hepatitis e virus ( hev ) , a member of the genus hepevirus , is a non - enveloped virus with a positive - stranded rna genome approximately 7.2 kb in length .
hev has been the cause of waterborne outbreaks of hepatitis in asia and africa and is a major cause of sporadic hepatitis in these regions .
acute hev infection primarily affects young adults and is generally self - limiting and resolves in 1 - 6 weeks , except in women during late pregnancy , among whom 20% mortality has been reported ; chronic hev infection has recently been reported in transplant recipients ( 1 ) .
it has been hypothesized that zoonosis is involved in the transmission of hev ( 2 ) .
hepatitis e virus and antibodies to hev have been found in a wide variety of animals ( 3 - 6 ) .
hepatitis e viruses were divided into 4 distinct genotypes : genotype 1 , 2 , 3 , and 4 .
genotypes 1 and 2 have been identified exclusively in humans , while genotypes 3 and 4 have been found in humans and several species of animals .
genotypes 1 and 2 have been isolated in asia , africa , and america ; genotype 4 has been identified only in asia ; genotype 3 has been found almost worldwide ( 7 , 8) . since 2000 , genotype 4 hev has become the dominant cause of hepatitis e disease in china ( 9 , 10 ) .
genotype 4 was proposed to be further classified into 24 subtypes ( 11 ) . in the present study , we identified 52 genotype 4 hepatitis e viruses to subtype the hev strains in eastern china .
in the present study , we aimed to investigate the subtype of hev prevalent in eastern china , and phylogenetically analyze the relationship between different hev isolates prevalent in this area .
a total of 125 human serum samples , which were tested to be positive for anti - hev igm using commercial elisas ( wan tai pharmaceutical , china ) , were collected from local centers for disease control and prevention ( cdc ) in jiangsu province by jiangsu cdc from 2010 to 2013 .
a total of 290 swine fecal samples were obtained from pigs ( aged 8 to 26 weeks old ) in zhenjiang city ( 80 samples ) of jiangsu province , anhui province ( 90 samples ) and shanghai city ( 120 samples ) from 2010 to 2012 .
the three areas have the sample culture and interregional mobility of people is very common .
all the fecal samples were converted to 10% ( w / v ) suspensions in 0.01 m phosphate buffered saline ( pbs ) ( ph 7.2 - 7.4 ) .
fecal sample suspensions were then clarified by centrifugation at 10,000 g for 10 minutes and 100 l aliquots of the clarified material was used for viral rna extraction .
total rna was extracted from 100 l of serum sample or clarified fecal suspension , using trizol ( invitrogen , usa ) .
reverse transcription was performed using the takara rna polymerase chain reaction ( pcr ) kit ( takara , japan ) according to the manufacturer s protocol .
briefly , a 348 bp segment of open reading frame 2 ( orf2 ) , was amplified using primers e1 ( 5-aattatgcycagtaycgrgttg-3 ) and e2 ( 5-cccttrtcytgctgmgcattctc-3 ) for the first round of pcr and primers e3 ( 5-gtwatgctytgcatwcatggct-3 ) and e4 ( 5-agccgacgaaatcaattctgtc-3 ) for the second round .
the pcr cycling conditions for both rounds consisted of 35 cycles of denaturation for 30 seconds at 94c , annealing for 30 seconds at 50c , and extension for 40 seconds at 72c .
the expected dna band specific for the hev was excised from the gel , purified with the axyprep dna gel extraction kit ( axygen , usa ) and cloned into a pmd t - vector ( takara , japan ) .
both strands of the inserted dna amplicons were sequenced in a dna analyzer ( applied biosystems 3730 dna analyzer ; invitrogen , usa ) .
phylogenetic trees were constructed using the neighbor - joining method and evaluated using the interior branch test method with mega software version 5.0 .
prototype hev strains used as references in the analysis and their genbank accession numbers are shown in the phylogenetic tree .
a total of 125 human serum samples , which were tested to be positive for anti - hev igm using commercial elisas ( wan tai pharmaceutical , china ) , were collected from local centers for disease control and prevention ( cdc ) in jiangsu province by jiangsu cdc from 2010 to 2013 .
