Abstract: Objective To investigate the synergistic effect of hepatitis E virus (HEV)-encoded microRNA-A6 (miR-A6) on viral replication in full-length HEV transiently transfected-human hepatocellular carcinoma cell line (HepG2).Methods miR-A6 was synthesized according to the reported sequence. Technologies of recombinant polymerase chain reaction (PCR), gene cloning and in vitro transcription were used to obtain a high concentration of HEV RNA. Proportional mixed HEV RNA and miR-A6 were transiently transfected into HepG2 using pulse current. Immunoglobulin G (IgG) secretion and HEV RNA load in supernatants of cultured HepG2 were measured at hour 24, 48 and 96 after transfection, respectively. Replication of HEV was compared between HepG2 cells with and without miR-A6 transfection. Moreover, anti-A6 was further used to confirm the effect of miR-A6 on the replication of HEV.Results Compared with that in HepG2 transfected with HEV RNA alone, lg HEV RNA increased by 1.57 and 2.44 at hour 48 after co-transfection of miR-A6 and HEV RNA with mass ratio of 0.5∶1 and 1∶1, respectively. Detection of IgG in supernatants was prior to HEV RNA. There was no obvious difference of replication in vitro between HEV type Ⅰ and type Ⅳ, in which HEV RNA replication both peaked up at hour 48 after transfection. The expression of HEV-IgG and HEV RNA were inhibited by 47.12% and 43.3% after addition of anti-A6.Conclusion miR-A6 could significantly increase the replication ability of HEV RNA in full-length HEV transiently transfected HepG2 in vitro, which may lay a foundation for further research on HEV molecular virology and phenotype resistance.

Key words: microRNA, Hepatitis E virus, Full-length amplification, Transient transfection, Replication ability