Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7–15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications.
Citation: Hakze-van der Honing RW, van Coillie E, Antonis AFG, van der Poel WHM (2011) First Isolation of Hepatitis E Virus Genotype 4 in Europe through Swine Surveillance in the Netherlands and Belgium. PLoS ONE 6(8): e22673. https://doi.org/10.1371/journal.pone.0022673
Editor: Tara C. Smith, University of Iowa, United States of America
Received: January 21, 2011; Accepted: July 1, 2011; Published: August 1, 2011
Copyright: © 2011 Hakze-van der Honing et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was commissioned by the Dutch Ministry of Agriculture Nature and Foods as part of the Wageningen University and Research Centre internal research program “Kennisbasis”. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Hepatitis E Virus (HEV) is a small, spherical, non-enveloped single stranded, RNA virus of 27–34 nm with a genome of about 7.2 kb . It is classified as a Hepevirus in the family of Hepeviridae . The RNA contains a short 5′untranslated region (UTR), three overlapping open reading frames (ORF1, ORF2, and ORF3) and a short 3′UTR with a polyA-tail , . The ORF1 encodes nonstructural proteins, ORF2 encodes the capsid protein, and ORF3 encodes a small cytoskeleton-associated phosphoprotein .
HEV sequences can be classified in four different genotypes, 1, 2, 3 and 4. Genotype1 and 2 circulate primarily in humans, and cause the majority of HEV infections and epidemics in Asia, Africa and Mexico . The relatively conserved genotypes 1 and 2 are subclassified in five (1a–1e) and two (2a–2b) subtypes respectively . Genotype 3 and 4 are primarily detected in swine, genotype 3 is mainly seen in the United States, several European countries and Japan, and genotype 4 is mainly found in Asia . Genotype 3 and 4 are responsible for sporadic cases of acute Hepatitis E in humans. These genotypes are more diverse and are divided in ten (3a–3j) and seven (4a–4g) subtypes .
Hepatitis E virus is an important cause of acute human hepatitis in regions with inadequate water supplies and poor sanitary conditions . The major transmission route of HEV infections is fecal-oral , usually through contaminated water. HEV causes liver inflammation, fever and jaundice in humans. The incubation period of HEV in cases where clinical symptoms arise, is 2 to 8 weeks. Chronic HEV infections have been reported but do not occur frequently . In outbreaks in general the mortality is not really high (1%) , except in pregnant women, where this may reach 25% .
HEV is assumed to be a zoonotic agent: Swine have been successfully infected with human HEV ,  and rhesus monkeys (experimental surrogate for human) have been infected with a swine HEV strain . Eating raw swine liver and consumption of wild boar and deer has led to human cases of HEV genotype 3 , , . Also, in countries previous thought to be non-endemic, 11 to 51% of subgroups like swine workers have antibodies to HEV and a general population seroprevalence of 2 to 25% has been recorded , , , , , . Furthermore, swine origin HEVs are closely related to the human strains that were found in the sporadic human cases of acute Hepatitis E of genotypes 3 and 4 , , . These observations indicate that HEV genotypes 3 and 4 infect across species, and that swine are a potential animal reservoir , , .
Research groups in the Netherlands have characterized different HEV strains in swine, and according to the subtyping suggested by Lu et al. 2005  found subtypes 3a,3c and 3f, and since 2006 also 3e. Most strains where subtyped as 3c and 3f , , , . In Belgium characterization of HEV strains circulating in swine was not yet reported. New HEV strains may emerge and behave different in their host. Therefore it is important to monitor the circulating strains in swine. If one particular strain becomes dominant, this may indicate that such strain is more virulent and possibly more infectious for humans. In addition, to have an insight in the potential exposure of humans to HEVs from swine we wanted to determine the actual prevalence of HEVs in swine production. To estimate approximately the percentage HEV positive pigs that could enter pork industry we tested pigs in slaughterhouses in the Netherlands and Belgium, using a real-time RT-PCR for testing of fecal samples. HEV positive samples were characterized on the ORF2 region of the virus RNA and then phylogenetically compared with known human and swine sequences.