a total of 290 swine fecal samples were obtained from pigs ( aged 8 to 26 weeks old ) in zhenjiang city ( 80 samples ) of jiangsu province , anhui province ( 90 samples ) and shanghai city ( 120 samples ) from 2010 to 2012 .
the three areas have the sample culture and interregional mobility of people is very common .
all the fecal samples were converted to 10% ( w / v ) suspensions in 0.01 m phosphate buffered saline ( pbs ) ( ph 7.2 - 7.4 ) .
fecal sample suspensions were then clarified by centrifugation at 10,000 g for 10 minutes and 100 l aliquots of the clarified material was used for viral rna extraction .
total rna was extracted from 100 l of serum sample or clarified fecal suspension , using trizol ( invitrogen , usa ) .
reverse transcription was performed using the takara rna polymerase chain reaction ( pcr ) kit ( takara , japan ) according to the manufacturer s protocol .
briefly , a 348 bp segment of open reading frame 2 ( orf2 ) , was amplified using primers e1 ( 5-aattatgcycagtaycgrgttg-3 ) and e2 ( 5-cccttrtcytgctgmgcattctc-3 ) for the first round of pcr and primers e3 ( 5-gtwatgctytgcatwcatggct-3 ) and e4 ( 5-agccgacgaaatcaattctgtc-3 ) for the second round .
the pcr cycling conditions for both rounds consisted of 35 cycles of denaturation for 30 seconds at 94c , annealing for 30 seconds at 50c , and extension for 40 seconds at 72c .
the expected dna band specific for the hev was excised from the gel , purified with the axyprep dna gel extraction kit ( axygen , usa ) and cloned into a pmd t - vector ( takara , japan ) .
both strands of the inserted dna amplicons were sequenced in a dna analyzer ( applied biosystems 3730 dna analyzer ; invitrogen , usa ) .
phylogenetic trees were constructed using the neighbor - joining method and evaluated using the interior branch test method with mega software version 5.0 .
prototype hev strains used as references in the analysis and their genbank accession numbers are shown in the phylogenetic tree .
our results indicated that 19.2% ( 24.125 ) of the serum samples were positive for hev rna , while 9.7% ( 28.290 ) of the swine fecal samples were positive for hev rna .
sequence analysis based on the pcr - amplified products ( primer sequences were removed ) indicated that the 52 hev strains , including 24 from humans and 28 from pigs , all belonged to genotype 4 hev , which suggested that genotype 4 was the main genotype prevalent in human and swine population in this area during our sample collecting time .
the 52 genotype 4 hev sequences shared more than 81.5% - 100% sequence identities and showed 49 distinct nucleotide sequences . in order to identify the subtype of the 49 genotype 4 hev strains ,
phylogenetic analysis was performed based on the nucleotide sequences in the present study and other referenced hev strains retrieved from genbank and results were shown in figure 1 .
other 20 well - characterized subtypes of genotype 4 hevs in a previous report ( 11 ) were also included in the phylogenetic analysis , where the genbank accession were not marked with black or squares .
results indicated that the 49 genotype 4 hev sequences in the present study were divided into 6 different subtypes , belonging to subtypes 4b , 4a , 4c , 4e , 4 g and a new subtype , respectively . the phylogenetic tree was produced with a 348 bp orf2 sequence alignments of 49 strains in the present study and the reference sequences from genbank , using the neighbor - joining method and evaluated using the interior branch test method with mega 5 software .
the percent bootstrap support is shown by values at the branch nodes of the tree .
the white squares indicated the sequences from pigs and the black indicated the sequences from humans .
twenty - one of the 49 ( 42.9% ) genotype 4 strains were subtyped as 4b and shared 92.5% - 99.1% sequence similarity among themselves .
the 21 subtype 4b strains included 12 strains from pigs and 9 strains from human populations .
eight strains in the present study felt into the cluster of subtype 4a , and shared 91.1% - 98.6% identities among themselves , including four strains from humans and four strains from pigs .
