The Antiviral Effects And Mechanism Of Immunosuppressants On Influenza B Virus

Background: Rejection reaction in solid organ transplantation is the main factor affecting the postoperative survival rate of transplanted organs.The use of immunosuppressive agents can effectively prevent the occurrence of rejection reactions and improve the success rate of transplantation.However,patients undergoing solid organ transplantation have severely impaired immune function due to long-term use of immunosuppressive drugs,which puts them at a higher risk of viral infections compared to healthy individuals.In particular,influenza virus infection is one of the important causes of complications in organ transplant recipients.Globally,influenza-related diseases are mainly caused by influenza A and B viruses,with the latter accounting for one-third of the total cases and having a high fatality rate.Influenza B virus infection can cause complications in organ transplant recipients and increase the failure rate of transplantation.Hence,simultaneous use of immunosuppressive management and antiviral treatment is imperative for this patient population during viral infection treatment.Objective: Immunosuppressive agents exert their effects by inhibiting the immune response.In this study,we first used influenza B virus-infected cells as a model to detect the innate immune response mechanism activated by influenza B virus infection.We then tested the antiviral effects of clinically used immunosuppressive agents and explored their mechanisms of action.We also explored the antiviral mechanisms of immunosuppressive agents with antiviral effects and combined them with antiviral drugs to determine the efficacy of combination therapy.In this research provide important references data for the immunosuppressive management and antiviral treatment of organ transplant recipients at risk of influenza B virus infection.Methods: We used influenza B virus-infected MDCK cells as a model in vitro and used real-time quantitative PCR to detect the activation of the interferon signaling pathway,the expression of interferon-stimulated genes,and the antiviral effects of endogenous interferon produced by influenza B virus-infected cells.We used influenza B virus-infected A549 cells as an vitro model to simulate respiratory epithelial cells and used real-time quantitative PCR and Western blot to detect the RNA replication level of viral after drug treatment and the expression of influenza B virus nucleoprotein(NP)and hemagglutinin(HA)proteins.We used plaque assays to detect changes in virus titers in the A549 cells supernatant and used hemagglutination assays to determine the efficacy of combination therapy.We analyzed the synergistic effects of drugs using the Synergy Finder2.0 software.Results: Influenza B virus infection of MDCK cells can effectively activate the JAK-STAT signaling pathway mediated by type I and III interferons,to achieve the immune response of cells anti-virus.Screening of immunosuppressive agents with antiviral effects showed that three types of immunosuppressive agents,including calcineurin inhibitors,glucocorticoids,and mammalian target of rapamycin inhibitors,had no significant effect on virus proliferation.Mycophenolic acid(MPA)and 6-thioguanine(6-TG)significantly inhibited the proliferation of influenza B virus.MPA significantly inhibited influenza B virus RNA replication and the expression of NP and HA proteins,and the addition of exogenous guanosine weakened this antiviral effect,indicating that MPA’s antiviral effect is achieved by inhibiting the activity of inosine monophosphate dehydrogenase and reducing intracellular guanosine synthesis.6-TG significantly inhibited the expression of HA protein and affected the assembly of virus particles,but had no effect on virus RNA replication.Treatment with the proteasome inhibitor MG132 reduced the inhibitory effect of 6-TG on HA expression.We confirmed that 6-TG inhibits HA protein expression through the ubiquitin-proteasome pathway by demonstrating the interaction between HA protein and ubiquitin through immunoprecipitation.When MPA and 6-TG were combined with the antiviral drug oseltamivir,MPA and oseltamivir showed antagonistic effects,while 6-TG and oseltamivir showed synergistic effects.Conclusions: Influenza B virus-infected cells effectively activate the JAK-STAT signaling pathway mediated by type I and type III interferon to play an antiviral role.After adding different types of immunosuppressants to suppress cellular immune response,most immunosuppressants have no significant inhibitory effect on virus reproduction.Among some commonly used anti-cell proliferation drugs,MPA inhibits the proliferation of influenza B virus by inhibiting the activity of IMPDH and reducing the pathway of intracellular guanosine synthesis.6-TG inhibits the proliferation of influenza B virus by promoting the degradation of HA protein by the ubiquitinproteasome pathway.The combined use of MPA and the antiviral drug oseltamivir produces an antagonistic drug effects;the combined use of 6-TG and oseltamivir produces a synergistic drug effects.