildlife animals. A total of 1038 animal serum samples (from 246 bovines, 249 swine, 98 equines, and 445 wild boars) were tested using hemagglutination inhibition and virus neutralization assays. The results confirm bovines as the primary reservoir for IDV, with high seroprevalence for both D/660 (87%) and D/OK (80%) strains. Swine and equines demonstrated limited exposure, suggesting they are infrequent spillover hosts. Notably, wild boars showed high seroprevalence, especially for the D/660 lineage (80%), indicating their potential role in a wildlife transmission cycle. Continuous surveillance in both livestock and wildlife is essential to monitor the spread and evolution of IDV.

Influenza D virus (IDV) is an emerging pathogen with zoonotic potential. Epidemiological and serological evidence suggests that cattle serve as the natural reservoir, with periodic spillover events to other animal hosts. This study evaluates the in vitro activity of lemon juice (LJ) and lemon essential oil (LEO) against two IDV lineages (D/660 and D/OK). Compound cytotoxicity was evaluated on MDCK cells by measuring cell viability with a fluorescein diacetate assay. Viral suspensions were pre-treated with the compounds, and the viral titers were determined by TCID₅₀. Immunofluorescence analysis was performed to evaluate viral nucleoprotein expression. Then, the potential morphological changes in treated viral particles were investigated with transmission electron microscopy. Both LJ and LEO showed significant dose- and time-dependent antiviral effects, leading to a rapid reduction in viral titers at higher concentrations and at non-cytotoxic levels. Moreover, at the highest non-cytotoxic dilutions, prolonged exposure (30–60 min) resulted in a reduction of approximately 1–1.5 log₁₀ TCID₅₀/mL in viral titers. Moreover, treatments reduced viral nucleoprotein expression and were associated with morphological alterations of viral particles observed by transmission electron microscopy, providing supportive evidence for a possible direct effect on virion integrity. These findings suggest that lemon-derived compounds possess promising properties against IDV and may represent potential natural agents for disinfectant or virucidal applications.