Modelling Lyssavirus Infections in Human Stem Cell-Derived Neural Cultures

Abstract

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods.

Establishment of Human-Induced Pluripotent Stem Cell-Derived Neurons—A Promising In Vitro Model for a Molecular Study of Rabies Virus and Host Interaction

“…Like other neurotropic viruses reported to preferentially infect NPCs, RABV-TH significantly induced NPC apoptosis ( Figure 4 A). However, apoptosis was not observed in infected neurons and astrocytes ( Figure 4 B), correlating with previous reports describing a lack of apoptosis in hiPSC-derived neurons [ 17 ] and postmortem brains of humans infected with RABV [ 33 ]. These findings suggest that RABV modulates cell death at various degrees in different neural cell types.…”

“…This seemingly abortive replication in astrocytes is in agreement with other in vitro and in vivo findings, which demonstrated a pivotal role of astrocytes in inhibiting RABV infection or spreading of the virus in the CNS through an activation of interferon-beta (IFN-β) and mitochondrial antiviral-signaling protein signaling pathway (MAVS) [ 30 , 31 ]. Our hiPSC-derived neurons were susceptible to RABV infection as reported by other groups [ 17 ], confirming that our system is applicable for in vitro molecular studies of RABV infection in the nervous system.…”

“…However, this ex vivo platform faces ethical difficulties, provides cells with limited proliferative capacity, and may not represent adult human neurons’ biology. The use of human-induced pluripotent stem cells (hiPSC) provides an alternative solution to overcome these issues and, therefore, enables an ease-of-use model for studying virus–host interactions at cellular and molecular levels [ 16 , 17 , 18 ].…”

“…In addition, the application of hiPSC technology in virology was applicable for in vitro modeling of neurotropic virus infections [ 16 , 22 ]. Previously, hiPSC-derived neurons were proposed as a model for investigating biological responses to RABV infection, focusing on differentially expressed inflammatory cytokines during infection with different RABV strains [ 17 ].…”

Machine Learning Identifies Cellular and Exosomal MicroRNA Signatures of Lyssavirus Infection in Human Stem Cell-Derived Neurons

“…MicroRNA expression profiling has been documented in mouse brain tissue following infection with rabies virus (Zhao et al, 2012a;Zhao et al, 2012b;Shi et al, 2014), although analysis of lyssavirus-induced miRNA changes in human neurons has not been explored due to limitations in experimental platforms available. Previously, we have generated and characterized human stem cell-derived neural models for the investigation of rabies pathogenesis (Sundaramoorthy et al, 2020a). This approach allows ethical, reproducible, high-throughput, and relevant investigations of lyssavirus infections directly on a human cell system.…”