in may 2009 , hepatitis  splenomegaly syndrome affected a flock of 37-week - old broiler breeder hens in shandong , china . affected chickens had regressive ovaries , extensive necrosis and hemorrhage of the liver , and enlarged liver and spleen . antibodies against avian hev orf2 were detected in 80 of 94 serum samples from the same chicken flock , according to elisa ( 5,10 ) with the truncated orf2 protein used by guo et al ( 10 ) and chicken serum diluted 1:100 in 0.5% tween-20 phosphate - buffered saline containing 2.5% nonfat dry milk and 10% escherichia coli lysate . on the basis of previous results , we used a cutoff optical density of 0.43 ( 11 ) . using a published method ( 12 ) , we detected an avian hev orf2 rna gene with 242 bp in 7 of 10 fecal and 5 of 8 bile samples . from the bile samples that were positive for the avian hev orf2 gene , we used nested rt - pcr with 5 overlapping fragments to amplify the near - complete genomic sequence of avian hev . primers were designed on the basis of the other 4 avian hev near - complete sequences in genbank ( table 1 ) . the rt - pcr conditions and reaction mixture were designed according to the superscript ii one - step rt - pcr system instructions ( invitrogen , carlsbad , ca , usa ) . to identify the extreme 3 genomic sequence , we used a modified race ( 3 rapid amplification of cdna ends ) technique . the sense primer f5 ( table 1 ) was chosen from the orf2 region , and the antisense primers included a commercially available anchored adaptor primer and an amplification primer ( invitrogen ) . using inner pcr primers , we sequenced the pcr products of 5 fragments in both directions ( table 1 ) ; the sequence data were collected by an abi3730 genetic analyzer ( jinsite biotech co. , nanjing , china ) . * primers f1 - 1 to r1 - 2 , f2 - 1 to r2 - 2 , f3 - 1 to r3 - 2 , and f4 - 1 to r4 - 2 were used to amplify the first , second , third , and fourth fragment of the near - complete avian hepatitis e virus ( hev ) genome . primers f5 , amplification primer ( auap ) , and adapter primer ( ap ) were used to amplify the extreme 3 genomic sequence . primers r1 - 1 , r2 - 1 , r3 - 1 , r4 - 1 , and ap are also reverse transcription primers . sequences of primers were designed according to the sequences of 4 other known avian hev strains . positions of primers located in the complete genome are shown according to the europe avian hev isolate . commercial primer ( invitrogen , carlsbad , ca , usa ) of nonviral origin . we assembled the near - complete genome of avian hev , which was 6,660 nt long including the 3 poly a tail , by using 5 overlapping fragments sequences and lasergene 7.0 editseq computer programs ( dnastar , madison , wi , usa ) and designated it china avian hev ( cahev ) . cahev contained a complete orf1 gene encoding a nonstructural protein of 1,522 aa , an orf2 gene encoding a capsid protein of 606 aa , an orf3 gene encoding a cytoskeleton - associated phosphoprotein of 87 aa , and a 3 noncoding region of 121 nt . the near - complete genomic and different region sequence analyses performed by using clustalw ( www.clustal.org ) and lasergene 7.0 megalign software indicated that cahev shared the highest identity ( 98.3% ) with european avian hev isolate ( eahev ) and 82.0%82.6% with 3 other avian hev isolates ( table 2 ) . moreover , compared with the different regions of 4 other avian hev strains , the orf1 gene of cahev shared 80.7%98.3% nt and 92.7%98.8% aa sequence identities , the orf2 gene shared 84.1%98.5% nt and 98.3%99.7% aa sequence identities , the orf3 gene shared 93.9%98.9% nt and 88.6% 97.7% aa identities , and the 3 noncoding region shared 78.9%97.6% nt identities ( table 2 ) . * hev , hepatitis e virus ; orf , open reading frame ; ncr , noncoding region . avirulent ahev  and prototype ahev are avian hev isolates from the united states , genbank accession nos . ef206691 and am535004 , respectively . aahev , eahev , and cahev are avian hev isolates from australia , europe , and china , genbank accession nos . orf1 of cahev contained most mutations compared with prototype avian hev ( prototype ahev ) ; 5 , 16 , and 29 nonsilent mutations occurred in the methyltransferase , helicase , and rna - dependent rna polymerase ( rdrp ) functional domains , respectively ( data not shown ) . however , only 2 mutations occurred in motif vii of rdrp domain ( figure 1 , panel a ) , which contains 8 motifs responsible for virus replication ( 13 ) . the 2 mutations in motif vii of the cahev rdrp domain are l(1432)m and i(1434)v . australian avian hev isolate ( aahev ) also has the mutation in the latter position and was a transition from i(1433 ) to t ( figure 1 , panel a ) . this position is well conserved among mammalian hev isolates by the presence of v , which is the same as cahev ( figure 1 , panel a ) . amino acid sequence comparison of motif vii in the open reading frame ( orf ) 1 rna - dependent rna polymerase ( rdrp ) region of avian , human , and swine hepatitis e viruses ( hevs ) ( a ) , antigenic domain ii ( b ) , and antigenic domain iv ( c ) in the orf2 region of avian hev . residues that are conserved among avian hev ( ahev ) isolates are shown as the consensus above the sequences ; residues that are conserved in the hev strains are not shown . genbank accession numbers of human and swine hev ( shev ) strains are m73218 ( burma ) , af076239 ( hyderabad ) , d11092 ( china xingjiang ) , m74506 ( mexico ) , af082843 ( prototype shev ) , fj527832 ( china shanghai ) , ab291955 ( japan shinagawa ) , aj272108 ( china t1 ) , ab480825 ( japan he - jf5 ) , and fj763142 ( korea ) . genbank accession numbers of avian hev strains are ef206691 (  avirulent ahev  from the united states ) , am535004 ( prototype ahev from the united states ) , am943647 ( ahev from australia [ aahev ] ) , am943646 ( ahev from europe [ eahev ] ) , and gu954430 ( ahev from china [ cahev ] ) . boxes indicate mutations of cahev compared with different hev strains . in the orf2 region , 6 nonsilent mutations ( c4r , r5 g , g27s , t42a , t303v , and q473 m ) were determined for cahev and compared with prototype ahev . one mutation of q(473)m , in the antigenic domain ii , was seen in eahev and in cahev ( figure 1 , panel b ) . because this domain is unique to avian hev , as predicted by haqshenas et al . ( 10 ) , this point mutation may change the antigenicity of the epitopes in domain ii of the capsid protein . in antigenic domain iv , a mutation of r(600)k occurred in the  avirulent ahev  compared with other 4 avian hev strains , including cahev , from the sick chickens ( figure 1 , panel c ) . this mutation may affect the virulence of avian hev as speculated by billam et al . the 3 putative n - linked glycosylation sites ( nls , nst , and ngs ) are shared between prototype ahev and cahev ( data not shown ) . however , the second site is nnt in avirulent ahev  and aahev strains and is eliminated in the eahev strain ( 9 ) . in human and swine hev strains , these sites are nls ( 1 ) , nlt ( 2 ) , and nls ( 3 ) . recently , the potential n - linked glycosylation in orf2 was shown to prevent formation of infectious particles , but its role in other functions of hev , e.g. , virus virulence and cell tropism , remain to be elucidated ( 15 ) . in the orf3 gene , including only 83 aa , 10 nonsilent mutations were found compared with the prototype ahev , and 9 mutations were the same as eahev ( data not shown ) . phylogenetic trees of the near full - length sequence of avian and mammalian hev strains were constructed by using the neighbor - joining distance method and lasergene 7.0 software . a bootstrap test of 1,000 replicates was used to evaluate the reliability of the groups . avian hev was segregated into a distinct branch separate from mammalian hev ; according to the genotype separation corresponding to their geographic origin suggested by bilic et al . ( 9 ) , cahev belongs to avian hev genotype 3 ( figure 2 ) . phylogenetic trees based on the near - complete genomic sequences of avian hepatitis e virus ( hev ) and 10 human and swine hev isolates . the trees were constructed by the neighbor - joining method with 1,000 bootstrap replicates using lasergene 7.0 ( dnastar , madison , wi , usa ) . the length of each pair of branches represents the distance between sequence pairs ; the units at the bottom of the tree indicate the number of substitution events . avian hev infection of a chicken flock in shandong , china , was identified by detection of avian hev orf2 antibodies and viral rna . a near - complete avian hev genome from the flock was determined , and sequence analysis indicated that this avian hev strain displayed the highest identity ( 98.3% ) with eahev and belonged to avian hev genotype 3 .
avian hepatitis e virus ( hev ) has been identified in chickens ; however , only 4 complete or near - complete genomic sequences have been reported . 
 we found that the near - complete genomic sequence of avian hev in chickens from china shared the highest identity ( 98.3% ) with avian hev from europe and belonged to avian hev genotype 3 .