Quantifast

Swabs were briefly vortexed in 500 µL PBS and viral RNA was extracted from 140 µL in accordance with the manufacturer’s instructions (QIamp viral RNA; Qiagen, Toronto, ON, Canada). RNA extractions were performed within the 48 h following sampling (swabs were kept at 4 °C in the meantime). RT-qPCR was performed on 96-well plates with a final volume of 20 µL in a Light Cycler system (Roche, Penzberg, Germany). The mixes were prepared in accordance with the manufacturer’s instructions (QuantiNova Probe; Qiagen, Toronto, ON, Canada) with 2 µL of RNA and primers that targeted the E-gene. To obtain human-derived viral RNA and perform phylogenetic analysis, the owner from case 3 self-performed a nasal swab which was treated as a feline one. To check whether the material in the oropharyngeal and rectal swabs originated from felines, RT-qPCR targeting the feline 40S ribosomal protein S7 (RPS7) gene was also performed. SARS-CoV-2-positive swabs were further tested for human ribosomal protein L30 (RPL30) mRNA expression, to rule out human contamination. RPS7 and RPL30 RT-qPCR were performed on 96-well plates in a final volume of 10 µL in a Light Cycler system (Roche, Penzberg, Germany). Mixes were prepared according to the manufacturer’s instructions (QuantiFast; Qiagen, Toronto, ON, Canada). The primers’ sequences are listed in Supplementary Table S2.

Total RNA was extracted from snap-frozen tissues or cultured cells using either the mirVana^TM micro-RNA isolation kit (ThermoFisher AM1560) according to the manufacturer’s instructions, or using TRI reagent (Sigma T9424). 1-2 µg of total RNA was used for cDNA synthesis with RevertAid H-Minus reverse transcriptase (Thermo Scientific EP0451) and a mix of oligo-d(T)₁₈ (ThermoFisher FERSO132) and random primers (ThermoFisher FERSO142). Quantitative (q)PCR was performed using the QuantiFast (Qiagen 25057), QuantiNova (Qiagen 208057) or SsoAdvanced Universal SYBR Green Supermix (BioRad 1725274) and intron-spanning primer pairs on a QuantStudio 3 (ThermoFisher) or CFX384 (Bio-Rad) real-time PCR thermocycler. All primers were tested for efficiency and correct product amplification prior to use. In the few cases where primers were non-intron-spanning, RNA was DNAse treated before cDNA synthesis. Expression levels were normalized against housekeeping gene Sdha and are displayed as mean relative to the WT control samples; error bars indicate standard error of the means (SEM) of at least three independent biological replicates. Where appropriate, Student’s t test or ANOVA was performed to calculate the statistical significance of expression differences (p < 0.05) using GraphPad Prism software (9.4.1); all p-values are provided in Supplementary Data 1.