Materials and Methods
Swine fecal samples in The Netherlands were collected as part of an ongoing surveillance for bacterial research. From September 16, 2008, to November 24, 2008, 101 individual fecal pig samples were collected from different pig slaughter houses (and all from different farms). All samples were collected from fattening pigs of 5 to 6 months of age, and after sampling stored for maximum one week at 4°C until testing.
Swine fecal samples in Belgium, from 23 different fattening farms were collected at a slaughterhouse; five samples from each farm, in total 115 samples. The samples were collected from fattening pigs 5 to 6 months of age. Fecal samples were resuspended in phosphate buffered saline and 10% glycerol in a (1∶ 3 dilution), and stored at −70°Cuntil testing.
Molecular Detection of HEV
The fecal samples were mixed to a final dilution of 1∶10 in PBS. For the Dutch samples, 100 mg fecal material was mixed with 900 µl PBS and for Belgium samples 300 mg of faeces was mixed with 700 µl PBS. The 1∶10 fecal suspensions were mixed thoroughly and centrifuged in a table top centrifuge for 10 minutes at 1000× g. Two-hundred µl of supernatant was used to extract RNA with the High Pure RNA isolation kit (Roche, Mannheim, Germany). RNA was used immediately for HEV RT-PCR or stored at −70°Cuntil further testing. HEV detection by real-time RT-PCR was performed on undiluted RNA samples with the primers JVHEVF and JVHEVR as described by Jothikumar et al. .
Sequencing and Phylogenetic Analysis
Fecal samples positive for HEV RNA by real-time RT-PCR were amplified using a nested RT-PCR format targeting an ORF2 fragment of HEV. Described briefly: for RT, 2 ul of 10 µM RH-HEV-Rnested primer was added to 6 µl RNA. The solution was heated for 10 minutes at 72°C and after cooling on ice, 12 µl of RTmix was added. The RT mix contained 4 µl 5× first strand buffer (Invitrogen, Breda, The Netherlands), 50 mM DTT, 0,5 mM dNTPmix(TaKaRa), 40 U RNasine (Promega, Madison, USA) and 200 U MMLV reverse transcriptase (Invitrogen, Breda, The Netherlands). The RT reaction was performed at a final volume of 20 µl. The mixture was incubated for 10 minutes at 20°C followed by 60 minutes at 42 C, heated for 5 minutes at 95°C and then placed on ice. Five µl of the RT mixture was added to the PCR mix. The first PCR was done with 15 pmol of each primer TqFwd: CTG TTY AAY CTT GCT GAC AC  and RH-HEV-Rnested: GAG ACA TAC ATA GGG TTG GT. The PCR mix contained 10× PCR buffer (Invitrogen, Breda, The Netherlands), 2 mM MgCl2, 0,2 mM dNTP and 2,5 U Taq DNA polymerase. The PCR reaction was performed in a final volume of 50 µl. Cycling conditions were denaturation at 95°C for 5 minutes followed by 55 amplification cycles (95°C, 1 minute, 56°C, 1 minute and 72°C, 1 minute 30 seconds), and finally heating at 72°C for 5 minutes. Five µl of the first PCR reaction is used in the nested PCR. In the nested PCR the primers ORF-s1 and ORF1-a1 were used , . With this PCR cloned fragment of 197 nucleotides was obtained. The PCR product was separated in a 1.5% agarose gel and visualized with UV after ethidium bromide staining. Positive RT-PCR products were excised from the gel, purified by using a Gel DNA Recoverykit (Zymo Research, CA, USA), and sequenced in both directions subsequently. Sequences were submitted to Genbank.
Nucleotide sequences were aligned and clustered using Bionumerics vs 5.1 (Applied Maths) using the Jukes and Cantor correction for evolutionary rate. Evolutionary trees were drawn using Neighbor-Joining clustering. The confidence values of the internal lineages were calculated by performance of 1000 bootstrap analyses.
