VALUTAZIONE DELL'ATTIVITÀ ANTIVIRALE DI COMPOSTI ANTI-CITOMEGALOVIRUS UMANO IN DIVERSI MODELLI DI CELLULE STAMINALI/PROGENITRICI NEURALI.

Background: Human cytomegalovirus (HCMV) infection is considered asymptomatic and harmless in immunocompetent individuals, whereas in neonates as well as in immunocompromised and immunosuppressed subjects it is a cause of morbidity and mortality and can lead to the development of cognitive impairment and transplant rejection, respectively. HCMV congenital infection (cCMVi) is the most common cause of congenital defects worldwide. The possible consequences of viral transmission to the fetus are associated with brain damages such as hearing and vision loss, microcephaly, developmental and motor delay (Britt, 2017). One of the main concerns nowadays is the identification of a treatment for cCMVi, since there is no approved antiviral drug or vaccine to prevent and treat the infection. Aim of the Study: This project aims at evaluating the efficacy of different antiviral drugs for the treatment of cCMVi by analyzing their activity in two different models of neural cells representing the target in vivo of cCMV, i.e. human embryonic stem cell (hESC)-derived Neural Stem Cells (NSCs) and Neuroepithelial Stem Cells (NESCs) obtained from an aborted fetus. Molecules with antiviral activity such as letermovir, manidipine, and nitazoxanide were tested at different concentrations and at various conditions and compared to ganciclovir as a control. The screening of antiviral drugs' efficacy in NSCs and NESCs is of paramount importance to identify pharmacological treatments for cCMVi and to decrease the development of neurological sequelae as a consequence of the infection. Results: As previously demonstrated for NSCs, HCMV was able to establish a productive infection in NESCs. This was demonstrated with the production of infectious viral particles by plaque assay, the expression of viral immediate early promoter-derived (MIEP), early (UL54) and late (UL55) genes by real-time qPCR, and the detection of early and late viral proteins (i.e. IEAs, gB and pp65) by western blot and immunofluorescence. The anti-HCMV activity was confirmed for ganciclovir and letermovir that showed relatively high Selectivity Index (SI) (>975 and >2000, respectively), whereas nitazoxanide was toxic in NESCs with a poor SI. Moreover, viral gene B2.7 quantification by qPCR confirmed the efficacy of the drugs in blocking viral DNA synthesis in both NESCs and NSCs. The infection led to the downregulation of key neural progenitor cell markers important for neurogenesis such as Doublecortin and Nestin in NSCs, and SOX2 in both cell lines. The transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARγ), critical in the developing brain, was also upregulated in both cell types. Finally, a preliminary analysis revealed that the dysregulation of PPARγ and SOX2 by HCMV infection is partially rescued by antiviral compounds ganciclovir and letermovir in NESCs whereas a similar rescue was observed only for PPARγ in NSCs. Conclusion: NSCs and NESCs are susceptible and permissive to HCMV replication and could represent an excellent model of disease-relevant cells during cCMVi to evaluate both antiviral and neuroprotective potential of candidate therapeutics. Given the high SI showed by letermovir in these systems, this approved drug deserves further investigation as a candidate antiviral for the treatment of cCMVi.