Modelling Congenital Cytomegalovirus infection in Neural progenitor cells and cerebral organoids for evaluating Antiviral Therapies and Neuroprotection strategies

Background: human cytomegalovirus (HCMV) is classified as a beta-herpesvirus and it is known for its ability to induce severe congenital birth defects such as microcephaly, vision and hearing loss, and cognitive impairments. Congenital CMV (cCMV) is a major cause of neurodevelopmental abnormalities and can lead to CNS damage in the fetus. Several aspects of cCMV infection remain poorly characterized, including the primary factors driving neuropathogenesis in the developing brain during the infection alongside with a targeted treatment. Aim: aim of this thesis work was to employ a 2D dynamic model of neurogenesis, consisting of neural progenitors differentiating into neurons, to characterize HCMV infection, with the specific aim of evaluating the efficacy of approved and investigational antiviral drugs as potential therapeutic options for cCMV. To add complexity to this model, a 3D platform of cerebral organoids that closely mimic in vivo fetal brain development, was optimized and applied to test antiviral compounds in HCMV-infected organoids. Methods: Differentiating neural progenitor cells were infected with HCMV TB40/E clinical-like strain and subsequently treated with anti-HCMV drugs, including ganciclovir (GCV), letermovir (LTV), nitazoxanide (NTZ), and OZ418. The assessment of the effective concentration at half-maximal response (EC50) was performed by virus yield reduction assays. In parallel, the cytotoxicity of the drugs was tested by MTT assays. Dysregulation of genes involved in neurogenesis and neurons development, i.e. Peroxisome Proliferator-Activated Receptor gamma (PPARγ), Doublecortin (DCX) and β-tubulin (TUBB3), upon HCMV infection along with a potential rescue by antiviral treatment, were assessed by qPCR. Thirty-day old cerebral organoids were infected with two strains of HCMV, i.e., TB40E-pp65-YFP and the clinical strain VR1814 and treated for 21 days with antiviral drugs. Fluorescence signals from infected and uninfected organoids were measured at different time points and production of infectious viral particles and viral proteins expression were analyzed at 21days post infection (dpi) by plaque assays and Western blot, respectively. Results: The anti-HCMV activity of tested compounds in infected neural progenitors was confirmed for GCV and LTV that showed a high Selectivity Index (SI), whereas NTZ resulted toxic and OZ418 did not inhibit the production of viral progeny at the concentrations tested. Infection of differentiating neurons with HCMV led to an upregulation of PPARγ, and a downregulation of both DCX and TUBBIII. GCV treatment restored PPARγ to normal levels but did not affect DCX and TUBBIII expression. Thirty-day old cerebral organoids were efficiently infected with both HCMV strains. TB40E-pp65-YFP infected organoids showed widespread regions of pp65+ cells expanding during the time course of infection. Live imaging of TB40E-pp65-YFP infected organoids, treated with antiviral drugs, demonstrated that GCV, LTV and OZ418 effectively reduced the number of YFP+ cells. All tested drugs did not affect organoid morphology and resulted in a significant reduction of viral proteins after 21 dpi. Plaque assay revealed the release of infectious viral particles only in the supernatant of VR1814-infected organoids. Western blot analysis of VR1814-infected organoids exhibited higher levels of immediate early antigens (IEA), gB, and UL44 proteins compared to TB40E-pp65-YFP-infected organoids. Antiviral treatments with all drugs tested remarkably reduced viral protein expression in organoids infected with both viruses. Conclusion: this study emphasizes the importance of in vitro differentiating progenitors and cerebral organoids as disease-relevant models to characterize HCMV congenital infection of the developing brain and investigate both the antiviral and neuroprotective potential of therapeutic candidates against cCMV.