Epidemiology Study Of Hepatitis E And Vitro-Synthesized RNA Expression Research Followed Genome CDNA Clone Of Hepatitis E Virus Isolated From Jilin Province

To determine the extent of genetic variations and the prevalence of animals HEV infection in JiLin, we developed some methods for the study of HEV in clinical samples, such as ELISA (Enzyme linked immunosorbent assay), RT-PCR, gene cloning, DNA sequencanalysis, indirect immunofluorescence analysis. Using these methods, anti-HEV antibody was tested in sera collected from animals and human with anti-HEV ELISA. HEV RNA was detected by Immune-capture Multiple RT-PCR that was established. The positive PCR products were cloned and sequenced. These sequences were compared with the reported HEV isolates. And Changchun Isolate Ch-S-1 was sequenced completely. For farther study, HEV full-length cDNA clone was constructed, and transcriped in vitro, and its expression in HepG2.To investigate the prevalence of HEV in animals in Ji Lin.The anti-HEV antibody was detested in sera collected from animals with anti-HEV ELISA . HEV RNA was detected by Immune-capture Multiple RT-PCR. PCR products were cloned into pMD18-T vector, and sequenced .These sequences were compared with the reported HEV isolates. It indicated that 427 / 493 swine, 122 / 266 cattle, 70/ 93 sheep, 348/798 deer, 18/369 chicken and 31 /197 horse were positive for anti-HEV antibody. And 5/493 swine samples were positive for HEV RNA. The sequence analysis showed that the nucleotide identity was 91.2～99.1% among them and 77.8%～82.3%,77.2%～78.1%,77.2%～99.1% and 85.2%～95.2% identity to genotypeⅠ-Ⅳrespectively. These data suggest that the seroprevalence of HEV is much higher in pigs than in other animals. HEV sequences isolated from pigs belong to genotypeⅣand closest to isolates from patients with acute hepatitis in Japan .They belong to the same branch through the analysis of phylogenic analysis.The anti-HEV antibody and HEV RNA was also tested in sera collected from human 1621 /7514 samples were positive for anti-HEV. The seroprevalence of anti-HEV was 21.57%.And the seroprevalence of male was significantly higher than that of female .The seroprevalence in persons with age below 20 years old was about 1.00%.Fifteen sera samples of patient were positive for HEV RNA. The sequence analysis showed that the nucleotide identity was 90.3%～99.4% among them, and 81.3%～84% nucleotide identity to CCC220 isolate. The Results indicated that this study area isa place of endemic of hepatitis E, the infection happened mainly among the adult with the age increasing. The prevalence in male of HEV is higher than in female. HEV sequences isolated from persons belong to genotypeⅣand closest to isolates from swine in Jilin. They belong to the same branch through the phylogenic analysis.To analyze the full length sequence of Ch-S-1, isolated from a swine bile in Changchun,4 overlapping fragments of genome were amplified with reverse transcription nested polymerase chain reaction (RT-nPCR) and the 5' and 3'ends were amplified with RACE method. The PCR products were cloned into pMD18-T vector and sequenced. The result showed that Ch-S-1(GenBank ID EF077630) genome consisted of 7261nt excluding the poly (A) tail ,and contained three ORFs(ORF1-3) that encoded proteins of 1706, 674 and 114aa respectively. Sequence analysis revealed that the overall nucleotide identity of Ch-S-1 was 68.9%～72.6% and 83.5%-89.8% with genotype I-III and IV respectively. Compared with genotypes I-III and IV,the ORF 1 of Ch-S-1 shared 78.8%～86.9% and 93.7%～97.6% identity at amino acid level ,the ORF2 shared 87.5%～92.4% and 95.9～98.2% identity at amino acid level and the ORF3 shared 75.2%～84.0% and 97.5%～99.2% identity at amino acid level. The phylogenic tree constructed based on the full genomic sequence confirmed that Ch-S-1 belong to genotype IV and were most closely related to JYI-ChiSai01C, a prototype human HEV strain of genotype IV isolated from patients with acute hepatitis in Japan. Findings of this study, further supported the notion that hepatitis E is zoonosis.HEV has been very difficult to grow in cell culture, so only the most basic knowledge of its molecular biology has been obtained. In order to develop methods of intervention, it is essential to understand the biology of the virus. So a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of a JiLin epidemic isolate was constructed. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. We demonstrated its expression in the transfected cells by strand-specific reverse transcription-PCR and indirect immunofluorescence analysis(IFA).