The Antiviral Effects And Mechanism Of Immunosuppressants On Influenza A(H1N1) Virus In Vitro

Background The influenza virus belongs to the Orthomyxoviridae family,and its infection causes acute respiratory infectious disease known as the flu.Influenza A virus(IAV)is prevalent seasonally every year.Although influenza is usually self-limiting,it can lead to severe cases and even death in special populations.Immunosuppressants are drugs that have immunosuppressive effects and are commonly used to prevent rejection after organ transplantation or to treat autoimmune diseases.The immunosuppressive effect of immunosuppressants,while bringing therapeutic effects,increases the risk of viral infection and severe illness after infection.Objective In this study,IAV-infected cells were used as an infection model to evaluate the antiviral effects of clinically used immunosuppressants,explore their antiviral mechanisms,assess the combined effects of these antiviral immunosuppressants with antiviral drugs,and provide an experimental basis for clinical prevention and treatment of IAV infections in patients undergoing immunosuppressive treatment.Methods The infection model was established by using IAV to infect A549 cells.Using Real-Time PCR to quantify the level of IAV RNA,the virus growth curve was constructed and the inhibitory effect of drugs on virus was evaluated.The IAV viral protein expression was examined by western blot,and plaque assay and Hemagglutination assay were performed to detect changes in the virus titer in the culture supernatant.Synergy Finder 2.0 software was used to analyze the effects of drugs synergy,and Graph Pad Prism 9.0 software was used for data analysis.Results Most clinically used immunosuppressants showed no significant inhibitory effect on influenza virus proliferation.Mycophenolic acid(MPA)can significantly reduce IAV RNA replication and viral protein expression,ultimately achieving inhibition of IAV viral particle assembly and release.The addition of exogenous guanosine weakened the antiviral effect of MPA,indicating that MPA can reduce intracellular guanine nucleotide synthesis by inhibiting IMPDH enzyme activity.6-Thioguanine(6-TG)showed a weak inhibitory effect on IAV RNA replication but can significantly reduce viral hemagglutinin protein expression at a high concentration,thereby affecting IAV viral particle assembly.The proteasome inhibitor MG-132 can block the decrease in hemagglutinin protein induced by 6-TG,indicating that 6-TG can induce ubiquitin-proteasome pathway degradation of hemagglutinin protein.Filgotinib effectively inhibited IAV RNA replication,thereby reducing viral protein expression and viral particle production.Since Tofacitinib showed no significant effect on IAV RNA replication,JAK inhibition made no contribution to the antiviral effect of filgotinib.With the increase of filgotinib dose,the m RNA and protein expression of matrix protein 2(M2)decreased.According to previous research,it suggested that filgotinib can inhibit M2 m RNA splicing and interfere with M2 protein expression.The combination of the three antiviral immunosuppressants and the antiviral drug Oseltamivir showed that the three drugs showed a superposition or synergistic enhancement of antiviral effects when combined with oseltamivir.Conclusions1.The majority of clinically used immunosuppressants have no inhibitory effect on the proliferation of influenza virus.2.MPA inhibits de novo synthesis of intracellular purines,reduces the raw materials for virus RNA replication and transcription,and shows inhibition effect on IAV proliferation.3.6-TG promotes ubiquitin-proteasome degradation of viral glycoproteins,achieving inhibition of IAV proliferation.4.Filgotinib can inhibit the splicing of IAV M2 m RNA,thereby inhibiting the expression of M2 protein and achieving inhibition of IAV proliferation.5.When combined with the anti-influenza drug oseltamivir,MPA,6-TG and filgotinib all show synergistic or additive anti-influenza virus effects.