Rene Ryll, Martin Eiden, Elisa Heuser, Markus Weinhardt, Madlen Ziege, Dirk Hoeper, Martin H. Groschup, Gerald Heckel, Reimar Johne, Rainer G. Ulrich

• Rabbit associated genotype 3 hepatitis E virus (HEV) strains were detected in feral, pet and farm rabbits in different parts of the world since 2009 and recently also in human patients. Here, we report a serological and molecular survey on 72 feral rabbits, collected along a rural-urban transect in and next to Frankfurt am Main, Central Germany. ELISA investigations revealed in 25 of 72 (34.7%) animals HEV-specific antibodies. HEV derived RNA was detected in 18 of 72 (25%) animals by reverse transcription-polymerase chain reaction assay. The complete genomes from two rabbitHEV-strains, one from a rural site and the other from an inner-city area, were generated by a combination of high-throughput sequencing, a primer walking approach and 5′- and 3′- rapid amplification of cDNA ends. Phylogenetic analysis of open reading frame (ORF)1-derived partial and complete ORF1/ORF2 concatenated coding sequences indicated their similarity to rabbit-associated HEV strains. The partial sequences revealed one cluster of closely-related rabbitHEVRabbit associated genotype 3 hepatitis E virus (HEV) strains were detected in feral, pet and farm rabbits in different parts of the world since 2009 and recently also in human patients. Here, we report a serological and molecular survey on 72 feral rabbits, collected along a rural-urban transect in and next to Frankfurt am Main, Central Germany. ELISA investigations revealed in 25 of 72 (34.7%) animals HEV-specific antibodies. HEV derived RNA was detected in 18 of 72 (25%) animals by reverse transcription-polymerase chain reaction assay. The complete genomes from two rabbitHEV-strains, one from a rural site and the other from an inner-city area, were generated by a combination of high-throughput sequencing, a primer walking approach and 5′- and 3′- rapid amplification of cDNA ends. Phylogenetic analysis of open reading frame (ORF)1-derived partial and complete ORF1/ORF2 concatenated coding sequences indicated their similarity to rabbit-associated HEV strains. The partial sequences revealed one cluster of closely-related rabbitHEV sequences from the urban trapping sites that is well separated from several clusters representing rabbitHEV sequences from rural trapping sites. The complete genome sequences of the two novel strains indicated similarities of 75.6–86.4% to the other 17 rabbitHEV sequences; the amino acid sequence identity of the concatenated ORF1/ORF2-encoded proteins reached 89.0–93.1%. The detection of rabbitHEV in an inner-city area with a high human population density suggests a high risk of potential human infection with the zoonotic rabbitHEV, either by direct or indirect contact with infected animals. Therefore, future investigations on the occurrence and frequency of human infections with rabbitHEV are warranted in populations with different contact to rabbits.…