The Antiviral Activity Of Aedes Aegypti Cecropin A Against Zika Virus

Background:Zika virus,a member of flaviviridae,is an enveloped,positive single-stranded RNA virus,it infects humans mostly through bites of Ae.aegypti species.Despite the overwhelming similarity in the structure and the replication strategy of ZIKV with other flaviviruses,the downstream diseases caused by these viruses are remarkably different.ZIKV,unlike most other flaviviruses,traverses the placental barrier and causes robust infection in the fetal brains,resulting in teratogenicity or fetal demise.Effective treatment for ZIKV infection remains unsolved,and a preventative vaccine is not yet available.Ae.aegypti act as many types of virus,such as DENV,ZIKV and Chikungya as well as other yellow fever.While infection of vertebrates causes serious disease,the presence of these viruses in mosquitoes is non-pathogenic and results in life-long persistent infection.This difference may reflect the capacity of insects to own a highly effective innate immune response to control invading microbes.This immune response includes the rapid production of potent antimicrobial peptides effective against bacteria and fungi.There are three families of AMPs in mosquitoes,including defensins,cecropin and gambicins,all of them have the functions of direct sterilization and/or regulating immune response.Cecropin from Ae.aegypti have variety of types,including cecropin A(cecropin A1 and A2),cecropin B(cecropin B1 and B2),and cecropin N.Among them,the antiviral function and mechanism of cecropin A have not been described.This study focused on the function and molecular mechanism of cecropin A against ZIKV virus.Purpose:To elucidate the effect of cecropin A,an antimicrobial peptide of Ae.aegypti,against Zika virus,and to explore its antiviral mechanism.Methods:1.The safety analysis of cecropin ACells were seeded into 96-well cell culture plates and treated with fresh growth medium containing different concentrations of cecropin A for 48 h at 37℃.At the experimental end point,10 μl CCK-8 was added to the cultures and incubated for 30 min.The absorbance at 570 nm was recorded through microplate reader.C57BL/6J mice were assigned randomly into eight groups and injected with cecropin A at different concentrations(10,20,40mg/kg)or PBS as control.Assessed the histopathological changes after 24 h,and detect the levels of alanine aminotransferase and creatinine in the serum of mice.2.The inhibitory activity of cecropin A on ZIKV infection in vitroCells(1×105)were seeded into a 24-well plate the day before infection.Then,ZIKV(MOI=1)was mixed with different concentrations of cecropin A(5,10 and 20μM)in serum-free DMEM and added to cells.After incubation for 2 h at 37℃,the mixture was replaced with 2%FBS-DMEM,added the corresponding concentrations of cecropin A at the same time.After 48 h,the level of viral RNA was detected by Real-time PCR,and the expression of viral protein(NS3 and E)was detected by western Blot and immunofluorescence,the virus titer of cell culture supernatant was determined by plaque assay.3.Detect the combination of cecropin A and ZIKV particles by ELISA assayHigh-affifinity binding plates were coated with 2μg/mL Cecropin A or BSA control and then 1×106 PFU virus was added,and incubated at 4℃ for 30 min,wells were exposed to anti-ZIKV E protein antibody,after 1 h,washed with PBS,then wells were exposed to HRP-labeled secondary antibody,and TMB.The absorbance at 450 nm was recorded through microplate reader.4.The role of cecropin A in destroying the integrity of ZIKV envelopBriefly,about 1×103 PFU of ZIKV was incubated with cecropin A at 37℃ for 2 h.The released genomic RNA from the cecropin A treated ZIKV particles was then digested with micrococcal nuclease at 37℃ for 4 h.After inactivation of the residual RNase at 70℃ for 30 min,the undigested genomic RNA in the intact viral particles was extracted using the Qiagen QIAamp Viral RNA Mini Kit.ZIKV RNA genome was quantified by Real-time PCR.5.The effect of cecropin A to the expression of viral receptorCecropin A or PBS pretreated with Vero cells at 37℃ for 2 h,then the expression of AXL,Tyro3 and TiM-1 was detected by Western Blot in the condition of ZIKV infection(MOI=1)and non-infection,respectively.6.The inhibition of cecropin A to the exosomes induced by ZIKV infectionZIKV(MOI=1)or PBS was used to incubate with Vero cells,cecropin A or PBS was added into the group of ZIKV infection at the same time,after incubation at 37℃for 