| Hepatitis E virus (HEV), is the pathogen of hepatitis E and one of the major pathogens that cause viral acute hepatitis. Hepatitis E virus is prevalent in developing countries, and sporadic epidemic in developed countries. Now, HEV infection is an important public health and safety concern in many developing countries, however, there is no specific treatment for hepatitis E. Therefore, prevention of HEV infection is the key strategy for HEV treatment. In the present study, the sero epidemiology and molecular epidemiology for pregnant women, chlidren and several animals in Kunming city of Yunnan province we investigated. Phylogenetic relationship of HEV strains endemic in Kunming city were analyized. Truncated capsid protein of HEV ORF2is cloned and expressed in Prokaryotic cell (E.coli), and the antigenicity was analyzed.In this paper:1) The anti-HEV IgG antibodies in sera samples collected from234pregnant women,90children and42tree shrews were determined by ELISA, and anti-HEV IgM antibodies of139pregnant women were determined as well.2) Total RNA of HEV sero-positive pregnant women and168general populations was extracted and HEV RNA was detected by RT-nPCR. Meanwhile, the feces samples of48peacocks,58Larus ridibundus,12tree shrews and180pigs were detected for HEV RNA.3) The nucleotide sequences HEV strains isolated in this study were blasted in NCBI, and the phylogenetic relationship were analysized. Complete genome sequence of one HEV strain endemic in Kunming city was obtained by RT-nPCR, and the molecular characterization were conducted.4) The partial capsid protein gene (ORF2) were amplified and inserted into prokaryotic expression vector pFN18the to construct a recombinant expression verctor pFN18the/ORF2. The trunted capsid protein were expressed in E. coli and purified, its antigenicity was evaluated.Results of this study indicated that:1) The prevalence rate of anti-HEV IgG among pregnant women in Kunming city was11.55%, and3.33%in children (the overall prevalence rate in pregnant women and children was9.26%). There is significant different between pregnant women and children was significant (p<0.05). Pregnant women in their third trimester of pregnancy have the highest HEV IgG positive rate (13.79%),10.31%in their second trimester of pregnancy, and10%in women in their first trimester of pregnancy. The prevalence of anti-HEV IgG was16%among pregnant women of20-25years old,2.94%among pregnant women more than30years old. In children, the prevalence rate of anti-HEV IgG in boys was higher (3.92%) than that in girls (2.56%); but the prevalence in infant less than one year old was4.26%. Tthe prevalence of anti-HEV IgM among pregnant women was1.44%. All the42sera samples collected from tree shrews were negative for anti-HEV IgG antibodies.2) Nine (9/180,5%) feces samples of pigs were positive to HEV RNA by RT-nPCR, but none of peacocks (48), Larus ridibundus (58) and tree shrews (12), even these sero-positive sera and feces samples of168general population.3) Phylogenetic analysis and homologous evolution analysis showed that These HEV strain isolated in this study belongs to HEV genotype4, The complete genome sequence of one epidemic HEV strain in Kunming was obtained by RT-nPCR, which sharing96.2%homology with Xinjiang strain Gu19961(swine).4) ORF2recombinant prokaryotic expression vector pFN18the/ORF2was constructed and fusion expressed with Halo tag in E. coli KRX. The purified (>90%) ORF2protein showed a well reaction with anti-HEV IgG antibody analysized by Western-Blot.In conclusion, the investigation of HEV prevalence in people and animals indicated that people in Kunming city are at a high risk for HEV infection. HEV endomic in Kunming city is primarily HEV genotype4, and one complete genome sequence was obtained, which laid a foundation of further study on HEV prevention and vaccine development. Meanwhile, the trunted recombinant ORF2protein with fine antigenicity was expressed successfully, which will benefit to HEV diagnosis and antigen studies. |
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