Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is currently recognized as one of the major causes of acute human hepatitis worldwide. In Europe, the increasing number of hepatitis E cases is mainly associated with the consumption of animal food products or contact with infected animals. Dogs and cats have been suggested as a zoonotic source of HEV infection. The aim of this study was to assess Orthohepevirus circulation, including HEV-A, HEV-B and HEV-C species, in sympatric urban cats and dogs in southern Spain. Between 2017 and 2020, blood samples were collected from 144 stray cats and 152 dogs, both strays and pets. The presence of antibodies against HEV were tested using a double-antigen sandwich ELISA and seropositive samples were further analyzed by western blot. A RT-PCR was performed to detect RNA of Orthohepevirus species (HEV-A, HEV-B and HEV-C). A total of 19 (6.4%; 95%CI: 3.6-9.2) of the 296 animals tested showed anti-HEV antibodies by ELISA. Seropositivity was significantly higher in dogs (9.9%; 15/152; 95%CI: 5.1-14.6) than in cats (2.8%; 4/144; 95%CI: 0.1-5.5). Ten out of the 18 ELISA-positive animals that could be further analyzed by western blot, reacted against HEV-3 and/or HEV-C1 antigens, which suggest circulation of both genotypes in urban cats and dogs in the study area. However, HEV-A, HEV-B and HEV-C RNA was not detected in any of the tested sera. This is the first study to assess HEV circulation in both stray cats and dogs in Europe. Our results provide evidence of HEV exposure in sympatric urban cat and dog populations in southern Spain. Further studies are needed to determine the role of these species in the epidemiology of HEV. This article is protected by copyright. All rights reserved

Hepatitis E (HE) is an important emerging disease in European countries. To analyse the role of equids as potential reservoirs for HE virus (HEV), we determined the prevalence of HEV infection in 861 equines from 464 herds in Spain. HEV RNA in serum was detected in 0.4% (3/692) of horses, 1.2% (1/86) of donkeys and 3.6% (3/83) of mules. Phylogenetic analysis identified the zoonotic genotype 3 as being closely related to viral human and swine strains. In this first report on HEV in equids in Europe, we confirm the susceptibility of horses, donkeys and mules to HEV infection. The low prevalence detected indicates that equids may be considered spillover hosts rather than true reservoirs.