Human stem cell-derived models for the characterization of the neuroprotective activity of antiviral treatments against HCMV

Human cytomegalovirus(HCMV) is a member of the Herpesviridae family.It is an opportunistic infectious agent,which becomes latent in the host after primary infection.Infection is common around the globe and dependent on the socio-economic standards of the different countries.In immunocompetent individuals,most HCMV infections develop as asymptomatic or very mild,while in immunocompromised patients,it can induce severe complications.Moreover,HMCV is the most common cause of congenital infections.HCMV congenital infection (cCMV) can be caused either by primary infection,re-infection or reactivation of the virus in the mother, and can give rise to several neurodevelopmental disabilities in the newborns.Despite the growing need of an effective treatment for this pathogen,no effective drug or vaccine has been approved yet.The complexity of the problem is due to the strict species-specificity of the virus, limiting the use of experimental animals and the difficulty in recapitulating the developing brain.The aim of this study was to characterize HCMV infection in Neural Epithelial Stem Cells(NESCs) derived from an aborted foetus and in pluripotent stem cells-derived Neural Stem Cells (NSCs) and induced neurons(iNeurons), to study the neuroprotective activity of antiviral drugs, such as ganciclovir,letermovir,nitazoxanide,OZ418,as potential treatments against cCMV.Human embryonic stem cells(hESCs) H9 cell line was employed to derive Neural Stem Cells(NSCs) and terminally differentiated neurons (iNeurons).NESCs and NSCs were infected at a MOI 3 with HCMV TB40/E strain.iNeurons were infected at MOI 1 at the stage of Neural Progenitor Cells(NPCs),and completed their differentiation during the infection period.Infection of cells was verified by observing cytopathic effect, by targeting viral immediate early(IEs) proteins through immunofluorescence,and by evaluating viral transcripts by quantitative Real-Time PCR.After every infection,cells were treated with different approved or candidate anti-HCMV drugs:ganciclovir (GCV,50 μM), letermovir(LTV,10 μM),nitazoxanide(NTZ,30 μM),OZ418(30 μM).Cytotoxicity of drugs was previously evaluated by MTT assay,to calculate the half-maximal cytotoxic concentration.Effective concentration was evaluated by half-maximal effective concentration(EC50) in virus yield assays.RNA was extracted from infected-treated cells and reverse transcribed, to evaluate the differences in relative expression of key genes of neurogenesis,stemness and neural differentiation by qRT-PCR.Immunofluorescence analysis was used to characterized obtained NSCs and iNeurons,and to evaluate the reduction of HCMV-infected iNeurons upon drug treatment.Neural Stem Cells(NSCs) and induced Neurons(iNeu) were correctly generated and showed proper markers,specific for their stage of differentiation.All cell lines could be infected with HCMV, being both susceptible and permissive.Infection with HCMV demonstrated a dysregulation of key markers of neurogenesis (e.g. Doublecortin,Nestin,SOX2,Pax6,PPAR and FOXG1).Data acquired from relative gene expression level analysis of infected NESCs and NSCs, showed neuroprotective activity of the drugs for some of neurogenesis markers,although statistically significant only for SOX2 and NESTIN treated with ganciclovir.NTZ seemed to be toxic in Neural Progenitor Cells(NPCs),disrupting the differentiation towards iNeurons,while the other drugs did not interfere with the development into neurons and showed different activity in reducing the number of cells infected by the virus.Dysregulation of PPARby HCMV infection in neurons was partially rescued by antiviral compound ganciclovir.Neural stem cells and differentiating neurons represent a valuable platform to study cCMV,since they recapitulate the differentiation stages of cells in the developing brain,which are the targets of HCMV infection.