Background: A characteristic feature of many infections is that only a portion of exposed individuals develop clinical disease. These include mosquito-borne flaviviruses such as West Nile virus (WNV), Zika virus (ZIKV) and Usutu virus (USUV) infections, which generally cause mild illness or asymptomatic infections in humans. Nonetheless, WNV can cause serious neuroinvasive diseases in less than 1% of infected patients, mainly elderly and immunocompromised subjects; ZIKV may cause fetal microcephaly in about 5% of infections acquired during pregnancy and 1 in 10,000 infected adults develop Guillain-Barré syndrome; USUV seems less pathogenic than WNV and most human infections described so far were asymptomatic, with rare cases of encephalitis or meningitis. Aim of the study: The different infection outcomes or progression to severe disease can be partly explained by host genetic variations, but the genetic traits associated with susceptibly to severe infection remain poorly understood. Aim of this study was to develop a patient-specific in vitro platform, based on human induced pluripotent stem cells (hiPSCs), to investigate the mechanisms of variations in human susceptibility to severe flavivirus infection. Methods: iPSCs were generated from erythroblasts of two blood donors with asymptomatic WNV infection (controls) and from two patients who developed WNV encephalitis but had no co-morbidity or other risk factors (cases). Patient-specific iPSCs were differentiated into neural stem cells (NSCs) and infected with WNV lineage 1 (GU011992), ZIKV Asian lineage (KU853013), and USUV lineage Europe 1 (AY453411) at different MOIs. Time course experiments were performed to evaluate viral replication kinetics in infected NSCs, cell viability and cell death following infection, and expression of genes involved in antiviral innate immunity. Next-generation sequencing of 2,600 genes related to immune system in iPSCs of cases and controls was performed to detects mutations potentially associated with increased susceptibility to neuroinvasive disease. Results: USUV and WNV replicated more efficiently, yielding 10 and 100-fold higher viral load and inducing 40% and 70% higher cell mortality, respectively, in NSCs derived from cases than in NSCs derived from controls. WNV induced 3-fold higher caspase 3 activity in infected NSC derived from encephalitis patients than in NSCs derived from asymptomatic donors. Several genes involved in the antiviral IFN pathway were significantly upregulated after USUV, ZIKV and WNV infection (in particular, type 3 IFNs genes), but the general trend indicated an attenuated response in NSCs derived from WNV encephalitis cases, which showed significantly lower mRNA levels of IFN pathway regulators such as TLR3, MAVS and IRF7. Exome sequencing analysis identified heterozygous inactivating mutations in the PSIP1 and DDX58 genes of cases, but not in controls, as polymorphism in other genes that could play a role in disease susceptibility. Conclusions: Patient-specific iPSCs are useful tools to model individual susceptibility to viral infectious diseases and allowed to demonstrate that WNV and USUV and, to a lesser extent, ZIKV, replicated more efficiently and induced more cell death and apoptosis in NSCs derived from patients with WNV encephalitis than in cells derived from blood donors with asymptomatic infection. This increased susceptibility to neurotropic flaviviruses was associated with a significantly attenuated innate antiviral response. Exome sequencing revealed inactivating mutations in genes that represent good candidates for further investigation.

In vitro modelling of patient-specific susceptibility to neurotropic flavivirus infection by using induced pluripotent stem cells / Riccetti, Silvia. - (2019 Oct 26).

In vitro modelling of patient-specific susceptibility to neurotropic flavivirus infection by using induced pluripotent stem cells

Riccetti, Silvia
2019

Abstract

Background: A characteristic feature of many infections is that only a portion of exposed individuals develop clinical disease. These include mosquito-borne flaviviruses such as West Nile virus (WNV), Zika virus (ZIKV) and Usutu virus (USUV) infections, which generally cause mild illness or asymptomatic infections in humans. Nonetheless, WNV can cause serious neuroinvasive diseases in less than 1% of infected patients, mainly elderly and immunocompromised subjects; ZIKV may cause fetal microcephaly in about 5% of infections acquired during pregnancy and 1 in 10,000 infected adults develop Guillain-Barré syndrome; USUV seems less pathogenic than WNV and most human infections described so far were asymptomatic, with rare cases of encephalitis or meningitis. Aim of the study: The different infection outcomes or progression to severe disease can be partly explained by host genetic variations, but the genetic traits associated with susceptibly to severe infection remain poorly understood. Aim of this study was to develop a patient-specific in vitro platform, based on human induced pluripotent stem cells (hiPSCs), to investigate the mechanisms of variations in human susceptibility to severe flavivirus infection. Methods: iPSCs were generated from erythroblasts of two blood donors with asymptomatic WNV infection (controls) and from two patients who developed WNV encephalitis but had no co-morbidity or other risk factors (cases). Patient-specific iPSCs were differentiated into neural stem cells (NSCs) and infected with WNV lineage 1 (GU011992), ZIKV Asian lineage (KU853013), and USUV lineage Europe 1 (AY453411) at different MOIs. Time course experiments were performed to evaluate viral replication kinetics in infected NSCs, cell viability and cell death following infection, and expression of genes involved in antiviral innate immunity. Next-generation sequencing of 2,600 genes related to immune system in iPSCs of cases and controls was performed to detects mutations potentially associated with increased susceptibility to neuroinvasive disease. Results: USUV and WNV replicated more efficiently, yielding 10 and 100-fold higher viral load and inducing 40% and 70% higher cell mortality, respectively, in NSCs derived from cases than in NSCs derived from controls. WNV induced 3-fold higher caspase 3 activity in infected NSC derived from encephalitis patients than in NSCs derived from asymptomatic donors. Several genes involved in the antiviral IFN pathway were significantly upregulated after USUV, ZIKV and WNV infection (in particular, type 3 IFNs genes), but the general trend indicated an attenuated response in NSCs derived from WNV encephalitis cases, which showed significantly lower mRNA levels of IFN pathway regulators such as TLR3, MAVS and IRF7. Exome sequencing analysis identified heterozygous inactivating mutations in the PSIP1 and DDX58 genes of cases, but not in controls, as polymorphism in other genes that could play a role in disease susceptibility. Conclusions: Patient-specific iPSCs are useful tools to model individual susceptibility to viral infectious diseases and allowed to demonstrate that WNV and USUV and, to a lesser extent, ZIKV, replicated more efficiently and induced more cell death and apoptosis in NSCs derived from patients with WNV encephalitis than in cells derived from blood donors with asymptomatic infection. This increased susceptibility to neurotropic flaviviruses was associated with a significantly attenuated innate antiviral response. Exome sequencing revealed inactivating mutations in genes that represent good candidates for further investigation.
26-ott-2019
flavivirus/flavivirus suscettibilità individuale/individual susceptibility cellule staminali umane pluripotenti indotte/ human induced pluripotent stem cells
In vitro modelling of patient-specific susceptibility to neurotropic flavivirus infection by using induced pluripotent stem cells / Riccetti, Silvia. - (2019 Oct 26).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3422230
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