Histopathological Analysis of SARS-CoV-2 in Lungs

For histopathology and in situ-hybridization (ISH), lungs were processed as described² . Left lung lobes were immersion-fixed in 10% formalin, pH 7.0, for 48 h, embedded in paraffin, and cut into 2 µm sections. Hematoxylin and eosin (HE) staining and in situ-hybridizations were performed as described^{7 (link)} using the ViewRNA™ ISH Tissue Assay Kit (Invitrogen) following the manufacturer’s instructions with minor adjustments. SARS-CoV-2 RNA was localized with probes detecting N gene sequences (NCBI database NC_045512.2, nucleotides 28,274–9533, assay ID: VPNKRHM). An irrelevant probe for detection of pneumolysin was used to control for sequence-specific binding⁴ . Amplifier and label probe hybridizations were performed following the manufacturer’s instructions using fast red as chromogen with hemalaun counterstain. Tissues were histopathologically evaluated by board-certified veterinary pathologists (KD, ADG) in a blinded fashion following standardized recommendations^{9 (link)}, including pneumonia-specific scoring parameters^{41 (link)} as described for SARS-CoV-2 infection in hamsters² .