The Study Of Epidemiology Of Hepatitis E In Wuhan Region

【Objective】Hepatitis E is a disease caused by hepatitis E virus and is transmitted through the digestive tract. Hepatitis E usually occurs in developing countries where health conditions are relatively poor, but an epidemiological survey showed that Hepatitis E can also occur in developed countries among people without a history of travel to areas of high incidence. In recent years, the number of Hepatitis E patients has shown an upward trend. Many developed countries have reported native cases of Hepatitis E. Its mortality rate is much higher than that of Hepatitis A and Hepatitis B, especially in pregnant women whose mortality rate reaches 20-30%. So far, there is no animal and cell model for hepatitis E virus and its pathogenesis is not clear. In addition, diagnostic criteria and reagent is lacking. Therefore, we hope that we can get some clues through the analysis of a large number of patients with hepatitis E.【Methods】1. This study included 210 hepatitis E patients from Department of Infectious Diseases of Wuhan Tongji Hospital within the period of January 2007 to December 2008. The serum samples from all of the patients with hepatitis E was collected for HEV RNA detection, antibody against hepatitis E virus and biochemical indicator. Use the comparison between deceased group and survival group, univariate and multivariate logistic regression to screen the risk factors for death of hepatitis E. Use the risk factors to construct the probability of death equation for hepatitis E. 2. Use the p239 antigen corresponding to aa368-aa606 of the open reading frame 2 of a genotype 1 strain of hepatitis E virus to construct the indirect enzyme linked immunosorbent assay to detect anti HEV IgA. The cut off value is defined by receiver operating characteristic curve analysis. Then, use the 2210 serum samples with elimination of hepatitis E virus infection as the negative control panel and serum samples collected from 245 hepatitis E patients as positive control panel. The specificity, sensitivity and the value of clinical application was evaluated.3. To construct eukaryotic expression vectors for expressing genotypeⅠhepatitis E virus and genotypeⅣhepatitis E virus recombinant open reading frame 3 fusion protein. Determine the cellular localization of hepatitis E virus open reading frame 3 fusion protein by laser scanning confocal microscope. To detect the affect of proliferation of HepG2 cell line with or without actinomycin D caused by hepatitis E virus recombinant open reading frame 3. To detect the change of NF-κB of HepG2 cell line transfected with hepatitis E virus recombinant open reading frame 3 by western blotting.【Results】1. The sera of 78 patients (37.14%) were positive for HEV RNA. They were all shown to be infected with genotype IV HEV by the bi- directional sequencing and phylogenetic analysis. The total mortality is 10%. Among the 210 of sporadic HE patients,179 cases were male (85.2%) and 31 cases were female (14.8%); the ratio of male to female was approximately 5.8:1. In 2007 and 2008, the cases of HE in the first quarter (January-March) accounted for 45.9%(56/122) and 56.8%(50/88) of the whole year. TBil, BUN, and INR are the major risk factors (adjusted OR=1.0009,1.178 and 9.216, respectively) for death in patients with HE. The predictive values of both HEPOD in the first week of sickness were poor, but it improved in the second week and was better able to predict the prognosis of hepatitis E (AUC=0.957).2. We constructed the anti-HEV IgA indirect ELISA assay to evaluate the significance of anti-HEV IgA. The specificity of anti-HEV IgA was 99.6%. Among 245 AHE patients, 84 samples from 84 patients were positive for HEV RNA. The positive rate of anti-HEV IgA, anti-HEV IgM and anti-HEV IgG in 84 samples positive for HEV RNA was 96.3%,97.6% and 88.1% respectively and no sample was negative for anti-HEV IgA and anti-HEV IgM simultaneously. Among 245 AHE patients, we found 9 samples collected from 9 patients in acute period were negative for anti-HEV IgM but positive for anti-HEV IgA and 2 samples were positive for HEV RNA.3. We construct eukaryotic expression vectors(pEGFP-N1 and pcDNA3.1-HisC) for expressing genotype I hepatitis E virus and genotype IV hepatitis E virus recombinant open reading frame 3 fusion protein. The hepatitis E virus open reading frame 3 fusion protein is only localizes in cytoplasm. There is no affect to proliferation of HepG2 cell line without actinomycin D caused by hepatitis E virus recombinant open reading frame 3 and can improve the proliferation of HepG2 cell line with actinomycin D. P65 expressed in cellular nucleus is more in HepG2 cell transfected with hepatitis E virus recombinant open reading frame 3 than HepG2 cell transfecteed with control plasmid.【Conclusion】1. The hepatitis E patients in Hubei province were all shown to be infected with genotype IV HEV. The first quarter (January-March) is the epidemic season for hepatitis E. TBil, BUN, and INR are the major risk factors for death in patients with HE. The predictive value of HEPOD in the second week was good.2. The anti-HEV IgA indirect ELISA assay was constructed. The specificity and sensitivity of anti-HEV IgA was good. Both specificity and sensitivity of combined detection of anti-HEV IgA and anti-HEV IgM was 100% that can improve the accuracy of diagnosis of hepatitis E.3. The hepatitis E virus open reading frame 3 fusion protein is only localizes in cytoplasm. Hepatitis E virus open reading frame 3 and can improve the proliferation of HepG2 cell line treated with actinomycin D maybe through activation of NFκB.