Fig. 2: Neuronal replication of SARS-CoV-2 in K18-hACE2 mice.

a, High-resolution Z-projection confocal images of the cortex of SARS-CoV-2-infected K18-hACE2 mice, after SARS-CoV-2 N protein (green) and NeuN (red) immunofluorescence detection, showing specific strong viral load in neuronal cells. b, Orthogonal projection of the cortical neuron indicated by an arrow in a, demonstrating the colocalization of the cytoplasmic SARS-CoV-2 and nuclear NeuN signals in the same neuronal cell. The confocal image corresponds to a single confocal plane (z = 10) of 661 nm Z-depth optic resolution. c–f, Microscopy confocal images showing the colocalization of SARS-CoV-2 N protein (green) with the neuronal subtype-specific markers (red): CaMKII (c), parvalbumin (d), ChAT (e) and tyrosine hydroxylase (f), showing the viral infection of cortical glutamatergic-CaMKII⁺ and GABAergic-parvalbumin⁺ neurons, cholinergic-ChAT⁺ neurons of the basal forebrain and mesencephalic dopaminergic-tyrosine hydroxylase⁺ neurons. The insets are depicted at higher magnification on the right. The arrows label some examples of SARS-CoV-2-infected neurons. Nuclei were counterstained with DAPI (blue). Immunofluorescence staining was performed in 3 independent experiments obtaining similar results, analyzing SARS-CoV-2-infected mice at 6 dpi, n = 6 (3 females and 3 males). In e, AC, anterior commissure.