Preliminary Study On The Interaction Between HEV And Platelets

Objectives:To explore whether hepatitis E virus(HEV)can infect platelets,increase platelet activation and exosomes secretion.Whether platelet-derived exosomes can mediate HEV transmission.Methods:Our study established a HEV amplification culture system and HEV RNA qRT-PCR detection method to collect HEV and detect HEV nucleic acid concentration.The healthy human washed platelets were co-cultured with HEV.The HEV RNA load in platelets was detected by qRT-PCR and the growth curve was drawn.The HEV ORF2 protein in platelets was detected by indirect immunofluorescence;flow cytometry was used to detect CD41a and CD62p expression on platelet surface;platelet-derived exosomes incubated with HEV or PBS were isolated and purified by differential centrifugation and immunomagnetic beads,and HEV RNA and HEV antigens were detected by qRT-PCR and ELISA,respectively;the PLC/PRF/5 cells were infected with two groups of exosomes,and qRT-PCR and indirect immunofluorescence were used to detect HEV RNA and HEV ORF2 proteins in PLC/PRF/5 cells,respectively.Results:The results showed that when HEV infection was found to be 4x 109 copies/mL,and the incubation time was 24h,the highest total genome copy of platelet-associated HEV was(2.34±0.39)×107 copies/mL;the total genome copy of platelet-associated HEV on day 1 was lower than day 0,and the difference was statistically significant(P<0.001).On day 1 to day 5,the platelet-associated virus genome copy was stable at 107copies/mL essentially;it was found that the CD41a and CD62p double positive cells were increased by about 10-20%with time.At each time,the experimental group increased by an average of about 4%(P<0.01)compared with the control group;the exosomes secreted by the platelets incubated with HEV containing HEV nucleic acid and protein components,and the PLC/PRF/5 cells infected with positive exosomes were found to be positive for HEV nucleic acid and protein in PLC/PRF/5 cells.Conclusion:The results indicate that HEV can enter platelets,but the mechanism of HEV entering platelets and whether it replicates in platelets needs further study;HEV can increase platelet activation and exosomes secretion;the exosomes secreted by platelets incubated with HEV can mediate HEV transmission in PLC/PRF/5 cells.