Additional sequencing of genotype 4 isolate
For further characterization of the genotype 4 isolates additional sequencing was performed on isolate BeSW67HEV4-2008. On the basis of an alignment of 20 HEV genotype 4 full length sequences (data not shown) the primers HEVF1 GGCCTCACWACTGCTATTGAGC(57), HEVR1 GCRTCYTCRGARGCRfTTCCA(1129) and HEVF1nest GCCTTGGCGAATGCTGTG (105), HEVR1nest GYCTGTCCCATATATGCAGGGAC(991) were used to amplify an 887 bp fragment. For RT, 2 µl of 10 µM HEVR1 primer was added to 6 µl RNA. The solution was heated 10 minutes at 72°C and after cooling on ice, 12 µl of RTmix was added. The RT-mix contained 4 µl 5× first strand buffer (Invitrogen, Breda, The Netherlands), 50 mM DTT, 0,5 mM dNTPmix(TaKaRa), 40 U RNasine (Promega, Madison, USA) and 200 U MMLV reverse transcriptase (Invitrogen, Breda, The Netherlands). The RT reaction was performed at a final volume of 20 µl. The mixture was incubated for 10 minutes at 20°C followed by 60 minutes at 42 C, heated for 5 minutes at 95°C and then placed on ice. Five µl of the RT mixture was added to the PCR mix. The first PCR was done with 1 µM HEVF1 and HEVR1 primer, the PCR mix contains 10× pcr buffer (Invitrogen, Breda, The Netherlands), 1,5 mM MgCl2, 0,2 mM dNTP and 2,5 U Taq DNA polymerase. The PCR reaction was performed in a final volume of 50 µl. Cycling conditions were 93°C for 3 minutes and 45 amplification cycles (95°C, 30 seconds, 56°C, 30 seconds and 72°C 1 minute 30 seconds), followed by 7 minutes 72 C. Five µl of the first PCR reaction was used in the nested PCR. In the nested PCR the primers HEVFnest1 and HEVRnest1 were used, the cycling conditions were the same as the first PCR round. The PCR product was separated in a 1.5% agarose gel and visualized with UV after ethidium bromide staining. The product was excised from the gel, purified by using a Gel DNA Recoverykit (Zymo Research, CA, USA), and sequenced in both directions subsequently.
Experimental infection in pigs
To test the infectivity of the hepatitis E virus genotype 4 isolate an experimental infection in pigs was performed. The infection experiments in pigs were approved by the ethics committee of the Animal Sciences Group, part of the Wageningen University and Research Centre, prior to the execution of the experiments (reference number 2009229/2009153.c/EXIHEV, approval date: Dec 10th 2009). The review of animal experiments by this body is fully in compliance with European ethical requirements for animal experiments.
To obtain the inocula one of the HEV genotype 4 positive (RT-PCR) fecal samples from the surveillance study was diluted 1∶10 and centrifuged at 1500× g. Subsequently supernatants were filtered using a 5.0, a 0.45 and a 0.22 micropore filter, respectively. The obtained volume of 4.2 ml and was administered intravenously (Day 0) in two SPF piglets (nrs. 4392 and 4393) (2.1 ml i.v in each pig) of around 4 weeks old. All piglets used for experimental inoculation had been tested previously to be free of HEV (RT-PCR) and HEV specific antibodies (ELISA). A third piglet (4394) was not inoculated and served as a sentinel to detect HEV transmission from the inoculated animals. All three piglets were housed in one pen and were observed clinically and temperatured rectally every day. Blood samples were taken at day 0, 7, 9, 11, 14, 18, 21 and 28 post inoculation. In serum samples Alanine aminotranferase (ALT) and aspartate aminotransferase (AST) levels were analysed longitudinally by a spectrophotometric method in an automated analyser (HumaStar 89, Instruchemie, Delfzijl, The Netherlands) in all three piglets (4392, 4393 and 4394). The reference values in normal pigs used for ALT were 31–58 U/l and for AST 32–84 U/l. Fecal samples were obtained by taking rectal swabs at day 0, 7, 9, 11, 14, 16, 18, 21 and 28 post inoculation. All fecal samples were subjected to QPCR testing. In the last phase of the experiment all three piglets were euthanized to be subjected to pathological examinations and tissue sampling. The first piglet was euthanized after 2 weeks in the acute phase of the infection in order to obtain HEV positive tissue samples. The second piglet together with the sentinel piglet were euthanized and subjected to post mortem investigations 4 weeks post infection. Post mortem samples were taken of urine, feces, liver, kidney, and colon. Macroscopic pathological examinations were done in all three animals and all fecal samples, urine and liver, kidney and colon tissue samples were tested for HEV RNA by QRT-PCR (Table 1.). Excreted HEV was characterized through sequencing of PCR amplification product. All remaining samples were stored at −80°C for future studies.
