Screening Of Candidate Drugs Against Tick-borne Encephalitis Virus

Background and ObjectiveTick-borne encephalitis is a neurological disease caused by tick-borne encephalitis virus(TBEV).Based on the World Health Organization reports,there are nearly 12000clinical cases each year all over the word.TBE is mainly prevalent in Europe,Russia and the northeast and northwest regions of China.Despite the availability of vaccines for TBE prevention,the incidence of TBE is on the rise due to vaccine coverage is insufficient for many regions.Moreover,with climate change,expanding tick habitats,increasing travel activities and so on,the incidence of TBE increasing globally.Currently,there is no specific antiviral drugs for TBEV treatment.Therefore,there is an urgent need to develop effective anti-TBEV drugs.Cepharanthine is one of the members of bisbenzylisoquinoline alkaloids,with anti-inflammatory,antioxidant,anti-parasitic,antivirus and other biological activities.Since 1950,Cepharanthine has been used to treated various diseases,such as leukopenia,alopecia,snake bite and so on.Studies have shown that Cepharanthine suppresses nuclear factor-kappa B(NF-κB)activation,production of cytokine and expression of cyclooxygenase,all of which are crucial to viral replication and inflammatory response.Ribavirin is a synthetic nucleoside with broad-spectrum antiviral activity,such as Zika virus,Yellow fever virus,Dengue virus,Hepatitis C virus and so on.According to reports,Ribavirin can also promote production of a human myxovirus resistance protein A(Mx A).Mx A is an antiviral protein,about 70 KDa,with broad-spectrum antiviral activity.Many animals are infected with TBEV,such as golden hamsters,Kunming mice,C57BL/6 mice,BALB/c mice and so on.Antiviral drugs need to further verify antiviral effect in animal models.Therefore,an appropriate animal infection model is an important basis for evaluating the effect of antiviral drugs in vivo.Through FDA-approved drug library to preliminary screening of anti-TBEV drugs in this study.The anti-TBEV effects of Cepharanthine and Ribavirin were verified at cellular level.BALB/c mice model of TBEV infection was used to evaluate anti-TBEV effect of Cepharanthine in vivo.This study aims to provide potential drug candidates for the treatment of TBE.Methods and ResultsIn TBEV-infected human hepatoma cell Huh-7,through high-throughput screening of 2580 drugs on the FDA-approved drug library and preliminary screening of 209 anti-TBEV drugs by immunofluorescence.Combined the existing research of drug and description of effect in the FDA-approved drug library,13 drugs were selected to reconfirm the effect of inhibiting TBEV in human lung cancer cell A549.Immunofluorescence assay showed Cepharanthine,Ribavirin,Puromycin 2HCl and Nitazoxanide have inhibitory effects for TBEV.The cytotoxicity of four drugs were detected on A549,Human neuroblastoma cell SH-SY5Y and African green monkey kidney cell Vero by MTS assay.Studies have shown that the cytotoxicity of the four drugs is cell-type dependent.Low cytotoxicity of Cepharanthine and Ribavirin were observed on A549 cells,high cytotoxicity of Puromycin 2HCl and Nitazoxanide were observed on A549 cells.A549 and SH-SY5Y cells infected with TBEV were treated with Cepharanthine by co-treatment,pre-treatment and post-treatment.Cytopathic effect(CPE)showed that Cepharanthine had obvious protective effect on TBEV-infected cells under co-treatment and pre-treatment.Plaque experiments showed that,compared with TBEV-infected cells,Cepharanthine significantly reduced virus titer in the supernatant of TBEV-infected cells under co-treatment and pre-treatment(P<0.05)in a concentration-dependent manner.Quantitative real time-PCR(q RT-PCR)showed that,compared with TBEV-infected cells,Cepharanthine significantly reduced the level of TBEV RNA in the two cells infected with TBEV under co-treatment and pre-treatment(P<0.05)in a concentration-dependent manner.Compared with cells without virus infection,the expression of C/EBP homologous protein(CCAAT/enhancer-binding protein homologous protein,CHOP)m RNA was significantly increased after TBEV-infected cells(P<0.05).Compared with TBEV-infected cells,Cepharanthine significantly reduced CHOP m RNA expression in the two cells