Hepatitis E virus (HEV) is a zoonotic virus for which pigs are the primary animal reservoir. To investigate whether HEV occurs in mink in Denmark, we screened feces and tissues from domestic and wild mink. Our finding of a novel HEV variant supports previous findings of HEV variants in a variety of species

Batts

Bilic

Bodewes

Breum

Drexler

Huang

Jesper S. Krog

Johne

Lars E. Larsen

Meng

Purcell

Smith

Solvej Ø. Breum

Trine H. Jensen

Uttenthal

Yoon

Emerging Infectious Diseases

PubMed

Krog, Jesper Schak

Breum, Solvej Østergaard

Jensen, Trine Hammer

Larsen, Lars Erik

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 19, 2017Hepatitis E Virus Variant in Farmed Mink, DenmarkKrog, Jesper Schak; Breum, Solvej Østergaard; Jensen, Trine Hammer; Larsen, Lars ErikPublished in:Emerging Infectious Diseases (Print Edition)Link to article, DOI:10.3201/eid1912.130614Publication date:2013Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Krog, J. S., Breum, S. Ø., Jensen, T. H., & Larsen, L. E. (2013). Hepatitis E Virus Variant in Farmed Mink,Denmark. Emerging Infectious Diseases (Print Edition), 19(12), 2028-2030. DOI: 10.3201/eid1912.130614DISPATCHES2028	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	12,	December	2013Hepatitis E Virus Variant in Farmed Mink, DenmarkJesper S. Krog, Solvej Ø. Breum,  Trine H. Jensen, and Lars E. LarsenHepatitis	 E	 virus	 (HEV)	 is	 a	 zoonotic	 virus	 for	which	pigs	are	the	primary	animal	reservoir.	To	investigate	wheth-er	HEV	occurs	in	mink	in	Denmark,	we	screened	feces	and	tissues	from	domestic	and	wild	mink.	Our	finding	of	a	novel	HEV	variant	supports	previous	findings	of	HEV	variants	in	a	variety	of	species.Hepatitis E virus (HEV, family Hepeviridae) is a main cause of acute liver inflammation in humans. It is a nonenveloped RNA virus with a positive-sense genome of ≈7.2 kb. In 1997, HEV was discovered in pigs (1), and sev-eral studies have since shown that HEV is endemic in pigs and that pigs probably are a major animal reservoir. HEV traditionally had been divided into 4 primary genotypes (G1–G4). G1 and G2 have been found only in humans. G3 has been found globally in a wide range of mammals, in-cluding humans, pigs, deer, rabbits, and mongooses. G4, like G3, has an animal reservoir and has been found in hu-mans, pigs, and wild boars (2).Along with the human and porcine variants, avian HEV (aHEV) has been characterized. It is widespread globally and has been proposed to comprise 3 genotypes (3). Since 2010, several novel HEV variants have been de-scribed in red foxes, cutthroat trout, rats, bats, and ferrets (4–8). All new variants clearly differed from HEV G1–G4, aHEV, and each other. HEV is highly prevalent among pigs in Denmark; 92% of herds are seropositive, and ≈50% of investigated herds had pigs positive for HEV RNA (9). Because HEV is highly prevalent in swine in Denmark, animals fed offal from Danish slaughterhouses will be ex-posed to HEV. Production of mink fur is a major industry in Denmark, and mink are routinely fed a mixed diet, which often includes swine offal. Inappropriate heat-treated swine offal has previously been shown to be the source of swine-related influenza A virus infection in mink (10,11). Thus, we aimed to investigate whether mink in Denmark are in-fected with HEV G1–G4 or other HEV variants by screen-ing fecal and tissue samples from domestic and wild mink.The StudyInitially, 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 sequenc-ing 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 re-verse transcription PCR (RT-PCR) (online Technical Ap-pendix, wwwnc.cdc.gov/EID/articlepdfs/19/12/13-0614-Techapp1.pdf). The initially tested 85 fecal samples and an additional 233 fecal samples from farmed mink, to-gether 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-infect-ed mink were all submitted for diagnostic examination; all had histories of diarrhea in the herd. Three submis-sions were from herds having mink enteritis virus. Lipi-dosis, 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 Jut-land, Denmark, with a minimum distance of 80 km be-tween 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 (online Technical Appendix Figure). The high homology in this region is not surprisingbecause the gene encodes the RNA poly-merase. We initially tried to uncover a larger fragment by primer walking, but the limited amount of material pro-hibited this.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% iden-tical to the closest classical HEV genotype (G3 and G4) and 76% and 69% identical with ferret and rat HEVs, re-spectively. 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).Author	affiliations:	Technical	University	of	Denmark,	Frederiksberg,	 Denmark	 (J.S.	 Krog,	 S.Ø.	 Breum,	 L.E.	 Larsen);	 and	 Aalborg	 University/Aalborg	Zoo,	Aalborg,	Denmark	(T.H.	Jensen)DOI:	http://dx.doi.org/10.3201/eid1912.130614HEV	in	Mink,	Denmark	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	12,	December	2013	 2029ConclusionsWe 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 circu-lating 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 phy-logenetic resemblance of mink HEV with these nonzoo-notic 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 vi-rus, 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 experimen-tally infectedd. However, the rat and ferret HEV variants induced almost no histologic signs in rats after experimen-tal 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 infec-tions. 