Study On Antiviral Efficiency And Mechanism Of FNC Against Coxsackievirus B3

ObjectiveViral myocarditis（VMC）,an immune response prevalently triggered by Coxsackievirus B3（CVB3）,manifests as cardiomyocytes necrosis,inflammatory cell infiltration.VMC can further progresses to chronic myocarditis,dilated cardiomyopathy（DCM）and congestive heart failure,which can seriously lead to acute heart failure and even sudden death,especially in adolescents and children.However,the pathogenesis of CVB3-induced myocardial injury is unclear,accurate treatments and efficient medicines are still lacking.It’s extremely significant to explore the pathogenesis of viral myocarditis in order to find safer and more effective therapeutic drugs for clinical use.FNC,2’-deoxy-2’-β-fluoro-4’-azide-nucleoside,a novel nucleoside analogue shown brilliant suppression on HIV and HBV virus in previous study.Nevertheless,researches concerning FNC on CVB3 has never been implemented yet.The study figures out antiviral efficiency of FNC on CVB3 and therapeutic effect on myocardial necrosis and inflammatory infiltration by established viral myocarditis model in vitro and vivo,wich provide theoretical basis for finding more effective and low toxicity antiviral medicine.Method1 In vitro study1.1 Pharmacodynamic experimentTCID₅₀ of CVB3 was calculated by end point dilution assay.Viral myocarditis model in vitro was constructed by Hela cells in good condition infected with CVB3.MTT assay and three different action modes were used to examine antiviral efficiency and mechanism of FNC on CVB3.1.2 Mechanism StudyAccording to effective dose of FNC,RT-PCR assay was performed to detect expression of genes related to virus,immune and inflammation in vitro.Western Blot was acted to detect the influence on expression of NF-κB protein.2 In vivo study2.1 Pharmacodynamic experimentMale Balb/c mice were intraperitoneally injected with CVB3 virus dilution to establish VMC model in vivo.Mental state and weight of mice were observing during a week while animals were intragastric administrated with medicine.Blood collected from eyeballs was used to measure level changes of lactic dehydrogenase（LDH）,creatine kinase（CK）and aspartate transaminase（AST）in serum by ELISA method.Myocardial histopathology was performed to verify the protective effect of FNC on myocardium in VMC mice.2.2 Mechanism StudyRT-PCR assay was used to detect the effect of FNC on expression of viral gene in mice heart,pancreas and liver.ELISA assay was taken to examine the influences on secretion of inflammatory cytokines IL-4,IL-6 and TNF-αin serum.Result1 In vitro study1.1 Pharmacodynamic resultCVB3 virus titer TCID₅₀ was 10^-7.8.TC₅₀ of FNC was 858.517±44.099μg/ml,IC₅₀ was 1.834±1.711 ng/ml,so the TI was 4.681×10⁵ while TC₅₀ of ribavirin was209.117±46.042μg/m,IC₅₀ was 7.343±3.028μg/ml,TI was 28.478.Results showed that FNC can exert good anti-CVB3 activity in three ways:prevention,treatment and direct killing of virus,especially the treatment action.1.2 Mechanism resultRT-PCR results showed that FNC significantly inhibited expression of CVB3virus,immune and inflammation-related genes at the gene level in vitro study.Western Blot experiments showed FNC can effectively inhibit growth of NF-κB protein and exert anti-inflammatory effects at protein level.2 In vivo study2.1 Pharmacodynamic resultVMC mouse model was successfully established by intraperitoneal injection of CVB3 dillution.FNC can effectively relieve symptoms of viral myocarditis and inhibit expression of serum myocardial enzymes.In addition,pathological sections demonstrated the improvement of myocardial necrosis and inflammatory infiltration of FNC.2.2 Mechanism resultFNC not only inhibited replication of CVB3 gene in heart,pancreas and liver of myocarditis mice effectively,but also control the expression of inflammatory factors IL-4,IL-6 and TNF-αin serum at the same time.Conclusion1.FNC has high antiviral activity against CVB3 both in vitro and in vivo.2.FNC exerts anti-CVB3 efficicency primarily by inhibiting viral replication and alleviating inflammation caused by viral infection.3.FNC inhibits the replication of CVB3 by entering in the cell and preventing virus biosynthesis.4.FNC supresses inflammatory response may by down-regulating innate immune recognition receptors,inhibiting NF-κB signaling pathway and decreasing the expression of inflammatory factors.