| Hepatitis E virus (HEV), the pathogen of Hepatitis E, is transmitted primarily by the fecal-oral route and will result in sporadic infection, outbreaks or epidemics in human beings. Mammalian HEV consists of at least four recognized genotypes, namely genotype 1-4 (HEV-1～4). HEV-1～2 are restricted to human, while HEV-3～4, the zoonotic HEV, could infect human, swine, deer, mongoose and other mammals. Swine is the main animal reservoir.China is highly endemic for Hepatitis E. There was a large outbreak of hepatitis E in Xinjiang from 1986 to 1988, leading to 119280 cases with the attack rate of 2.96%. According to the bulletins for the mandatorily reported infectious diseases issued by the Ministry of Health of China, Hepatitis E was increasing from 2000 to 2010. Thus, Hepatitis E has been considered as an important public health issue.In order to determine the distribution of time, space and population, genotype and phylogenetic characteristic of HEV strains between human and swine in Eastern and Southern China, as well as to find out biological footprints of the evolutional dynamics and history in HEV gene sequences, we collected a total of 357 sera of sporadic hepatitis E cases,252 swine feces samples and 3000 swine bile samples in Shanghai, Deqing (Zhejiang Province), Haimen (Jiangsu Province), Anqing (Anhui Province) and Nanning (Guangxi Province) from 2005 to 2009. By using nested reverse transcription polymerase chain reaction (RT-PCR), all the samples were tested for 821 nucleotide (nt) of RNA-dependent RNA polymerase (RDRp) within HEV RNA Opening Reading Frame (ORF) 1. PCR products were sequenced bidirectionally. The aligned sequences and their identities were calculated according to the studied areas and species. A phylogenetic tree constructed in the Maximum Likelihood method was used to analyze the genotype and phylogenetic relationship of human and swine HEV isolates. The variation of sample sequences and reference sequences in the GenBank was also analyzed to figure out the evolutionary pressure, evolutionary rate, evolutionary divergent time and migration history of HEV. It was found that the male patients were more than the female ones with the male-to-female sex ratio of 2.10:1. The average age of patients was (49.2±15.9) years old. Seasonal variation of the sporadic hepatitis E cases was demonstrated by the concentrated cases between February and April. The isolation rate of HEV among hepatitis E cases in Shanghai, Zhejiang, Anhui, Jiangsu and Guangxi was 48.09%, 43.48%,46.59%,77.11% and 28.13% respectively, showing a significant difference among these areas (χ2=30.77, v=4, P＜0.001). Human sample HEV shared 66.54%～70.49%,63.50%～67.65%,69.45%～72.74% and 83.68%～85.63% nucleotide sequence identities with reference HEV-1-4 respectively. All human sample HEV isolates were belonged to HEV-4. The isolation rate of swine HEV in Shanghai, Zhejiang, Anhui, Jiangsu and Guangxi was 5.00%,2.44%,4.00%,6.40% and 1.50% respectively, showing a significant difference among these areas (χ2=19.18, v=4, P=0.001). There was no obvious seasonal variation for the isolation rate of swine HEV. Swine sample HEV shared 66.47%～70.42%,63.74%～67.87%,69.69%～72.96% and 83.55%～85.52% nucleotide sequence identities with reference HEV-1-4 respectively. Two swine HEV isolates (pCN08Jiangsu32 and pCN06Zhejiang7) from Jiangsu and Zhejiang were belonged to HEV-3, while others were belonged to HEV-4. Meanwhile, the nucleotide-substitution mechanism both of human and swine HEV obeys TIM2+G model. The shape parameter of Gamma distribution a for human and swine HEV was 0.1550 and 0.1670 respectively.Nucleotide sequence identities of human HEV within Shanghai, Zhejiang, Anhui, Jiangsu and Guangxi were 84.61%,91.23%,86.83%,88.56% and 94.38% respectively. There were two areas, Zhejiang or Guangxi, where the nucleotide sequence identities of human HEV within the area were higher than the ones between the area and any of other areas, showing geography aggregation. Nucleotide sequence identities of swine HEV from Shanghai, Zhejiang, Anhui, Jiangsu and Guangxi were 88.48%,83.83%,87.43%,89.12% and 90.84% respectively. Only the nucleotide sequence identities of swine HEV within Guangxi were higher than the ones between the area and any of other areas, showing geography aggregation. Furthermore, neither the nucleotide sequence identities within human HEV or swine HEV were higher than the ones between human HEV and swine HEV. HEV from Guangxi clustering together in an evolutionary branch showed the geography aggregation in the phylogenetic tree. Human and swine sample HEV isolates scattered in different evolutionary branches without significant species aggregation. As a result, HEV may be transmitted between human and swine.Based on the analysis of HEV RDRp 821 nt of 303 sample sequences and 134 reference sequences, it was detected that the substitution rate of the third positions of codons was higher than the others, and HEV sequence had the codon usage bias during its replication. It was also indicated that there might be genotype specificity of substitutions in 516-618 nt between HEV-3 and HEV-4 and RDRp 821 nt of HEV-3-4 were dominated by neutrally selected sites. The molecular evolutionary rate of HEV was about (2.07～4.66)×10-3 per site per year. A phylogentic tree constructed in a Bayesian Inference method revealed that the evolutionary divergent time of HEV-1, HEV-3 and HEV-4 was 1958,1921 and 1933 respectively, and the most recent common ancestor of all the analyzed HEV isolates dated back to 1860. Six migration events were further discovered among human HEV in Eastern China:Anhuiâ†'Jiangsu, Jiangsuâ†'Anhui, Jiangsuâ†'Shanghai, Shanghaiâ†'Jiangsu, Shanghaiâ†'Zhejiang and Shanghaiâ†'Anhui. |
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