Development Of NSP3-based Rotavirus Vector

Objective:To establish a rotavirus（SA11 strain）reverse genetics manipulation platform,and use the established reverse genetic platform to study the feasibility of rotavirus non-structural protein NSP3 insertion into exogenous genes,and to lay the foundation for rotavirus as a vector for research.Methods:1.In vitro transfection of BHK/T7-9 cells with rotavirus reverse genetics 12plasmid system was used to rescue r SA11 and r SA11/NSP3-EGFP viruses,and the recombinant viruses were biologically characterized by microscopic observation of cell lesions,Western blotting,nucleic acid silver staining,etching assay,electron microscopy and virus one-step growth curve;2.Homologous recombinant construction of plasmid p T7-NSP3-No V-P dimer/SA11 and in vitro transfection of r SA11/NSP3-No V-P dimer for virus rescue.Results:1.r SA11 virus was successfully rescued,compared with wild-type SA11,typical lesions were observed microscopically after r SA11 virus inoculation;VP6 protein expression was identified by Western blotting;r SA11 virus showed a typical 4-2-3-2 distribution by nucleic acid silver staining;r SA11 virus formed obvious empty spots by etching experiment;clear rotavirus particles were observed by electron microscopy.A clear rotavirus particle could be seen;TCID₅₀one-step growth curve revealed that the virus started to proliferate at12 h and reached the plateau stage of virus multiplication at 36 h.2.r SA11/NSP3-EGFP virus was successfully rescued,and compared with wild-type SA11 virus,typical lesions and EGFP expression could be seen microscopically after r SA11/NSP3-EGFP virus inoculation;Western blotting showed that both VP6 protein and EGFP were expressed;r SA11/NSP3-EGFP virus showed typical group A rotavirus nucleic acid bands by nucleic acid silver staining,and the migration of NSP3 bands was observed;etching experiment showed that the number of empty spots of r SA11/NSP3-EGFP virus was slightly less than that of wild-type SA11 virus;electron microscopy One-step growth curve by TCID₅₀method showed that the virus started to proliferate at 12 h and reached the plateau of virus multiplication at 36 h,but its virus titer was slightly lower than that of wild-type SA11 virus.3.The plasmid p T7-NSP3-No V-P dimer/SA11 was successfully constructed,and virus rescue was performed on r SA11/NSP3-No V-P dimer.Virus rescue was performed,but several attempts were made,and no relevant virus was rescued.Conclusion:Two strains of r SA11 and r SA11/NSP3-EGFP viruses were obtained;the rescued r SA11 viruses had similar biological properties to wild-type SA11 viruses;the titers of rescued r SA11/NSP3-EGFP viruses were lower than those of wild-type SA11 viruses;the plasmid p T7-NSP3-No V-P dimer/SA11 was successfully constructed and which was subjected to multiple experiments and did not rescue the associated virus.This provides a basis for the study of NSP3 insertion of exogenous genes,and this study has important scientific value and public health significance for exploring RV virulence,pathogenicity and vector vaccine development.