infected with TBEV under co-treatment and pre-treatment(P<0.05)in a concentration-dependent manner.Enzyme linked immunosorbent assay(ELISA)showed that,compared with cells without virus infection,the expression of tumor necrosis factorα(TNF-α),interleukin 11(IL-11),interleukin 1β(IL-1β)was significantly increased after TBEV-infected cells(P<0.05).Compared with TBEV-infected cells,Cepharanthine significantly inhibited the expression of TNF-α,IL-11 and IL-1βin A549 cells infected with TBEV(P<0.05),and inhibited the expression of IL-11 in SH-SY5Y cells infected with TBEV(P<0.05)under co-treatment and pre-treatment.A549 and SH-SY5Y cells infected with TBEV were treated with Ribavirin by co-treatment,pre-treatment and post-treatment.CPE showed that Ribavirin had obvious protective effect on TBEV-infected cells under co-treatment and post-treatment.Plaque experiments showed that,compared with TBEV-infected cells,Ribavirin significantly reduced virus titer in the supernatant of TBEV-infected cells under co-treatment and post-treatment(P<0.05)in a concentration-dependent manner.q RT-PCR showed that,compared with TBEV-infected cells,Ribavirin significantly reduced the level of TBEV RNA in the two cells infected with TBEV under co-treatment and post-treatment(P<0.05)in a concentration-dependent manner.Compared with cells without virus infection,the expression of Mx A m RNA increased after TBEV infection of A549 cells(P<0.05).Compared with TBEV-infected cells,Ribavirin significantly increased the expression of Mx A m RNA in A549 cells infected with TBEV under co-treatment and post-treatment(P<0.05)in a concentration-dependent manner.ELISA experiments showed that,compared with TBEV-infected cells,Ribavirin significantly inhibited the expression of TNF-αin A549 cells infected with TBEV by post-treatment manner(P<0.05).In this study,BALB/c mice model of TBEV infection were established by subcutaneous and intraperitoneal injection to evaluate the anti-TBEV effect of the drug in vivo.Various indicators of TBEV-infected mice were analyzed by symptoms of infection,survival rate,body weight,plaque assay,immunohistochemistry,hematoxylin-eosin staining and m RNA expression levels of related inflammatory cytokines.Mice infected with TBEV by subcutaneous and intraperitoneal injection showed obvious symptoms of infection,such as arched back,paralysis and so on.BALB/c mice infected with TBEV by subcutaneous injection,the survival rates of mice with challenge doses of 10~3 Plaque forming unit(PFU)and 10~4PFU were 40%and 20%,respectively.BALB/c mice infected with TBEV by intraperitoneal injection,the mice with challenge doses of 10~3PFU and 10~4PFU have a mortality rate of 100%.TBEV antigen were detected in brain,spleen and kidney on BALB/c mice infected with TBEV by subcutaneous and intraperitoneal injection,and caused pathological changes in brain.Mice infected with TBEV by subcutaneous injection,plaque assay showed that TBEV could be detected in the spleen on day 5 post infection and TBEV could be detected in the brain and kidney on day 7 post infection.Virus titers in the brain,spleen and kidney showed an increasing trend with time of infection.Mice infected with TBEV by subcutaneous and intraperitoneal injection,q RT-PCR showed that the m RNA expressions of TNF-αand interferonβin brain,spleen and kidney changed dynamically with infection time.These results show that TBEV can establish infection in mice by subcutaneous and intraperitoneal injection.Based on BALB/c mice model of TBEV infection,the protective effect of Cepharanthine on TBEV-infected mice was evaluated.Compared with TBEV-infected mice,the survival rate of mice is 66.7%,which significantly inhibited the expression of TBEV antigens in the brain,spleen and kidney,and attenuates pathological damage of brain.Studies have shown that Cepharanthine has protective effect on TBEV-infected mice by pre-administration.In summary,the anti-TBEV effect of Cepharanthine and ribavirin were confirmed on the cellular level in this study.It is preliminarily shown that Cepharanthine has protective effect on TBEV-infected BALB/c mice by pre-administration.The research on the mechanism of action will be strengthen in the future to provide potential drug candidate for the treatment of TBE.