RV-vsRNA1755 Targeting Rotavirus VP3 To Regulate Viral Replication

Rotavirus(RV)is one of the most important pathogens that cause diarrhea in infants and young children worldwide.During evolution,the virus formed a set of strategies that use host cell metabolic mechanisms to defend against host defenses,including encoding microRNA molecules.In the previous study,we screened the miRNAs that were differentially expressed after RV infection in the host cell MA104.After RV infection it was found that the micro RNA chr-1755 was significantly up-regulated,inferring that the molecule may play an important role in rotavirus replication.After studying the biological properties,we named it vsRNA 1755.The software predicted that rotavirus VP3 might be a potential target for vsRNA1755.Objective:The purpose of this study is to determine whether VP3 is the target of vsRNA1755,and to analyze the specific regulatory mechanism of vsRNA1755's effect on rotavirus replication by regulating its own genes.Methods:The prediction results of miRnada software show that VP3 is the target gene of vsRNA 1755,and the target relationship between vsRNA 1755 and the target protein VP3 is verified by the dual luciferase report detection system.A eukaryotic expression vector carrying VP3 was constructed in vitro,and MA104 cells were co-transfected with vsRNA1755 mimics.The expression of VP3 was detected by Western Blot and RT-PCR,and the targeting relationship between vsRNA1755 and VP3 was clarified.Further,in vitro synthesis of mimics(mimetics)and inhibitors(inhibitors)targeting vsRNA1755 molecules was transfected into MA104 cells,and the rotavirus replication status was detected by immunofluorescence,CPE time,NSP3 copy number,and detected by plaque method and qRT-PCR Progeny virus titers were evaluated for the effect of vsRNA1755 on virus replication.Synthesize agomir(in vivo mimic)and antagomir(in vivo inhibitor)for the vsRNA1755 molecule,and transfer them into Balb/c mice by intragastric administration to up-and down-regulate the expression level of vsRNA1755.The mice were infected with rotavirus SA11 strain 24 hours after gavage.Observe the symptoms and score diarrhea every 12 hours.Press the abdomen to collect the feces of the suckling mice,and then use colloidal gold and ELISA for antigen detection to evaluate the virus detoxification.Two infant rats were sacrificed in each group at 12h,24h,48h and 72h.The small intestine tissues of the infant rats were collected for paraffin section,HE staining,RNA extraction for virus copy number detection,and transmission electron microscopy to observe pathological changes of intestinal villi.Results:MiRNA sequencing analysis of the differential expression of MA104 cells after rotavirus infection confirmed that vsRNA1755 was the research object.Database prediction and dual fluorescein report experiments found that vsRNA1755 can specifically target rotavirus VP3 gene.Protein detection and fluorescence observation verified that vsRNA1755 could regulate RV VP3 expression level in infected cells and pEGFP-N2-VP3 vector.Immunofluorescence results show that vsRN A175 5 up-regulation(Mimics)can inhibit RV replication,CPE time observation test and NSP3 RT-qPCR viral nucleic acid PAGE electrophoresis detection results show that up-regulation vsRNA1755 can inhibit RV RNA replication efficiency,down-regulation vsRNA1755 can promote RV lesion speed.At the same time,plaque detection results showed that vsRNA1755 can also inhibit the titer of RV progeny virus.In vivo experiments found that after vsRNA1755 overexpression,the diarrhea of the suckling mice was reduced,the content of RV antigen in feces was reduced,and the copy number of RV in the small intestine was reduced.Histopathological observations revealed that after overexpression of vsRNA1755,the pathological changes of the small intestine tissue were alleviated,especially in the ileum.Conversely,after inhibiting the expression of vsRNA1755,the diarrhea time of the suckling mice is prolonged,the diarrhea is aggravated,the amount of detoxification in the feces is increased,the virus copy number in the small intestine is increased,and the pathological changes in the small intestine are aggravated,especially the ileal lesions are more serious.Conclusion:In vitro studies have shown that vsRNA1755 can inhibit the expression of viral VP3 protein by targeting rotavirus VP3 gene,and further inhibit the rotavirus replication efficiency,thereby regulating the rate of virus value-added.In vivo experiments have found that suckling mice can suppress rotavirus replication in the small intestine by intragastrically overexpressing vsRNA1755 and then attacking the virus.Up-regulation of the expression level of vsRNA1755 after rotavirus infection in the host is an active regulatory strategy made by the virus,and this regulatory strategy is to limit the rate of virus proliferation,in order to prevent the rapid death of the host cell and not support the virus to continue to proliferate.