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The present nation-wide survey provides insight in the epidemiology of HEV in Portugal and confirms that HEV is endemic in the Portuguese population.

Hepatitis E virus (HEV) genotype 3 is widespread in industrialized countries and widely recognized as a serious public health issue. HEV genotype 3 is excreted in both human and animal stools, having the potential to be introduced in aquatic environments through wastewater discharge. For this reason, waterborne transmission of the HEV genotype 3 is drawing increasing attention from the scientific community. Although studies in Portugal have shown that HEV circulates in both humans and swine, no study has yet focused on the presence of HEV in wastewater. Hence, a total of 60 water samples (influent and effluent) from 15 Wastewater Treatment Plants (WWTP) of different regions of Portugal were tested for the presence of HEV using a broad spectrum real-time RT-PCR with amplification within the ORF2 region of HEV genome. Positive samples were retested by nested RT-PCR with amplification within the ORF1 region, sequenced followed by phylogenetic analysis. HEV sequences were retrieved from two influent samples collected in December 2013 from WWTPs of North and Central of Portugal and classified in subgenotype 3i and 3f. This is the first study showing the presence of HEV in aquatic environments of Portugal.