Study On The Effects Of Antiviral Drugs Against Enterovirus 71 And Herpes Simplex Virus Type 2

EV71?Enterovirus,71?is the major cause of severe or fatal HFMD?Hand,foot and mouth disease?.It can enter central nervous system and lead to permanent damage.Symptomatic treatment and intravenous immunoglobulin are commonly used to treat the infection.There is no available antiviral drug against EV71 in clinical treatment.Therefore,it is of great importance to develop safe and effective antiviral drugs against EV71.Retro-2^cycl was first identified as an inhibitor of ricin toxin by HTS?Highthroughput screening?.It has also been reported to be active against other pathogens exploiting intracellular vesicle transport,which also participates in several processes in the EV71 lifecycle,including non-lytic release.It provides a rationale for testing Retro-2^cycl and its optimized derivative Retro-2.1 as inhibitors of EV71.Flavonoids is a category of compounds possessing the same maternal structure and a variety of biological activities.Several flavonoids have been reported to be active against EV71.However,as these reports were individually performed in different evaluation systems,the possibility of a horizontal comparison between the flavonoids was limited as a result.Besides,only in vitro antiviral activities of these compounds were evaluated,however,the efficacy in animal experiment is an important indicator to verify the potential of a drug.Therefore,antiviral activities of representative flavonoids were evaluated both in vitro and in vivo.HSV-2?Herpes simplex virus type 2?is the major cause of genital herpes and can be lifelong latent.Besides,HSV-2 infection can also increase the risk of developing cervical cancer and HIV?Human immunodeficiency virus?infection.Chemotherapy with acyclovir or its prodrugs is standard practice in the management of HSV-2 infection.However,the use of acyclovir is limited due to the emergence of drug resistance and severe adverse effects.Although some non-nucleoside inhibitors have been developed,most are not approved for HSV-2 infection treatment due to severe adverse effects.Therefore,it is of great urgency to develop safe and effective alternative antiviral drugs against HSV-2.ABMA was first identified as another kind of inhibitor against ricin toxin by HTS?High-throughput screening?.It has also been reported to be active against other pathogens exploiting intracellular vesicle transport,which also participates in several processes in the HSV-2 lifecycle,including endocytic entry and envelopment,providing a rationale for testing ABMA as an inhibitor of HSV-2.The study is divided into three parts:1.Antiviral effects of Retro-2^cycl and Retro-2.1 against EV71EV71 infection model was firstly established on 293 S cells,followed by CPE?cytopathic effect?and plaque formation assays.Retro-2^cycl and Retro-2.1 were demonstrated to be safe and effective inhibitors of EV71 with selective index values of > 39.81 and 5356.Then,Retro-2^cycl and Retro-2.1 were demonstrated to be effective in the late stage of the EV71 lifecycle,by interfering with progeny virus packaging and/or release in the time of addition assay.Besides,significant reductions in virus yield in the cell supernatants but not in the cells were observed when Retro-2^cycl and Retro-2.1 were added during the period in which progeny EV71 packaging and release take place?5-16 h post-infection?,demonstrating that Retro-2^cycl and Retro-2.1 exerted the antiviral effects against EV71 by blocking progeny virus release.This mechanism was further confirmed by the cell immunofluoresence assay in which EV71 accumulated in the cell cytoplasm instead of under the plasma membrane after treatment.Finally,EV71 lethal infection model was established on newborn BALB/c mice,followed by protective efficacy study of Retro-2^cycl(only Retro-2^cycl was chosen to be tested due to the poor solubility of Retro-2.1 in aqueous medium,and the optimization to improve the solubility of Retro-2.1 is ongoing).Retro-2^cycl could protect 90 % of newborn mice from lethal EV71 challenge at the dose of 10 mg/kg.As a conclusion,Retro-2^cycl and Retro-2.1 were demonstrated to be effective inhibitors of EV71 both in vitro and in vivo for the first time,and the mechanism of blocking progeny virus release exploiting intracellular vesicle transport was also clarified.As numerous derivatives of Retro-2^cycl have been synthesized,these analogues could be developed as potential lead compounds of anti-EV71 drug development.As Retro-2^cycl and Retro-2.1 act on a host target but not directly on the virus,it could be more difficult for the virus to develop drug resistance.Moreover,the study amplified the pathogen range which Retro-2^cycl and Retro-2.1 are effective against and provided a rationale for the development of Retro-2^cycl and Retro-2.1 as broadspectrum inhibitors,indicating that intracellular vesicle transport is likely to be developed as an effective antiviral target.2.Antiviral effects of flavonoids against EV71In vitro antiviral activity assays against EV71 were performed on 9 flavonoids to provide references for the doses used in animal experiments.All of these compounds had selective index values higher than 4,which are suitable for drug development.Among all these flavonoids,7-hydroxyflavone possesses the highest selective index value of 12215.79.In vivo antiviral activity assays against EV71 were performed with the appropriate drug doses.All of these compounds had significant protective efficacy against EV71.7-hydroxyflavone showed the best protective rate of 66.67 % at the dose of 0.2 mg/kg,isorhamnetin showed the best protective rate of 100% at the dose of 10 mg/kg,indicating excellent drug development potential.As a conclusion,isorhamnetin was identified to be an effective inhibitor of EV71.Protective efficacy of flavonoid against EV71 in newborn BALB/c mice was tested,and significant protective efficacies of 9 flavonoids were demonstrated.Antiviral activities of various flavonoids against EV71 were tested in the same evaluation system for the first time,which provided a basis for the comparisons among the compounds and demonstrated 7-hydroxyflavone as well as isorhamnetin to be effective flavonoids against EV71 with quite good potential to be developed.3.Antiviral effect of ABMA against HSV-2HSV-2 infection model was firstly established on Vero cells,followed by CPE?cytopathic effect?and plaque formation assays.ABMA was demonstrated to be a safe and effective inhibitor of HSV-2 with a selective index value of 20.93,which was confirmed by reduced virus DNA and protein content.Then,ABMA was demonstrated to be effective in both early and late stages of the HSV-2 lifecycle in the time of compound addition assay.Besides,ABMA was found to block HSV-2 entry in the early stage in the virus binding and entry assays.This mechanism was further confirmed by the cell immunofluorescence assay in which viruses accumulated around the plasma membrane instead of in or near the cell nuclei after treatment.Moreover,significant reductions were founded both in intracellular and extracellular virus titers when ABMA was added during the late stages of the HSV-2 lifecycle?6-18 h post-infection?.As the formation of both the capsids and the progeny infectious particles of HSV-2 gradually takes place from approximately 5 h post-infection onwards,the results also suggested that ABMA was most likely to hinder the HSV-2 packaging and egress process in the late stage.Finally,HSV-2 infection model was firstly established on female BALB/c mice,followed by protective efficacy study of ABMA.ABMA could improve the survival rate of the mice from 8.33 % to 50 % at the dose of 5 mg/kg.As a conclusion,ABMA was identified as an effective inhibitor of HSV-2 both in vitro and in vivo for the first time,and the mechanism of blocking virus entry and late stages in the HSV-2 lifecycle exploiting intracellular vesicle transport was also clarified,providing a potential lead compound for anti-HSV-2 drug development.As ABMA acts on a host target but not directly on the virus,it could be more difficult for the virus to develop drug resistance.Moreover,the results amplified the pathogen range which ABMA is effective against and provided a rationale for the development of ABMA as a broad-spectrum inhibitor,indicating that intracellular vesicle transport is likely to be an effective antiviral target.