Study On The Impact Of Rotavirus Infection On The Expression Level Of RLRs And Its Adaptor Protein,IRFs And IFN-λ

Rotavirus,as a double-stranded RNA(dsRNA)virus of the family Reoviridae,is the main aetiological agent of severe dehydrating diarrhea in infants and young children worldwide.Rotavirus mainly infects small intestinal epithelial cells.Evidence has been accumulated that innate immune responses are critical as a first line of defense,limiting rotavirus replication and disease in the host.Moreover,innate responses are potentially important for the intensity and longevity of adaptive responses.When rotavirus infect the host,firstly,it triggers the first line of defense,mainly induces the expression of some cytokines and chemokines.Interferons(IFNs)are the most important cytokines,and it plays a potential roles in innate immune responses to resist exogenous pathogens.Up to now,IFNs can be divided into three types.Type ⅠIFNs,including IFN-α and IFN-β,they can be a key component of the host defense against viral infection,they possess the powerful antiviral properties.Type Ⅱ IFNs,including IFN-y.Recently,it has been shown that Type Ⅲ IFNs(IFN-λ),including IFN-λ1(IL-29),IFN-λ2(IL-28A),IFN-λ3(IL-28B)and IFN-λ4,are also critical in innate immune responses.Type Ⅲ IFNs(IFN-λ)is much more potent than Type Ⅰ IFNs at controlling rotavirus.The secretion of Type Ⅲ IFNs by rotavirus infection may involve some genes,following rotavirus infection in the cells,RLRs(RIG-I/MDA5)can recognize its dsRNA,then stimulate a sets of cascade,leading to the induction of Type Ⅲ IFNs.Some related genes are involved in this process,including RLRs and its adapter protein(MAVS)and IRFs.In order to investigate the impact of rotavirus infection on the expression of RLRs(RIG-1 and MDA5)and its adaptor protein(MAVS),IRFs and IFN-λ.In-cell western Assay was performed to determine the relative expression level of some related genes,including RIG-I(DDX58),MDA5,MAVS and IRP1/3/7/9.Activated rotavirus strain CC0812-1 isolated by our own laboratory was used to infect RV group intestinal epithelial HT29 cell lines which was planted into 96-well cell plate with a titer of 0.6TCID50/cell and NC group was infected by PBS.At 6hpi,12hpi,18hpi and 24hpi,respectively,all cells were fixed with 3.7%formalehyde,then cells were permeated with TritonX-100,use 5%non-fat milk to block the nonspecific protein.Cells were incubated with primary antibody diluted by PBS overnight,at last add the fluorescence-second antibody and DRAQ5 diluted by PBS.The results shown that at 6hpi,RIG-1 and MDA5 genes relative expression level was down-regulated by rotavirus infection,but as the time goes on,at 12hpi,18hpi and 24hpi those two genes relative expression level were up-regulated by rotavirus infection.At 6hpi,12hpi,18hpi and 24hpi,the relative expression level of MAVS gene was up-regulated by rotavirus infection.Only at 18hpi and 24hpi,the IRF1 relative expression level was changed by rotavirus infection,at 18hpi was up-regulated and down-regulated at 24hpi.For IRF3 gene,at 6hpi and 18hpi,the relative expression were not changed by rotavirus infection,but at 12hpi and 24 hpi,the relative expression of the gene were all down-regulated.IRF7 gene was up-regulated by rotavirus infection at 12hpi and 18hpi,but down-regulated at 24hpi.IRF9 gene only down-regulated by rotavirus infection at 24hpi,there is no change at 6hpi,12hpi and 18hpi.In order to calculate the expression level of IFN-λ1(IL-29),we use activated rotavirus CC0812-1 to infect intestinal epithelial HT29 cell lines with a titer of 0.6TCID50/cell,at 6hpi,12hpi,18hpi and 24hpi,harvest the culture medium which contains nothing but has DMEM basic medium,respectively.A Human IL-29 ELISA Kit was used to measure the content of IL-29 per 100μL.As the results shown,the concentration of IFN-λ1(IL-29)was increased 6.67 fold at 6hpi,7.25 fold at 12hpi,16.39 fold at 18hpi and 1.78fold 24hpi.The concentration of IFN-λ1(IL-29)reached the peak at 18hpi,it is 1049.40 ng/mL.727.10 ng/mL at 6hpi,790.50 ng/mL at 12hpi and 337.35 ng/mL at 24hpi,respectively.Trough the experimental results above,we found that rotavivus infection plays an important impact on the expression level of RLRs and its adaptor protein,IRFs and IFN-λ.