The Primary Study Of Entirely Plasmid-based Reverse Genetics System For Rotavirus

Group A rotavirus is one of the main pathogens causing infantile gastroenteritis worldwide.It causes 2 million hospitalized cases and 200,000 deaths each year,resulting in a serious social and economic burden.However,due to the lack of efficient reverse genetics system,the basic research progress of rotavirus replication,pathogenesis and immune mechanism is slow.In 2017,Komoto et al.established a reverse genetics system of rotavirus based on 14 plasmids.However,the system still has some problems,such as low efficiency and low virus titer.Besides the Komoto laboratory,no other laboratory was reported to successful use of the system for virus research.In order to establish a entirely plasmid-based reverse genetics system,we tried to construct a 14-plasmids reverse genetics system according to Komoto's idea,but co-transfected cells of the 14-plasmids failed to produce infectious virus particles and the expression of viral proteins was not detected.Subsequently,we optimized the 14 plasmids system.Firstly,we optimized the transfection cell line and transfection reagents,and determined that the cell line was BSR-T7/5 and the transfection reagent was XtremeGENE.In addition,we tried to add VP 1,VP2,VP3 and VP6 into the reverse genetics system because the infectious virus particles could be produced after the virus double-layer particles transduced into the cells.However,co-transfected of VP1,VP2,VP3,VP6 expression plasmids and 11 transcriptional plasmids could not obtain viral particles;in addition,considering that the number of 14 plasmids co-transfected was too large and the transfection efficiency was low,we designed and cloned 11 transcriptional plasmids of rotavirus to four.The expression of VP2 and VP6 was higher than that of 11 plasmids after co-transfection with four transcriptional plasmids.A few viroid particles were observed in the cells by electron microscopy,but no obvious viroplasm was observed.Detecting the expression of NSP2 protein,we found that there was no e xpression of NSP2 protein in cells,which may be the reason for the absence of viroplasm.In addition,we detected the transcription of the transcription plasmid and found that there was a non-viral sequence at the 3'end of the viral RNA transcribed by the transcription plasmid.The accuracy of 3'terminal sequence of viral gene will directly affect the packaging of genome and further affect the generation of viral particles.In general,The expression level of the optimized four plasmids system was higher than that of the 14 plasmids system,and a few viroid particles could be observed after co-transfection.In addition,we found that the formation of viroplasm and the accuracy of 3'terminal sequence of viral gene sequence directly affect the production of recombinant virus.Therefore,this study provides a new idea for the establishment of a more effi cient plasmid-based reverse genetics system.