48 h,exosomes in supernatant were collected,exosomal marker Alix,CD63,CD9 was detected by western blot.The particle size and concentrations were captured and analyzed with the NTA7.Cecropin A inhibited the infection of exosomes in Vero cellsThe exosomes secreted from the supernatant of virus-infected Vero cells were isolated and purified,and then it was used to infect Vero cells,cecropin A or PBS control was added at the same time,after 2 h of infection,the mixture was replaced with 2%FBS-DMEM,48 h later,the level of viral RNA was detected by Real-time PCR,and the expression of viral protein(NS3 and E)was detected by western Blot and immunofluorescence,the virus titer of cell culture supernatant was determined by plaque assay.8.Exosomes staining and uptakeDiD staining of exosomes:DiD dye was added to the exosome suspension,incubated at room temperature for 15 min,exosomes were precipitated by overspeed centrifugation at 31000 rpm for 1.5 h,then suspended with sterile PBS,and DiD labeled exosomes were obtained.DiD-labled exosomes was added into Vero cells,cecropin A and PBS was added at the same time,after incubation for 1.5 h,cell membrane and nucleus were stained with phalloidin and DAPI respectively.the integration of exosomes into cells was observed by immunofluorescence.9.In vivo imaging of miceMice were injected with 100μg of DiD-labeled exosomes and cecropin A simultaneously.Bioluminescence imaging was performed at 4 hours and 24 hours after injection.To determine the distribution of exosomes in organs,the dissected kidneys,spleens,lungs and brains were imaged at 24 h after injection.10.Cecropin A can destroy the exosomes directlyThe exosomes secreted from the supernatant of virus-infected cells were isolated and purified,then incubated with cecropin A or PBS at 37℃ for 2 h,the particle size and concentrations were captured and analyzed with the NTA,and the morphology of exosomes was observed by TEMResults:1.Cecropin A exhibited low toxicity to mammalian cells and miceCecropin A1 showed no cytotoxicity to Vero cells,RAW264.7 cells and mouse peritoneal macrophages when≤40,40,80 μM,respectively.Cecropin A2 showed no cytotoxicity to Vero cells,RAW264.7 cells and mouse peritoneal macrophages when<40,20,20 μM,respectively.Intraperitoneal injection of 10,20,40 mg/kg cecropin A did not cause acute toxicity in mice.2.Cecropin A significantly inhibited ZIKV replication in vitroIn ZIKV-infected Vero cells added with cecropin A,the results showed that cecropin A could inhibit the level of viral RNA,inhibit the expression of viral structural protein NS3 and envelop E protein markedly,and the virus titer in cell culture supernatant was also significantly reduced.3.Cecropin A was directly virucidal for ZIKV particlesAfter incubating ZIKV with cecropin A for 2 h,the infectivity of ZIKV was inhibited significantly,and resulted in the release of the viral genome.4.Cecropin A can not affect the expression of viral receptorCecropin A pretreated with cells can not affect the expression of AXL,Tyro3 and TiM-1 in the condition of ZIKV infection and non-infection,respectively.5.Cecropin A can inhibit the spread of ZIKV mediated by exosomesCecropin A can significantly reduce the accumulation level of exosomes in cell supernatants after ZIKV infection,inhibit the uptake of exosomes in Vero cells and the productive infections established by exosomes.In vivo imaging results of mice showed that cecropin A could significantly reduce the accumulation of exosomes in mice.Transmission electron microscopy results showed that direct treatment of exosomes with cecropin A would lead to the destruction of the exosome membrane.Conclusion:In this study,the antiviral activity and mechanism of antimicrobial peptide cecropin A of Ae.aegypti against ZIKV infection were explored.We confirmed that cecropin A can significantly inhibit ZIKV infection.And through the study of its antiviral mechanism,we found that:(1)cecropin A can play a direct role in the inactivation of ZIKV through breaking the envelope of ZIKV,Causing the viral genome to be released,(2)cecropin A has no significant effect on the expression of viral entry receptor.(3)cecropin A can block virus transmission from cell to cell by destroying exosomes.This study reveal the antiviral effect of cecropin A,a antimicrobial peptide ofAe.aegypti,these results provide novel candidates for the development of anti-ZIKV peptide drugs from mosquito,which can kill ZIKV and inhibit the tramission of ZIKV through exosomes.