**4. Conclusions**

In conclusion, our results confirm simultaneous circulation of WNV and USUV in the Czech Republic, so far limited to Southern Moravia. However, the first WNV-positive mosquitoes recently found in another region of the Czech Republic (Southern Bohemia) may indicate new WNV focus in the country [30]. Only a One-Health approach practicing interdisciplinary collaboration among local infection specialists, epidemiologists, veterinarians and entomologists, can bring benefit in the prevention and control of WNV in affected areas and also detect the introduction of WNV in previously virus-free areas in a timely manner.

**Author Contributions:** Conceptualization, H.Z., S.Š. and I.R.; funding acquisition, I.R.; investigation, H.Z., J.K., S.Š., P.S., H.K., R.S., P.H., P.H.J., E.T., H.L., O.Š., P.J., R.C., J.M. and I.R.; project administration, I.R.; supervision, H.Z. and I.R.; writing—original draft, H.Z., H.K., R.S., E.T., R.C. and I.R.; writing—review and editing, S.Š., P.S., R.S., E.T., H.L. and I.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was financially supported by the Ministry of Health of the Czech Republic (Reg. No. NV19-09-00036). All rights reserved. This work was also supported by the Ministry of Health, Czech Republic—conceptual development of research organization (FNBr, 65269705). Petra Strakova was supported by the Veterinary Research Institute (RVO: RO 0518).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Patient consent was waived: strictly anonymous epidemiological, laboratory and clinical findings were applied in the study.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