HEV prevalence in swine
In 15% of the101 individual Dutch fecal samples from slaughterhouses HEV was detected by real-time RT-PCR with the primer pair JVHEVF and JVHEVR. In Belgium 7% of the 115 individual samples were designated HEV positive using the same test protocol, and 5 of the 23 farms (21,7%) were designated HEV positive.
Sequencing and Phylogenetic Analysis
All HEV- positive samples were amplified by RT-PCR targeting the ORF 2 region and amplification products were sequenced subsequently. Of 11 (of 15) and 4 (of 8) earlier positive scored samples an HEV ORF2 amplification product was obtained. The ORF2 sequences (Genbank accession HQ842716–HQ842731) were compared with published sequences of HEV genotypes from humans and swine.
Through phylogenetic analysis using the ORF2 region, all eleven Dutch strains were characterized as genotype 3. These strains could be divided in 3 different subgroups, based on the classification scheme proposed by Lu et al . Most of the strains belonged to subgroup 3c, two belonged to 3a and one belonged to 3f. The analyzed HEV strains in swine were genetically closely related to HEV isolates earlier found in humans and swine in the Netherlands (Fig. 1A). In Belgium three (BeSW67HEV4, BeSW68HEV4 and BeSW69HEV4) of the four sequences were classified as genotype 4, (seeming subtype 4b). All of these three sequences came from fecal samples from one farm. These genotype 4 sequences showed the highest similarity (97%) to strain NN1, (DQ289450), which was obtained from a Hepatitis E virus infection in rural communities in South China (Fig. 1B). The remaining Belgium sequence (BeSW60HEV4) was classified as a G3f and was closely related to (95%) NLSW28 (AF33692) (Fig. 1A).
Additional sequencing of Genotype 4 isolate
In addition a part of ORF1 region (bp 105–991) of the isolate BeSW67HEV4 was amplified and sequenced. The obtained sequence was compared with published sequences of HEV genotypes from humans and swine. Through phylogenetic analysis using the ORF1 region, isolate BeSW67HEV4 was characterized as a genotype 4b (Fig. 2.). The sequenced part of isolate BeSW67HEV4 was closely related to the Chinese swine strain swDQ, 90,4% similarity. The ORF2 part gave 93% similarity with strain swDQ. A comparison with the NN1 strain was not possible for this ORF1 region since the sequence thereof was not available for the NN1 strain.
Experimental infection in pigs
To test the infectivity of the hepatitis E virus genotype 4 isolate an experimental infection in pigs was performed. After inoculation, pig fecal samples were collected and tested on day 7, 9, 11, 14, 16, 18, 21 and 28 post inoculation. RT-PCR detections in fecal samples are shown in Table 1. Piglet 4393 started excreting HEV in feces at day 9 post inoculation and Piglet 4392 at day 14 (Table 1). The inoculated piglets (4392 and 4393) demonstrated virus shedding for 3 and 4 days (Table 1.), but this was not observed in the contact (sentinel) piglet. After infection piglet 4393 showed a short peak of fever at day 7 (Fig. 3.). Piglets 4392 and 4394 did not show fever although temperatures of animal 4394 (the sentinel) were relatively higher (Fig. 3.) Elevations of ALT and AST levels were observed in inoculated piglets (4392 and 4393) but not in sentinel piglets (Fig. 4.). Apart from fever no overt clinical symptoms were observed.
