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bioRxiv · 10.64898/2026.09.22.753572

A multi-electrode array and immunofluorescence workflow to characterize impacts of HHV-6 infection in induced pluripotent stems cells differentiated to mixed neuronal endpoints

Bahramian, E.·Yang, X.·Bajpai, A.·Hernandez Garcia, J.·Ceballos, R. M.
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Abstract

Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions.

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Bahramian, E., Yang, X., Bajpai, A., Hernandez Garcia, J., Ceballos, R. M.. 2026-09-23. A multi-electrode array and immunofluorescence workflow to characterize impacts of HHV-6 infection in induced pluripotent stems cells differentiated to mixed neuronal endpoints. https://doi.org/10.64898/2026.09.22.753572

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