Part Ⅰ Construction of animal modelObjective In order to study the effect of respiratory syncytial virus nonstructural protein (RSV NS) on the airway of BALB/c mice, the first step was to establish a model for RSV and RSV NS expression in normal mice. We intented to establish animal models by intranasal instillation of RSV and recombinant RSV NS plasmid, and choose an optimal dose of plasmid transfection from 3 different doses for the following experiment.Methods First, we revived the RSV and let it proliferate, in order to get a virus titered at 1×107PFU/ml, meanwhile we extracted the recombinant plasmids for RSV NS1, NS2 that have already been designed by our team previously. Then we instilled the BALB/c mice with 50μl RSV and 8μg/4μg/2μg three different doses of recombinant RSV NS plasmids, establishing a mouse model of RSV infection and transfection of RSV NS plasmids. At 1,3,5,7days post instillation (dpi), mice were sacrificed and their lungs were harvested, left lungs were fixed by 4% paraformaldehyde for the analyses of histopathology and immunohistochemistry, and right lungs were for the extraction of RNA and following PCR progress, aming to verify the model construction.Results (1) Extraction of recombinant plasmids pNS1 and pNS2 was verified by agarose gel electrophoresis with positive expression of target genes. (2) After RSV infection, mouse lung tissue histopathology analysis revealed severe lung inflammation; the immunohistochemistry showed positive expression of RSV antigen. (3) After the transfection of recombinant plasmids, mouse lung tissue histopathology analysis revealed severe lung inflammation; the immunohistochemistry showed positive expression of pNS1-flag and pNS2-HA antigen, in a dose-dependent manner.Conclusion We have successfully established the animal model by intranasal instillation of RSV and recombinant RSV NS plasmid. And the expression of transfected plasmid was positively correlated with the transfection dose of plasmid, so we chose 4μg as the optimal transfection dose of each mouse for the following experiments. The successful construction of animal model has laid foundation for the following study of RSV NS proteins in vivo.Part ⅡRSV NS proteins have pathogenetic effect on miceObjective To explore the effect of respiratory syncytial virus nonstructural protein (RSV NS) on BALB/c mice, and the regulatory effects on the IFN signaling and the distribution of regulatory T cells(Treg), based on the construction of animal model.Methods We extracted the recombinant plasmids for RSV NS1, NS2 that have already been designed by our team previously. Then we instilled the BALB/c mice with 8μg/4μg/2μg three different doses of recombinant RSV NS plasmids, establishing a RSV NS plasmid transfection mouse model. At 1,3,5,7days post instillation (dpi), mice were sacrificed and their lungs and spleens were harvested, left lungs were fixed by 4% paraformaldehyde for the analyses of histopathology and immunohistochemistry, right lungs were for the extraction of RNA and following PCR, extraction of proteins and following Western blot; Spleens were used to produce the suspension of splenocytes for the following flow cytometry analyses of CD4+ CD25+ Foxp3+ regulatory T cells (Treg).Results (1) After the transfection of recombinant plasmids, mice showed an alteration of general state and significant loss of body weight. (2) NS1 inhibited the mRNA expression of IFN-β and Mxl in mice, while NS2 showed an suppressing effect briefly, and upregulated their expression levels later. (3) NS1 and NS2 both induced the expression of SOCS1 in mice. (4) NS1 inhibited the differentiation expression of CD4+ CD25+ Foxp3+ Treg, while NS2 first downregulated the differentiation expression of CD4+ CD25+ Foxp3+ Treg, and then upregulated its expression in later time.Conclusion RSV NS proteins have pathogenetic effect on mice, and this effect is achieved by inhibiting its endogenous IFN signaling pathway to suppress innate antiviral immune response as well as the interaction with CD4+ CD25+ Foxp3 Tregs.Part Ⅲ The effect and mechanism of inhibition of nonstuctural proteins on inflammatory reaction of respiratory tract in RSV infected miceObjective To explore the effect of inhibition of NS proteins on inflammatory reaction of respiratory tract in RSV infected mice and the related mechanisms.Methods First, we revived the RSV and let it proliferate, in order to get a virus titered at 1×107PFU/ml, and synthesized siRNA directed at RSV NS proteins. Then we instilled the BALB/c mice with 50μl RSV, besides, we instilled siRSV-NS 24h prior to RSV challenge. At 1,3,5,7days post infection (dpi), mice were sacrificed and their lungs and spleens were harvested, left lungs were fixed by 4% paraformaldehyde for the analyses of histopathology and immunohistochemistry, right lungs were for the extraction of proteins and following Western blot.Results (1) After the administration of RSV, mice exhibited change of general state and significant weight loss; mice received pretreatment of siRNA-NS showed significant increase of body weight compared with the RSV group. (2) The average optical density of lung tissue in mice received pretreatment of siRNA-NS was significant lighter compared with the RSV group. (3) The inflammation of respiratory tract in mice received pretreatment of siRNA-NS was significant attenuated compared with the RSV group. (4) siRNA-NS significantly inhibited the expression of SOCS1 in lung tissue of RSV infected mice.Conclusion Inhibiting RSV NS protein can obviously alleviate the harm airway inflammation caused by RSV infection in mice. siRNA-NS has obvious prevention and treatment effect of RSV infection. |