The average of the first 3 measurements before inoculation was taken as the baseline value.
a. ALT values in serum of experimentally HEV Gt4 infected piglets (nrs. 4392 and 4393) and sentinel piglet (4394). b. AST values in serum of experimentally HEV Gt4 infected piglets (nrs. 4392 and 4393) and sentinel piglet (4394).
Hepatitis E virus genotype 4 sequences obtained from the fecal samples of the inoculated piglets were all identical to the sequence of the HEV Gt4 strain in the inoculum and the primary HEV Gt4 field sample.
Fecal samples of pigs in slaughterhouses in the Netherlands and Belgium, were tested for HEV using a real-time RT-PCR. HEV positive samples were characterized on the ORF2 region of the virus' RNA and phylogenetically compared with known human and swine sequences. All HEV strains detected in the Netherlands were characterized as genotype 3. All of the analyzed HEV genotype 3 strains in swine were genetically closely related to HEV isolates earlier found in humans and swine in the Netherlands. In earlier phylogenetic analyses in the Netherlands ,  the most often detected genotype was 3f whereas in this study 3c was the most often detected subtype. The HEV strains found in the Belgium samples belonged to genotype 3f and genotype 4b. Since earlier information on HEV sequences from Belgium could not be retrieved comparison with such could not be made.
The detection of HEV genotype 4 in swine in Belgium was a remarkable finding. It was the first discovery of HEV genotype 4 strain in swine in Europe.
The virus readily infected swine and was isolated through swine inoculation.
In this study the virus was not naturally transmitted to a contact piglet, probably due to the short contact time.. However, it is likely that the virus will be transmitted in the field where exposures will be more frequent and last much longer. The introduction of HEV genotype 4 in swine in Europe could be an indication that genotype 4 viruses may spread over Europe and may be also to North America where genotype 3 prevalence is not much different from Europe. As a result, apart from zoonotic HEV genotype 3 in infections, we may expect zoonotic HEV genotype 4 infections in Europe as well. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications. In this study alanine aminotranferase (ALT) and aspartate aminotransferase (AST) levels in HEV Gt4 experimentally infected piglets showed elevations which were higher than earlier reported for experimental HEV Gt3 infections , and may indicate more severe liver damage caused by HEV4 infections compared to HEV3 infections.
It has remained unclear how HEV genotype 4 was introduced in swine in Europe. The closest sequence was found in China but the introduction route in Europe remains unknown. To date it can only be speculated what may have been the route of introduction. Possible introduction routes include: infected animals or infected human shedding and transmitting the virus into the swine population, HEV positive animal feed or animal produce, or contaminated foods or materials, and may be other. Given the present observations of a HEV genotype 4 sequence in man in Germany  and the observed HEV genotype 4 sequences in swine in Belgium in this study, it can be assumed that HEV genotype 4 circulates in swine in Europe. The German human strain V0716883  is closely related to the Japanese strain HE-JA3 (AB082548), genotype 4f, and there is no correlation between these genotype 4 strains. Looking at the dispersion of HEV genotype 3 in swine in Europe over the last decennium, a wider spread of HEV genotype 4 in swine in Europe may be expected. On the other hand, given the cross reactivity between HEV genotype 3 and genotype 4 strains , a rapid increase of HEV genotype 4 prevalence in swine in Europe may not be likely. In countries where both HEVGt3 and HEV Gt4 are detected these genotypes have been circulating together for a long time. Continued surveillance of HEV in swine will be needed to monitor changes in prevalences of the different genotypes.
This study indicates that a basic HEV surveillance in the animal reservoir can lead to the early detection of a previously undetected emerging zoonotic virus in the surveyed region. An observation that may have important public health implications.
This study was performed as part of the Wageningen University and Research Centre internal research program.
Conceived and designed the experiments: WHMvdP AA RHvdH EvC. Performed the experiments: WHMvdP AA RHvdH. Analyzed the data: WHMvdP RHvdH. Contributed reagents/materials/analysis tools: WHMvdP RHvdH EvC. Wrote the paper: RHvdH WHMvdP.
