First Trans-Border Serological Evidence of West Nile Virus Infection in Horses in Romania and Bulgaria

West Nile Virus (VNV) produces encephalitic West Nile Fever (WNF), a zoonotic disease that can affect birds, is a viral disease that can infect horses as well as other mammals, birds and humans as a result of infection by arbovirus -Orthoflavivirus nilense, from family Flaviviridae.Cases of dead affected wild birds during the summer season have been positively related to predicting WNV in human populations.We conducted the present study aiming to detect serological evidence of the WNV in horses in western and central Romania and in northern and southern Bulgaria.Previous studies conducted in neighboring countries -Serbia and Hungary, confirmed the presence of the virus in horse populations.Between July 2022 and July 2023, a total of 147 horse serums were collected from three counties in West Romania -Arad, Bihor and Timis, and one in central Romania -Sibiu.Additional sera were collected from Lovech municipality in North Bulgaria and Topolovgrad municipality in South Bulgaria.The presence of antibodies in the serum was detected using the commercial ELISA kit.In order to remove the cross-reactivity of the Usutu virus, another representative of the Mosquitoborne Japanese encephalitis virus group, an epitope-blocking ELISA kit with specificity to WNV's Glycoprotein E and absence of cross-reactivity with other Flaviviruses was used.The sera were further tested via RT-PCR for presence of the viral RNA.Seropositivity was confirmed for 45% of the sera in West Romania and for 63% of the sera in Central Romania; for 23% from North Bulgaria and 20% from South.All PCR results were negative, demonstrating an absence of the viral RNA in the horses' body at the time of sampling.The study confirms the presence of WNV in horse populations of all tested regions.The results from the different countries and areas demonstrate that there is no difference between the populations in forested areas and those in field areas.