sixty - three fecal samples were collected from laboratory ferrets ( mustela putorius furo ) at the national institute of infectious diseases , tokyo , japan , on may 24 , 2013 .
these ferrets had been imported from a farm in the united states for influenza research 7 days before sample collection .
fecal specimens were diluted with 10 mmol / l phosphate - buffered saline to prepare a 10% suspension , shaken at 4c for 1 h , and clarified by centrifugation at 10,000  g for 30 min .
the supernatant was passed through a 0.45-m membrane filter ( millipore , bedford , ma , usa ) , and stored at 80c until use .
rna was extracted by using the magna pure lc total nucleic acid isolation kit ( roche applied science , mannheim , germany ) according to the manufacturer s recommendations .
reverse transcription was performed by using the superscript ii rnase h reverse transcription procedure ( invitrogen , carlsbad , ca , usa ) and primer tx30sxn as described ( 14 ) . ferret hev rna was detected by using a nested , broad - spectrum reverse transcription pcr ( 15 ) .
sequences were similar to those detected in ferret serum samples in the united states ( 11 ) , which suggested that the laboratory ferrets were infected in the united states and then transported to japan .
rna from 2 ferret hevs was randomly selected , and the full - length genome was amplified by using reverse transcription pcr with primers based on nucleotide sequences derived from strains from the netherlands and united states ( table 1 ) . sequence of the 5-terminal noncoding regions of the genome was determined by using rapid amplification of cdna ends kits ( invitrogen ) according to the manufacturer s instructions .
all pcr products were purified by using the qiaquick pcr purification kit ( qiagen , valencia , ca , usa ) and cloned into the ta cloning vector pcr2.1 ( invitrogen ) .
nucleotide sequencing was conducted by using an abi 3130 genetic analyzer automated aequencer ( applied biosystems , foster city , ca , usa ) .
* values in parentheses indicate positions of the primer corresponding to the entire genome of hepatitis e virus ( jn998607 ) isolated from ferret . 
both ferret hev genomes consisted of 6,820 nt and a poly ( a ) tail ( genbank accession nos . ab890001 and ab890374 ) , and nucleotide sequence identity was 99.6% .
genomic structure of strains from the united states was similar to that of 2 strains from the netherlands .
the amino acid alignment of orf2 indicated that ferret hev orf2 has an additional 6 amino acids at the n terminus .
however , because the seventh codon is aug , it is unclear which codon was used for the orf2 translation initiation .
the orf1 of strains from the united states encodes 1,589 aa , which is 7 aa shorter than orf1 of both strains from the netherlands .
in addition , the ferret hev strains from the united states have 2 aa insertions between amino acid residues 596597 ( thr ) and 631632 ( ile ) and 9 aa deletions in amino acid residues 645653 ( cys - leu - arg - ser - ser - pro - lys - pro - pro ) , which corresponds to those of strains from the netherlands .
similar to ferret hev from the netherlands , an additional putative 183-aa orf 4 ( nt 30581 ) was found in strains from the united states .
ab890375ab890379 ) showed that nucleotide identities among them were 98.9%99.5% , which indicated that genetically similar ferret hev strains had circulated at the ferret farm in the united states .
nucleotide and deduced amino acid sequence identities between ferret hev from the united states and other hevs are shown in table 2 .
the entire genome of strains from the united states shared relatively high nucleotide sequence identities ( 82.4%82.5% ) with strains from the netherlands .
these trees showed that although strains from the united states were closely related to strains from the netherlands , they formed a new and distinct cluster ( figure ) .
we observed similar phylogenetic clustering when we analyzed nucleotide sequences of orf1 and orf3 separately .
although we can not conclude whether ferret hev from the united states is a new genotype , these results indicated that there is genetic variety in ferret hev .
researchers should also bear in mind that some laboratory ferrets are contaminated with ferret hev .
phylogenetic relationships among genotypes 14 and wild boar , rabbit , rat , avian , bat , and ferret isolates of hepatitis e virus .
phylogenetic trees with 1,000 bootstrap replicates were generated by using the neighbor - joining method ( njplot 2.3 , http://njplot.sharewarejunction.com/ ) based on a ) the entire genome and b ) open reading frame 2 .
we amplified the entire genome of 2 ferret hev strains isolated from laboratory ferrets imported from the united states .
nucleotide sequence comparisons showed that 2 ferret hev strains from the united states had high ( 99.6% ) identity and shared 98.6%100% identities with partial sequences of orf1 that were detected in the united states ( 11 ) , which indicated that genetically similar ferret hev was circulating in laboratory ferrets .
although nucleotide sequence identities of the entire genome for strains from the united states and the netherlands was 82.4%82.5% , orf2 showed relatively high amino acid identities ( 94.2%94.8% ) , which suggested that isolated from the united states and the netherlands share similar antigenicity .
ferret hev  like particles derived from 1 of the isolates from the netherlands were cross - reactive with serum from hev - infected laboratory ferrets in the united states ( 11 ) . in conclusion , we isolated and identified 2 ferret hev strains from laboratory ferrets imported from the united states .
 the complete genome of hepatitis e virus ( hev ) from laboratory ferrets imported from the united states was identified . 
 this virus shared only 82.4%82.5% nt sequence identities with strains from the netherlands , which indicated that the ferret hev genome is genetically diverse . 
 some laboratory ferrets were contaminated with hev . 