Influence of inoculation dose and route on EHDV-8 distribution and the induced immune response in experimentally infected cattle

Epizootic haemorrhagic disease virus serotype 8 (EHDV-8) is an emerging Culicoides-borne virus that was first detected in Europe in autumn 2022 in Italy and subsequently spread to Spain, Portugal and France. Despite its economic impact, little is known about its tissue distribution, persistence, and induction of immune responses in cattle. Therefore, we conducted an experimental infection study in groups of 3 cattle inoculated intradermally (ID; 10^6.2 or 10^5.2 TCID₅₀/animal) or subcutaneously (SC, 10^6.2 TCID₅₀/animal) with a Spanish EHDV-8 isolate. Viremia appeared at 3 days post-inoculation (dpi), peaked at 7-10 dpi, and persisted until the end of the study (21 dpi). Fever spiked at 8-9 dpi with a longer duration in the high-dose groups. No other clinical symptoms were noted, except in one animal from the SC high-dose group. This animal exhibited apathy, conjunctivitis, and swollen lymph nodes from 10 dpi onwards. At necropsy, lymph nodes and spleen contained higher viral loads than muscles, brain, and skin. The clinical animal displayed petechiae on the kidney with high viral loads (Ct value = 20), which was an outlier compared with all other organs tested. The virological data thus confirmed that blood, spleen and lymph nodes are the most suitable diagnostic samples. A strong humoral response (100% seroconversion) was observed in all the groups at 10 dpi. All the animals, except the one showing clinical signs, also exhibited a strong cellular immune response, with IFN-γ levels peaking at 5-7 dpi. In conclusion, this study demonstrated that the inoculation route and dose only minimally impacted viremia, viral spread and induced immune responses.