Comparative analysis of Zika virus and other Flavivirus infection in human neural cells

Background: Zika virus (ZIKV), West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that generally cause mild or asymptomatic disease in humans. However, ZIKV infection has been associated with fetal microcephaly and Guillain-Barrè syndrome in adults; WNV infection may evolve to severe neuroinvasive disease in the elderly and immunocompromised subjects; DENV may rarely cause neurological complications in infected individuals. In addition, another emerging mosquito-borne flavivirus, Usutu virus (USUV), which may cause fatal neuroinvasive disease in different bird species, has been recently shown to infect humans but its pathogenicity is unknown. Aim of the study: In the context of the recent outbreak of ZIKV in the Americas and the increasing evidences of an association between ZIKV infection and the occurrence of fetal microcephaly, aim of this study was to investigate the effect of ZIKV infection in human neural cells in comparison with other flavivirus infections. To this aim, ZIKV infectivity, replication kinetics, cytopathic effect (CPE), and induction of innate antiviral responses were investigated in human induced pluripotent stem cells (hiPSCs), hiPSCs-derived neural stem cells (NSCs) and neurons and compared with other flaviviruses, i.e., WNV, DENV and USUV. Methods: NSCs and neurons were differentiated from hiPSCs. hiPSCs, NSCs, and neurons were infected with isolates of ZIKV Asian lineage (KU853013), WNV lineage 2 (KF179640), DENV serotype 2, and USUV Europe lineage 1 (AY453411). Time course experiments were performed to evaluate viral load by qRT-PCR and TCID50, expression of host genes involved in antiviral innate immunity by qRT-PCR, expression of cell differentiation markers by IF and qRT-PCR, cell viability and cell death by flow cytometry. The impact of ZIKV on embryogenesis and neurogenesis was evaluated by infection of hiPSCs and NSCs during neural differentiation and embryo body formation. Results: ZIKV infected and replicated efficiently in NSCs, neurons and hiPSCs. Infection led to typical CPE and cell death by apoptosis. ZIKV infection of hiPSCs, NSCs, and neurons induced the expression of innate immune response genes, especially the cellular pattern recognition receptor (PRR) IFH1 gene (MDA5), IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) and 2 (IFIT2). Infected embryoid bodies were massively destroyed by ZIKV infection and infected hiPSCs and NSCs died before ending the neural differentiation process. ZIKV replication efficiency in NSCs was significantly higher than that of DENV-2 and USUV, but lower than that of WNV. In particular, WNV replicated more efficiently, induced more cell death and higher levels of antiviral gene expression than ZIKV in NSCs, neurons and hiPSCs. The induction of innate immune response genes in NSCs after infection with ZIKV and DENV-2 infection was milder than after infection with WNV and USUV, in agreement with the adaptation of these viruses to the human host and their ability to shut down the antiviral response. Conclusion: ZIKV infects and replicates efficiently in NSCs and induces cell death abrogating neural development, although less efficiently than WNV. Because of the similarities between flaviviruses in their interactions with host neural cells, it is conceivable that infection of other human cells, such as those involved in the extablishment of the blood-placenta barrier, are crucial for ZIKV-induced damage of the fetal brain.

Presupposti dello studio: Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV) e Usutu virus (USUV) sono trasmessi da zanzare ed appartengono al genere Flavivirus della famiglia Flaviviridae. L’infezione da ZIKV è associata a microcefalia fetale e sindrome di Guillan-Barrè; WNV può causare una grave sindrome neuroinvasiva nell’anziano e nei soggetti immunocompromessi; l’infezione da DENV raramente si associa a complicazioni neurologiche; USUV può causare una sindrome neuroinvasiva fatale in diverse specie di uccelli, è stato dimostrato che può infettare pure l’uomo, ma la sua patogenicità resta ancora da chiarire. Scopo: Alla luce della recente epidemia di ZIKV in America e di una probabile associazione tra l’infezione da ZIKV e lo sviluppo di microcefalia fetale, lo scopo di questo studio è stato confrontare l’infezione da ZIKV sulle cellule neurali umane con l’infezione da WNV, DENV e USUV. A tal fine, la cinetica di replicazione, l’effetto citopatico e l’immunità innata indotta dall’infezione virale sono state analizzate in cellule staminali pluripotenti indotte (hiPSCs), cellule staminali neurali derivate da iPSCs e neuroni. Materiali e metodi: Le NSCs ed i neuroni sono stati differenziati da hiPSCs. I diversi tipi cellulari sono stati infettati con l’isolato di ZIKV lignaggio asiatico (KU853013), WNV lignaggio 2 (KF179640), DENV sierotipo 2 e USUV lignaggio 1 europeo (AY453411). La carica virale è stata valutata a diversi tempi dall’infezione mediante qRT-PCR e TCID50, il livello di espressione dei geni coinvolti nell’immunità innata è stato analizzato mediante qRT-PCR e l’espressione dei markers di differenziamento cellulare mediante IF e qRT-PCR, la sopravvivenza cellulare e l’apoptosi mediante il saggio MTT e analisi dell’attivazione di caspasi-3. L’impatto dell’infezione da ZIKV sull’embriogenesi e la neurogenesi è stato valutato infettando le hiPSCs e le NSCs durante il differenziamento neurale e durante la formazione dei corpi embrioidi. Risultati: ZIKV era in grado di infettare e replicare efficientemente nelle NSCs, nei neuroni e nelle hiPSCs, causando un tipico effetto citopatico e morte cellulare per apoptosi. L’infezione ha indotto un significativo aumento dell’espressione dei geni dell’immunità innata, in particolare dei geni MDA5 (the cellular pattern recognition receptor (PRR) IFH1 gene), IFIT1 (IFN-induced protein with tetratricopeptide repeats 1) e IFIT2. I corpi embrioidi sono stati distrutti dal virus e le hiPSCs e le NSCs infettate sono morte prima di completare il differenziamento neurale. L’efficienza di replicazione di ZIKV nelle NSCs era maggiore rispetto a quella di DENV-2 e USUV, ma minore rispetto al WNV. Infatti, WNV replicava in modo più efficiente, induceva una maggiore morte cellulare e stimolava una più elevata risposta antivirale rispetto a ZIKV nei diversi tipi cellulari. Conclusione: ZIKV infetta e replica nelle NSCs, inducendo morte cellulare e impedendo lo sviluppo neurale, ma in modo meno efficiente rispetto al WNV. E’ probabile quindi che l’infezione di altri tipi cellulari sia determinante per il danno al sistema nervoso fetale indotto in modo specifico da ZIKV.