| Hepatitis E virus (HEV) is the cause of Hepatitis E (HE). Four genotypes (1-4) have been identified, of which, genotype 4 is the predominant genotype in China. HE is endemic in China and accounted for about 20% of acute viral hepatitis, the mortality rate of HE was 1.4%-5.3%, while it is～25% in pregnant women, which makes HE an important public health problem. A major obstacle that limited the HEV study in China is the lack of a stable and efficient culture system. Although in vitro HEV cell culture have been developed by Tanaka et al. and Shukla et al. in 2007 and 2010, respectively. It is hard for us to get their virus stocks. In this study, we used feces of monkeys with HEV genotype 4 infecrtion to prepare virus stocks and developed an in vitro culture system for HEV genotype 4 in PLC/PRF/5 cells. Using this system, we performed studies on some characteristics of HEV in cell culture.The primary HEV stock was derived from rhesus feces four weeks after inoculation with HEV genotype 4. 1.0×106 copies/ml HEV were inoculated with 15 candidate cell lines and HEV replicated and secreted only in PLC/PRF/5 cells. The inoculation time and temperature was optimized for HEV cell culture. When the inoculation time was 2,4,6 or 24 hours, the positive ratio (Relative Standard Deviation, RSD) of 1.0>×<106copies/ml HEV inoculated PLC/PRF/5 cells was 35%(RSD= 39%),60%(RSD= 23%),95% (RSD= 12%) and 100%(RSD= 0%), respectively. There were significant differences between the positive ratios of 2,4,6 hours inoculation times (p<0.05). When the inoculation temperature was 37℃ or 40℃, the positive ratio and RSD was 35%(RSD= 39%) and 55%(RSD= 20%), respectively. And there were no significant differences. Finally, the incubation time was determinated as 6 hour and the inoculation time was determinated as 40 ℃ or 37℃.Compared to MEM, when mixed DMEM/M199 was used as growth and maintenance medium for PLC/PRF/5 cells, the time of HEV RNA could be detected in culture supernatant moved up from 2 weeks to 1 weeks past inoculation and the time for pORF2 could be detected in culture supernatant moved up from 4 weeks to 2-3 weeks past inoculation. Analysis showed that there were 12 substances in DEMM/M199 medium might affect HEV replication. After test, cholesterol was determined that have the greatest impact. Using MEM containing 2μg/ml cholesterol as growth and maintenance medium, replication of HEV was equivalent to DMEM/M199 medium.There was no significant cytopathic effects observed in HEV inoculated PLC/PRF/5 cells. pORF2 positive fluorescence could be observed in the HEV inoculated PLC/PRF/5 cells by immunofluorescence staining. Compared with control cells by laser co-focal microscope, there were no significant differences in cell roundness and area for HEV inoculated PLC/PRF/5 cells. Infection ratio of HEV inoculated cells was 10.1%. Membrane associated virus particles could be observed in culture supernatant and HEV inoculated cells by transmission electron microscopy (TEM), and cross ELISA results showed that lipid membrane was associated with HEV virions in culture supernatant. The TEM results need to be further confirmed by immune electron microscopy. Neutralization assays showed that one monoclonal antibody (McAb) (McAb 34#) could neutralize HEV infection in PLC/PRF/5 cells, one human serum sample(9EM) with high-titer anti-HEV antibody and other two McAb(McAb 15# and 72#) had partly neutralization activity. Except epitope of one McAb 72# was conformational epitope, the epitopes of McAb 34# and 15# were located at 390aa-607aa of HEV genotype 1 pORF2 and 404aa-612aa of HEV genotype 4 pORF2. HEV was sub-cultured for five passages. There were no significant differences in HEV RNA increase speeds among five passages.There were four IFN-inducible genes up-regulated in HEV inoculated cells, including:IFI27 (7.07-fold), CMPK2 (4.03-fold), IFI6 (3.82-folds) and MX1 (2.75-fold). Therefore, the effect of IFN-α2b treatment on HEV replication was examined. Compared with control group, HEV replication was partly inhibited by IFN-α2b 1-7 days after treatment. After which, the HEV replication in IFN-α2b treated group recovered gradually and reached a equal level to control group 28 days after treatment. In this study, we established and optimized an in vitro culture system for HEV genotype 4. Using this system, we performed primary studies on HEV characteristics in cell culture. And in the subsequent work, we will carry out further and more detailed studies on HEV. |
PDF Full Text Request