- 1. Emerson SU, Purcell RH (2003) Hepatitis E virus. Rev Med Virol 13: 145–154.SU EmersonRH Purcell2003Hepatitis E virus.Rev Med Virol13145154
- 2. Emerson SU, Anderson D, Arankalle A, Meng XJ, Purdy M, et al. (2004) Genus Hepevirus. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. 2004. London: Elsevier/Academic Press. pp. 853–857.SU EmersonD. AndersonA. ArankalleXJ MengM. Purdy2004Genus Hepevirus.CM FauquetMA MayoJ. ManiloffU. DesselbergerLA BallVirus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses2004LondonElsevier/Academic Press853857
- 3. Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, et al. (1999) Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 185: 120–31.AW TamMM SmithME GuerraCC HuangDW Bradley1999Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome.Virology18512031
- 4. Meng XJ (2010) Recent advances in Hepatitis E virus. J Viral Hepat 1: 153–161.XJ Meng2010Recent advances in Hepatitis E virus.J Viral Hepat1153161
- 5. Lu L, Li C, Hagedorn CH (2006) Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol 16: 5–36.L. LuC. LiCH Hagedorn2006Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis.Rev Med Virol16536
- 6. Worm HC, Van der Poel WHM, Brandstätter G (2002) Hepatitis E: an overview. Microbes Infect 4: 657–666.HC WormWHM Van der PoelG. Brandstätter2002Hepatitis E: an overview.Microbes Infect4657666
- 7. Meng XJ, Halbur PG, Haynes JS, Tsareva TS, Bruna JD, et al. (1998) Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Arch Virol 143: 1405–1415.XJ MengPG HalburJS HaynesTS TsarevaJD Bruna1998Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV.Arch Virol14314051415
- 8. Kamar N, Selves J, Mansuy JM, Ouezzani L, Péron JM, Guitard J, et al. (2008) Hepatitis E virus and chronic hepatitis in organ-transplant receipients. N Engl J Med 358: 811–817.N. KamarJ. SelvesJM MansuyL. OuezzaniJM PéronJ. Guitard2008Hepatitis E virus and chronic hepatitis in organ-transplant receipients.N Engl J Med358811817
- 9. Aggarwal R, Naik S, Epidemiology of hepatitis E: current status (2009) J Gastroenterol Hepatol 24: 1484–1493.R. AggarwalS. NaikEpidemiology of hepatitis E: current status2009J Gastroenterol Hepatol2414841493
- 10. Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, et al. (1997) A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci 94: 9860–9865.XJ MengRH PurcellPG HalburJR LehmanDM Webb1997A novel virus in swine is closely related to the human hepatitis E virus.Proc Natl Acad Sci9498609865
- 11. Balayan MS, Usmanov RK, Zamyatina NA, Djumalieva DI, Karas FR (1990) Brief report: Experimental hepatitis E infection in domestic pigs. J Med Virol 32: 58–59.MS BalayanRK UsmanovNA ZamyatinaDI DjumalievaFR Karas1990Brief report: Experimental hepatitis E infection in domestic pigs.J Med Virol325859
- 12. Meng XJ, Halbur PG, Shapiro MS, Govindarajan S, Bruna JD, et al. (1998) Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. J Virol 72: 9714–9721.XJ MengPG HalburMS ShapiroS. GovindarajanJD Bruna1998Genetic and experimental evidence for cross-species infection by swine hepatitis E virus.J Virol7297149721
- 13. Colson P, Borentain P, Queyriaux B, Kaba M, Moal V, et al. (2010) Pig liver sausage as a source of hepatitis E virus transmission to humans. J Infect Dis 15;202(6): 825–34.P. ColsonP. BorentainB. QueyriauxM. KabaV. Moal2010Pig liver sausage as a source of hepatitis E virus transmission to humans.J Infect Dis15;202682534
