we obtained serum samples from wild boars , roe deer , red deer , and fallow deer during 2 hunting seasons ( season a , 20132014 ; season b , 20142015 ) and analyzed them by using an elisa ( i d screen hepatitis e indirect ; i d vet , grabels , france ) for hev - specific igg ( technical appendix figure 1 ) . of 339 serum samples , 81 ( 23.9% ) were positive for hev igg ; results from 1 sample ( 0.3% ) were questionable . although all wild deer samples tested negative , the proportion of antibody - positive wild boars increased significantly ( p = 0.018 ) from 13 ( 27.1% ; 95% ci 16.5537.65 ) of 48 in season a to 68 ( 51.5% ; 95% ci 44.3458.66 ) of 132 samples in season b , with a mean antibody prevalence of 45.0% ( table 1 ) . the capability of the elisa for detection of hev - specific antibodies in field serum samples from deer was demonstrated by testing of 153 deer serum samples from another hunting area , which led to 3 positive results ( data not shown ) . . we also tested liver and serum samples from 415 animals for the hev genome by using real - time reverse - transcription pcr ( rt - pcr ) ( technical appendix ) . hev rna was detected in 46 ( 11.1% ) animals : 39 ( 16.8% ) of 232 wild boars ( 6/95 [ 6.3% ] , from season a and 33/137 [ 24.1% ] from season b ) ; 5 ( 6.4% ) of 78 roe deer ; and 2 ( 2.4% ) of 83 red deer ( table 1 ) . testing of all available organs from the hev - positive wild boars revealed hev rna in > 89% of the samples . hev rna was detected in all tested muscle samples and in most of the other organ samples of hev - positive deer ( table 2 ) . comparison of viral loads in the organs revealed significantly higher genome copy numbers in wild boar liver ( median 2.26  10 genome equivalents[ge]/g ] ) compared with those for wild boar musculature ( median 4.37  10 ge / g ) or for deer liver ( median 2.22  10 ge / g ) and deer musculature ( median 5.25  10 ge / g)(figure 1 ; technical appendix figure 2 ) . however , the low number of positive deer samples limits the interpretation of the statistical results . * hev , hepatitis e virus . box plot comparison of hev rna load in wild boar and deer specimens from germany , 20132015 . boxes indicate first ( bottom ) and third quartile ; horizontal line within boxes indicate median ; error bars indicate minimum and maximum . a total of 39 of 46 samples were positive in a nested rt - pcr assay targeting the rna - dependent rna polymerase gene in the orf1 ( technical appendix ) that were suitable for sequencing . the amplicons showed nucleotide sequence identities to each other ranging from 73.6% to 100.0% . a phylogenetic tree set up for the samples together with hev subtype reference strains indicated that most sequences cluster in a clade containing subtypes 3c and 3i ( figure 2 ) . within this clade , hev sequences from wild boar and deer from both hunting seasons clustered very closely together . hev isolates from human hepatitis e cases from germany clustered near the wild boar and deer hev sequences ( nucleotide sequence identities up to 86.1% to a german 3f strain and 88.2% to a german 3c strain ) . phylogenetic relationship of hev sequences derived from wild boars and deer from germany , 20132015 . the tree is based on a 280-bp fragment of the orf1 ( rna - dependent rna polymerase gene ) region . the strain designations , animal species ( wb , wild boar ) , sample type , and sampling year ( season a , 20132014 ; season b : 20142015 ) are indicated for the novel strains . the genbank accession numbers , the corresponding hosts , the geographic origins and genotypes are indicated for selected additional strains . reference strains are given in bold ; the genotype of the novel strains is indicated at right . the tree is scaled in nucleotide substitution units and was constructed using megalign software ( clustalw [ http://www.ebi.ac.uk/tools/msa/clustalw2/ ] , iub residue weight table , 1,000 trials and 111 random seeds in bootstrap analysis ) . using a nested rt - pcr assay targeting the methyltransferase gene in the orf1 ( technical appendix ) , we sequenced a pcr product in 18/46 samples . a phylogenetic tree again showed grouping into hev subtype clade 3ci and subtype 3f ( technical appendix figure 3 ) . we detected hev - rna and hev - specific antibodies in a high percentage of wild boars , with a significant difference between the 2 hunting seasons . the detection rates are consistent with previous reports of infection of wild boars in germany ( 911 ) . the data underline the high importance of this animal species in the epidemiology of hev and indicate that wild boars likely represent a persistent reservoir for this virus . the detection of high amounts of hev rna in wild boar liver , other organs , and especially in muscle tissue highlights the high risk that hev can be transmitted to humans through the consumption of meat from these animals that has not been cooked properly . in contrast , only low percentages of samples from roe deer and red deer tested positive for hev in our study . data about hev infection in wild ruminants in europe are rare , but some reports have demonstrated hev infection in several deer species ( 12,13 ) . ( 14 ) reported serologic and molecular evidence for hev infection of the indigenous deer species in germany . we detected hev rna in liver , in several organs , and in muscle tissue of the infected deer species . sequence analysis showed a relationship of hev from deer with human hepatitis e cases from germany . in japan , consumption of deer meat could be linked to acute hepatitis e cases in humans ( 15 ) . taken together , deer are likely to represent a source of hev for humans , and consumption of undercooked deer meat should be considered a risk for acquiring hev infection . analysis of the detected hev sequences indicated that the same strains of genotype clade 3ci circulated in wild boar and deer species . this finding argues against specific hev strains exclusively circulating in deer species ; however , longer sequence parts or whole virus genomes should be analyzed in future studies to support this finding further . the consistently lower hev rna and antibody prevalence in deer than in wild boars indicates a primary circulation in wild boars and only accident transmission to deer . the hypothesis of spillover infections of deer is further supported by the consistent lower viral loads in tissues of infected deer . however , other authors classified deer as a true reservoir for hev ( 8) . further studies investigating more geographic areas over longer time , including the parallel analysis of different animal species , are necessary to unravel the epidemiology and transmission dynamics of hev in wildlife . materials and methods for analyzing serum samples from wild boars , roe deer , red deer , and fallow deer during 20132014 and 20142015 for hev - specific igg , germany .
to determine animal hepatitis e virus ( hev ) reservoirs , we analyzed serologic and molecular markers of hev infection among wild animals in germany . 
 we detected hev genotype 3 strains in inner organs and muscle tissues of a high percentage of wild boars and a lower percentage of deer , indicating a risk for foodborne infection of humans .