Initially, we screened 85 fecal samples collected during 2006 through mid-2012 from farmed mink by nested PCR; a broad panel of HEV variants was detected (6). One sample was positive, and subsequent sequencing and phylogenetic analysis showed that this virus represented a new HEV variant. To screen more samples for this new virus, we developed a specific real-time reverse transcription PCR (RT-PCR) (Technical Appendix). The initially tested 85 fecal samples and an additional 233 fecal samples from farmed mink, together with liver and fecal samples from 89 wild mink, were tested with this new and more sensitive assay. We identified 4 positive samples, all from farmed mink. In addition, screening with an HEV real-time RT-PCR (9) specific for G1–G4 found none positive. The HEV-infected mink were all submitted for diagnostic examination; all had histories of diarrhea in the herd. Three submissions were from herds having mink enteritis virus. Lipidosis, Aleutian mink disease virus, and catarrhal enteritis also were diagnosed in the mink (online Technical Appendix Table).

The 4 samples positive for the novel HEV variant were collected during 2008–2011 from herds across Jutland, Denmark, with a minimum distance of 80 km between the herds. The 4 PCR products obtained by the nested PCR, covering a region of 261 bp of the RdRp gene, were cloned and sequenced (GenBank accession nos. KC802090, KC802091, KC802092, and KC802093). The sequences were 98%–100% identical, with only 1 nonsynonymous mutation, resulting in a neutral amino acid change from isoleucine to valine (Technical Appendix Figure). The high homology in this region is not surprisingbecause the gene encodes the RNA polymerase. We initially tried to uncover a larger fragment by primer walking, but the limited amount of material prohibited this.

Figure

Figure. . Phylogenetic tree showing the relationship between the novel mink hepatitis E virus (HEV), other HEV variants, and the 4 known HEV genotypes based on 261 bp of the RdRp gene....

On the basis of the 261-bp fragment, we analyzed the phylogenetic relationship of this novel mink HEV variant to variants found in other animals (Figure). The mink HEV variant clustered with HEV variants found in ferrets and rats, which grouped in a separate branch that was clearly distinct from other previously described HEV variants. At nucleotide level, the mink HEV variant was ≈65% identical to the closest classical HEV genotype (G3 and G4) and 76% and 69% identical with ferret and rat HEVs, respectively. At the amino acid level, the homologies were more pronounced, showing ≈87% and ≈78% identity with ferret and rat HEV variants, respectively. The grouping of the HEV reference sequences in the analysis was identical with results of previously performed phylogenetic analysis on full-length sequences (12).

We detected a variant of HEV in 4 farmed mink from 4 geographically distinct locations in Denmark during a 3-year period, which indicates that the virus has been circulating among mink. Phylogenetic analysis showed that the virus was clearly distinct from, but closely related to, ferret and rat HEV variants recently reported from Germany and the United States (6,7,13).

It has not been possible to infect primates with rat or avian HEV variants (13,14). Thus, because of the phylogenetic resemblance of mink HEV with these nonzoonotic HEV variants, there are no indications that mink HEV can infect humans, although no human samples have been tested specifically for this virus. The zoonotic potential of HEV has been documented only in the case of G3 and G4, which were not found in mink. However, considering the relatively high HEV seroprevalence in humans, the possibility of other variants being zoonotic and cross-reacting with HEV G1–G4 in serologic assays cannot be ruled out.

The mink in this study were from herds that had mink enteritis virus, hepatic lipidosis, Aleutian mink disease virus, and catarrhal enteritis, all factors that could explain the conditions of the mink infected with HEV (15). However, it cannot be ruled out that the mink HEV variant contributed to the clinical signs of the mink HEV-positive animals. To determine whether the virus is indeed capable of inducing clinical signs in mink, the animals need to be experimentally infectedd. However, the rat and ferret HEV variants induced almost no histologic signs in rats after experimental infection, and the ferrets were described as not showing overt clinical signs (7,13). So far, only chickens infected with aHEV and humans infected with HEV G1–G4 have been described as being clinically affected by HEV infections. The possibility exists that the HEV variants recently reported in a variety of different species, including the 1 reported here, could evolve into disease-causing pathogens in animals and possibly also humans.