the subtype 4c included eight strains from the present study , which shared 87.8% - 99.0% sequence identities among themselves , including six human originated and swine originated strains .
interestingly , four strains in this study could not be subtyped based on the defined subtypes , but closely clustered with several other strains in genbank , forming a new subtype , sharing 92.2% - 95.4% sequence identities among themselves .
three strains including two strains from humans and one strain from pig clustered closely together and were subtyped into 4e .
an increasing number of evidences indicate that hev is enzootic and pigs were considered as the major reservoir for hev infections in human populations ( 9 , 13 , 14 ) .
the zoonotic transmission of hev from pigs to humans has been suggested all over the world .
genotype 4 hev was the main cause of hepatitis e in china and involved in zoonotic transmission in several areas of china ( 13 , 14 ) .
the hev isolates identified from the same geographic region tended to cluster together ( 15 , 16 ) .
however , recently several studies suggested the hev strain from a certain limited region may have several different subtypes ( 9 , 17 ) . in the current study , we identified 49 genotype 4 hev strains and phylogenetic analysis showed these isolates could be divided into six different subtypes , among which five subtypes contain isolates from both humans and pigs and the isolates from human and swine population closely clustered together , suggesting these five subtypes of genotype 4 hev were involved in cross - infection between human and pigs in this area .
the first swine strain of hev was isolated and characterized from a pig in the united states ( 2 ) .
subsequently , many hev samples from swine in over a dozen countries have been identified . in the present study , although we did not classify the swine fecal samples according to the pig age because of the label on part of the collection plastic bags were missing unexpectedly , the overall positive rate of hev rna ( 9.66% ) was in agreement with several previously reports in other areas of china ( 13 , 18 ) .
hepatitis e virus isolates from human or pigs were divided into four distinct genotypes according to sequence and phylogenetic analyses .
recent several reports indicated that genotype 3 hev was prevalent in swine and human populations in several regions of china ( 19 , 20 ) .
however , the hev strains in the present study all belonged to genotype 4 , which may owe to the different sampling site and time . in the present study , 42.9% ( 21.49 ) of the genotype 4 strains belonged to subtype 4b , including 12 strains from swine population and 9 strains from human , which suggested that subtype 4b is the main subtype prevalent in swine and human populations in this area .
phylogenetic analysis also revealed that the subtype 4b in the present study clustered with several other genotype 4 hevs isolated from eastern china , while the other subtypes in this study clustered with hevs from other regions in china or other countries , suggesting subtype 4b hevs are indigenous subtype of eastern china while the other subtypes may not be indigenous to this area .
 background : hepatitis e virus ( hev ) , a zoonotic pathogen , uses several species of animal as reservoirs . 
 swine is considered as the major reservoir for hev infection in humans . 
 genotype 4 hev is the dominant cause of hepatitis e disease in humans in china.objectives:although many researches revealed that genotype 4 hev is the main genotype that prevalent in eastern china , few researches have done to study the subtype of hev in this area . 
 thus , this study aimed to investigate the subtype of hev prevalent in eastern china.materials and methods : a total of 125 anti - hev igm positive human serum and 290 swine fecal samples were subjected to reverse transcription polymerase chain reaction ( rt - pcr ) screening of hev rna . 
 positive pcr products were sequenced and phylogenetically analyzed.results:from a total of 125 human serum samples , 19.2% ( 24.125 ) were positive , while 9.66% ( 28.290 ) of the 290 swine fecal samples were positive for hev rna . 
 phylogenetic analysis based on partial capsid gene showed that the 51 hev strains in the current study all belonged to genotype 4 , clustering into 6 different subtypes . 
 our results also revealed that some of hev isolates prevalent in the human and swine populations were classified into the same clusters.conclusions:genotype 4 hev in eastern china shows subtype diversity and some hev isolates are involved in cross - species transmission . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Samples
3.2. Detection of Hepatitis E Virus-RNA
3.3. Phylogenetic Analysis
4. Results
5. Discussion