Optimization of lytic herpes simplex virus infection in human induced pluripotent stem cell-derived cortical neurones

Herpes simplex virus (HSV)-1 infection of cortical neurones is a leading cause of encephalitis. Whilst we have substantial knowledge about the molecular virology of HSV-1 lytic infection in cells of the periphery, like keratinocytes or fibroblasts, we know much less about infection of human neurones owing to the challenges of working with neuronal cell-based models. Here, we demonstrate the use of a human induced pluripotent stem cell-derived cortical neurone model (i3Neurones) for HSV-1 infection. i3Neurones are highly scalable and can be rapidly and efficiently differentiated into an isogenic population of cortical glutamatergic neurones. We show that i3Neurones support the full HSV-1 lytic replication cycle. We present an optimized protocol for the infection of i3Neurones with HSV-1 that allows their synchronous infection at near-100% efficiency and optimized fixation methods that preserve organelle and neurite structure for immunocytochemistry analysis. Our study highlights i3Neurones as a robust, scalable platform for microscopy and biochemical studies of HSV-1 and other neurotropic pathogens.