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.AcknowledgmentsWe thank Mariann Chriél for supplying the samples from wild mink.This project was funded by the Ministry of Food, Agriculture and Fisheries of Denmark (project no. 3304-FVFP-09-F-011).Dr Krog was a PhD student at the National Veterinary Insti-tute of Denmark, and the work presented here is part of a thesis clarifying the zoonotic aspects of hepatitis E virus in Denmark. His primary research interests include HEV infection dynamics in pigs, food safety, and viral contamination of the environment.References  1. Meng X-J, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, et al. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci U S A. 1997;94:9860–5. http://dx.doi.org/10.1073/pnas.94.18.9860  2. Lu L, Li C, Hagedorn CH. Phylogenetic analysis of global hepa-titis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol. 2006;16:5–36. http://dx.doi.org/10.1002/rmv.482  3. Bilic I, Jaskulska B, Basic A, Morrow CJ, Hess M. Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes. J Gen Virol. 2009;90:863–73. http://dx.doi.org/10.1099/vir.0.007179-0  4. Batts W, Yun S, Hedrick R, Winton J. A novel member of the family Hepeviridae from cutthroat trout (Oncorhynchus clarkii). Virus Res. 2011;158:116–23. http://dx.doi.org/10.1016/j.virusres.2011.03.019Figure.	 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.	 The	CLC	Main	Workbench	software	(CLC	 bio,	 Aarhus,	 Denmark)	was	 used	 for	 the	 phylogenetic	analysis.	 Alignments	 were	made	 by	 using	 MUSCLE	algorithm	 (www.drive5.com/muscle/)	 and	 phylogenetic	tree	 was	 made	 by	 using	distance-based	 method	 with	the	 neighbor-joining	 algorithm	and	 bootstrap	 value	 of	 1,000.	Phylogenetic	 analysis	 with	other	 methods	 showed	 similar	results.	 Scale	 bar	 indicates	nucleotide	substitutions	per	site.DISPATCHES2030	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	19,	No.	12,	December	2013  5. Drexler JF, Seelen A, Corman VM, Fumie Tateno A, Cottontail V, Melim Zerbinati R, et al. Bats worldwide carry hepatitis E virus–related viruses that form a putative novel genus within the family Hepeviridae. J Virol. 2012;86:9134–47. http://dx.doi.org/10.1128/JVI.00800-12  6. Johne R, Plenge-Bönig A, Hess M, Ulrich RG, Reetz J, Schielke A. Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broad-spectrum RT-PCR. J Gen Virol. 2010;91:750–8. http://dx.doi.org/10.1099/vir.0.016584-0  7. Raj VS, Smits SL, Pas SD, Provacia LB, Moorman-Roest H, Osterhaus AD, et al. Novel hepatitis E virus in ferrets, the Netherlands. Emerg Infect Dis. 2012;18:1369–70. http://dx.doi.org/10.3201/eid1808.111659  8. Bodewes R, van der Giessen J, Haagmans BL, Osterhaus ADME, Smits SL. Identification of multiple novel viruses in feces of red foxes including a parvovirus and hepevirus. J Virol. 2013;87:7758–64. http://dx.doi.org/10.1128/JVI.00568-13  9. Breum SØ, Hjulsager CK, de Deus N, Segales J, Larsen LE. Hepatitis E virus is highly prevalent in the Danish pig population. Vet Microbiol. 2010;146:144–9. http://dx.doi.org/10.1016/j.vet-mic.2010.05.00210. Chriél M, Jensen TH, Hjulsager C, Larsen LE, Jørgensen PH, Harslund JL, et al. Consequences of outbreaks of influenza A virus in farmed mink (Neovison vison) in Denmark in 2009 and 2010. In: Larsen PF, Møller SH, Clausen T, Hammer AS, Lássen TM, Nielsen VH, et al., editors. Proceedings of the Xth International Scientific Congress in Fur Animal Production. Wageningen (the Netherlands): Wageningen Academic Publishers; 2012. p. 186–9.11. Yoon K-J, Schwartz K, Sun D, Zhang J, Hildebrandt H. Naturally occurring influenza A virus subtype H1N2 infection in a Midwest United States mink (Mustela vison) ranch. J Vet Diagn Invest. 2012;24:388–91. http://dx.doi.org/10.1177/104063871142834912. Smith DB, Purdy MA, Simmonds P. Genetic variability and the classification of hepatitis E virus. J Virol. 2013;87:4161–9. http://dx.doi.org/10.1128/JVI.02762-1213. Purcell RH, Engle RE, Rood MP, Kabrane-Lazizi Y, Nguyen HT, Govindarajan S, et al. Hepatitis E virus in rats, Los Angeles, California, USA. Emerg Infect Dis. 2011;17:2216–22. http://dx.doi.org/10.3201/eid1712.11048214. Huang FF, Sun ZF, Emerson SU, Purcell RH, Shivaprasad HL, Pierson FW, et al. Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. J Gen Virol. 2004;85:1609–18. http://dx.doi.org/10.1099/vir. 0.79841-015. Uttenthal A, Larsen S, Lund E, Bloom ME, Storgård T, Alexander-sen S. Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology. J Virol. 1990;64:2768–79 .Address for correspondence: Jesper S. Krog, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg, Denmark; email: jsck@vet.dtu.dkhttp://www2c.cdc.gov/podcasts/ player.asp?f=8626456Wild Birds and the  Urban Ecology of TicksDr. Sarah Hamer, Assistant Professor and Veterinary Ecologist with the College of Veterinary Medicine at Texas A&M University, discusses her investigation of ticks on wild birds in urban Chicago. 

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