- 14. Matsuda H, Okada K, Takahashi K, Mishiro S (2003) Severe hepatitis E virus infection after ingestion of uncooked liver from a wild boar. J Infect Dis 188: 944.H. MatsudaK. OkadaK. TakahashiS. Mishiro2003Severe hepatitis E virus infection after ingestion of uncooked liver from a wild boar.J Infect Dis188944
- 15. Tei S, Kitajima N, Takahashi K, Mishiro S (2003) Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362: 371–3.S. TeiN. KitajimaK. TakahashiS. Mishiro2003Zoonotic transmission of hepatitis E virus from deer to human beings.Lancet3623713
- 16. Bouwknegt M, Engel B, Herremans MM, Widdowson MA, Worm HC, et al. (2008) Bayesian estimation of hepatitis E virus seroprevalence for populations with different exposure levels to swine in The Netherlands. Epidemiol Infect 136: 567–576.M. BouwknegtB. EngelMM HerremansMA WiddowsonHC Worm2008Bayesian estimation of hepatitis E virus seroprevalence for populations with different exposure levels to swine in The Netherlands.Epidemiol Infect136567576
- 17. Drobeniuc J, Favorov MO, Shapiro CN, Bell BP, Mast EE, et al. (2001) Hepatitis E virus antibody prevalence among persons who work with swine. Journal of Infectious Diseases 184: 1594–1597.J. DrobeniucMO FavorovCN ShapiroBP BellEE Mast2001Hepatitis E virus antibody prevalence among persons who work with swine.Journal of Infectious Diseases18415941597
- 18. Kuniholm MH, Purcell RH, McQuillan GM, Engle RE, Wasley A, et al. (2009) Epidemiology of hepatitis E virus in the United States: results from the Third National Health and Nutrition Examination Survey, 1988–1994. J Infect Dis 1;200(1): 48–56.MH KuniholmRH PurcellGM McQuillanRE EngleA. Wasley2009Epidemiology of hepatitis E virus in the United States: results from the Third National Health and Nutrition Examination Survey, 1988–1994.J Infect Dis1;20014856
- 19. Meng XJ, Wiseman B, Elvinger F, Guenette DK, Toth TE, et al. (2002) Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. J Clin Microbiol 40(1): 117–22.XJ MengB. WisemanF. ElvingerDK GuenetteTE Toth2002Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries.J Clin Microbiol40111722
- 20. Olsen B, Axelsson-Olsson D, Thelin A, Weiland O (2006) Unexpected high prevalence of IgG antibodies to hepatitis E virus in Swedish pig farmers and controls. Scandinavian Journal of Infectious Diseases 38: 55–58.B. OlsenD. Axelsson-OlssonA. ThelinO. Weiland2006Unexpected high prevalence of IgG antibodies to hepatitis E virus in Swedish pig farmers and controls.Scandinavian Journal of Infectious Diseases385558
- 21. Withers MR, Correa MT, Morrow M, Stebbins ME, Seriwatana J, et al. (2002) Antibody levels to hepatitis E virus in North Carolina swine workers, non-swine workers, swine, and murids. American Journal of Tropical Medicine and Hygiene 66: 384–388.MR WithersMT CorreaM. MorrowME StebbinsJ. Seriwatana2002Antibody levels to hepatitis E virus in North Carolina swine workers, non-swine workers, swine, and murids.American Journal of Tropical Medicine and Hygiene66384388
- 22. Borgen K, Herremans MMPT, Duizer E, Vennema H, Rutjes SA, et al. (2008) Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; A Case Series 2004–2006. BMC Infect Dis 8: 61–71.K. BorgenMMPT HerremansE. DuizerH. VennemaSA Rutjes2008Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; A Case Series 2004–2006.BMC Infect Dis86171
- 23. Widdowson MA, Jaspers WJM, Van der Poel WHM, Verschoor F, de Roda Husman AM, et al. (2003) Cluster of cases of acute hepatitis associated with hepatitis E virus infection acquired in the Netherlands. Clin Infect Dis 36: 29–33.MA WiddowsonWJM JaspersWHM Van der PoelF. VerschoorAM de Roda Husman2003Cluster of cases of acute hepatitis associated with hepatitis E virus infection acquired in the Netherlands.Clin Infect Dis362933
