| Hepatitis E(HE) is a viral hepitis inflammation caused by hepatitis E virus(HEV), with the clinical and epidemiological features of acute viral hepatitis. The clinical symptoms of Hepatitis E are similar to the symptoms of Hepatitis A. Generally, the period of disease of patients with acute Hepatitis E lasts3to4weeks. In addition to the elevated level of aminotransferase inside the body, no significant complications would appear during the period. However, the study also shows that acute hepatitis E would pose threat to pregnant women. During pregnancy, the possibility of acute hepatitis E infection to pregnant women is higher than the rest populations, resulting in an abortion rate close to20%. The immunocompromised patients which are infected with HEV would deteriorate into chronic Hepatitis E. Part of the patients with chronic Hepatitis E, due to the rapid fibrosis of liver, would also deteriorate into liver cirrhosis, which eventually leads to decline of liver functions or even liver failure.Based on the existing studies, HEV totally has4genetypes. In general the genotype4HEV can infect animals and people. It is extremly popular in some areas with poor health condition and serious water-drinking problems, which causes huge social health hazard and economical loses. For the better detection and study of its biological activity, this paper did two aspects of studies.Firstly, according to several published sequences of genotype4swHEV in GeneBank, a pair of primers was designed and synthesized for the conserved open reading frame2(ORF2) of HEV, and a SYBR Green I real-time fluorescent quantitative PCR assay was established. The stability, specificity and sensitivity were evaluated. And meanwhile, the comparison of the Real-time RT-PCR with nested-RT-PCR was performed. The results indicate that the correlation coefficient(R2), the coefficient of variation (CV%) of intra-assay in the same test and the coefficient of variation (CV%) of inter-assay on different tests meet the requirement, which show good stability. Compared with the nested-RT-PCR, the Real-time RT-PCR which was established in this research is better for detection and quantitative analysis for genotype4swHEV.Secondly, with the aim of acquiring pure and high-loaded HEV virants liquid, after rapid centrifugation and filtration of genotype4swHEV positive fecal suspension, we used the fecal supernatant to infect human hepatocarcinoma cell line Smmc-7721, for the purification and replication of viral strains, which successfully established a cell culture system of genotype4swHEV in vitro. After incubation of positive fecal suspension, the massive apoptosis and disruption of infected cells could be found, and we proved that cell Smmc-7721supports replication of genotype4swHEV. The analysis of trend of HEV replication shows that under certain conditions and ranges, the higher incubation load of HEV is, the faster seepd and higher efficiency of viral replication will be. Meanwhile, continuous passaging cell culture for genotype4swHEV was passaged seven-times-in-row, with the highest viral load of HEV reaching2.05×107copies/mL after72days of incubation. Compared with conventional cell culture system of HEV, this established cell culture system in vitro which is based on cell Smmc-7721is better for enormous and continuous culture of genotype4swHEVThroughout this research, a stable real-time luorescent quantitative PCR assay and a cell culture system in vitro of genotype4swHEV has been initially established, which could be used for detection of genotype4swHEV and quantify of increase of genotype4swHEV in cell smmc-7721. This research is not only significant in detection and prevention of hepatitis E, but also conductive to the further research of HEV at molecular level. |
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