key: cord-0845614-bil454sg authors: Bauer, Lisa; Lendemeijer, Bas; Leijten, Lonneke; Embregts, Carmen W. E.; Rockx, Barry; Kushner, Steven A.; de Vrij, Femke M.S.; van Riel, Debby title: Replication kinetic, cell tropism and associated immune responses in SARS-CoV-2 and H5N1 virus infected human iPSC derived neural models date: 2021-03-16 journal: bioRxiv DOI: 10.1101/2021.03.15.435472 sha: 48f31ccfb07e7b6d366e1f7d0bdceeb095157246 doc_id: 845614 cord_uid: bil454sg Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite ACE2, TMPRSS2 and NPR1 expression in all cultures. However, this sparse infection did result in the production of type-III-interferons and IL-8. In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS. to study the neurotropism of viruses in vitro. Specifically, we directed hiPSC-colonies towards 68 embryoid bodies with differentiation into neural progenitor cells (NPCs) and subsequently 69 mature neural networks 24 . In addition, we also utilized a rapid neuronal differentiation protocol 70 based on forced overexpression of the transcription factor Ngn2 in hiPSCs ( Figure 1A ) 25,26 to 71 generate pure populations of neurons that we co-cultured with hiPSC-derived astrocytes. Using these specified CNS cell types, we directly compared the characteristics of SARS-CoV-73 2 with the highly pathogenic H5N1 influenza A virus, a virus with zoonotic potential which is 74 known to efficiently replicate in neural cells in vivo 27-32 and in vitro 33-36 . To investigate the replication efficiency of SARS-CoV-2, we utilized hiPSC-derived NPCs and 80 differentiated these to mature neural cultures ( Figure 1A ). NPCs and fully differentiated neural Next, we wanted to investigate whether SARS-CoV-2 infection induced neuronal apoptosis. Therefore, we infected Ngn2 co-cultures with SARS-CoV-2 and stained for the apoptosis 132 marker caspase-3. We again observed that SARS-CoV-2 infected only MAP2 + neurons. 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