- 24. Dalton HR, Thurairajah PH, Fellows HJ, Hussaini HS, Mitchell J, et al. (2007) Autochthonous hepatitis E in southwest England. J Viral Hepat 14: 304–309.HR DaltonPH ThurairajahHJ FellowsHS HussainiJ. Mitchell2007Autochthonous hepatitis E in southwest England.J Viral Hepat14304309
- 25. Meng XJ (2003) Swine hepatitis E virus: cross-species infection and risk in xenotransplantation. Curr Top Microbiol Immunol 278: 185–216.XJ Meng2003Swine hepatitis E virus: cross-species infection and risk in xenotransplantation.Curr Top Microbiol Immunol278185216
- 26. Widén F, Sundqvist L, Matyi-Toth A, Metreveli G, Belák S, et al. (2010) Molecular epidemiology of hepatitis E virus in humans, pigs and wild boars in Sweden. Epidemiol Infect 14: 1–11.F. WidénL. SundqvistA. Matyi-TothG. MetreveliS. Belák2010Molecular epidemiology of hepatitis E virus in humans, pigs and wild boars in Sweden.Epidemiol Infect14111
- 27. Van der Poel WHM, Verschoor F, van der Heide R, Herrera MI, Vivo A, et al. (2001) Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerg Infect Dis 7: 970–976.WHM Van der PoelF. VerschoorR. van der HeideMI HerreraA. Vivo2001Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands.Emerg Infect Dis7970976
- 28. Rutjes SA, Lodder W, Bouwknegt M, De Roda Husman AM (2007) Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RT-PCR. J Virol Methods 143: 112–116.SA RutjesW. LodderM. BouwknegtAM De Roda Husman2007Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RT-PCR.J Virol Methods143112116
- 29. Bouwknegt M, Lodder-Verschoor F, van der Poel WHM, Rutjes SA, de Roda Husman AM (2007) Hepatitis E virus RNA in commercial porcine livers in The Netherlands. J Food Prot 70(12): 2889–95.M. BouwknegtF. Lodder-VerschoorWHM van der PoelSA RutjesAM de Roda Husman2007Hepatitis E virus RNA in commercial porcine livers in The Netherlands.J Food Prot7012288995
- 30. Rutjes SA, Lodder WJ, Lodder-Verschoor F, van den Berg HH, Vennema H, et al. (2009) Sources of hepatitis E virus genotype 3 in The Netherlands. Emerg Infect Dis 15(3): 381–7.SA RutjesWJ LodderF. Lodder-VerschoorHH van den BergH. Vennema2009Sources of hepatitis E virus genotype 3 in The Netherlands.Emerg Infect Dis1533817
- 31. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR (2006) A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 131: 65–71.N. JothikumarTL CromeansBH RobertsonXJ MengVR Hill2006A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.J Virol Methods1316571
- 32. Gyarmati P, Mohammed N, Norder H, Blomberg J, Belák S, et al. (2007) Universal detection of hepatitis E virus by two real-time PCR assays:TaqMan® and Primer-Probe Energy Transfer. J Virol Methods 146: 226–235.P. GyarmatiN. MohammedH. NorderJ. BlombergS. Belák2007Universal detection of hepatitis E virus by two real-time PCR assays:TaqMan® and Primer-Probe Energy Transfer.J Virol Methods146226235
- 33. Schlauder GG, Desai SM, Zanetti AR, Tassopoulos NC, Mushahwar IK (1999) Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV. J Med Virol 57: 243–51.GG SchlauderSM DesaiAR ZanettiNC TassopoulosIK Mushahwar1999Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV.J Med Virol5724351
- 34. Wichmann O, Schimanski S, Koch J, Kohler M, Rothe C, et al. (2008) Phylogenetic and case-control study on hepatitis E virus infection in Germany. J Infect Dis 198: 1732–1741.O. WichmannS. SchimanskiJ. KochM. KohlerC. Rothe2008Phylogenetic and case-control study on hepatitis E virus infection in Germany.J Infect Dis19817321741