Qiamp viral rna

Viral/total RNA was extracted from plasma/sera and tissues, using Qiamp viral RNA (Qiagen) and RNeasy lipid tissue RNA extraction kit (Qiagen), respectively. Protocol as outlined by the kit was followed, except for tissue harvested from CTR rabbits. For these samples, Qiazol (Qiagen) was substituted with Trizol (Invitrogen). Tissue was homogenized in 1 mL of Qiazol or Trizol using an Omni-tip homogenizer (Omni) or stainless steel beads with a mixer mill (Retsch, Inc., Newtown, PA, USA), respectively. The remaining steps were performed according to the manufacturer’s protocol. Additional DNase I digest in solution was performed on extracted RNA, followed by additional RNA clean up with RNeasy RNA extraction kit (Qiagen). The absence of genomic DNA was confirmed by performing PCR without reverse-transcription on a selection of RNA samples from each extraction batch. Quality and concentration of extracted RNA was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA samples were kept at −80 °C.

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.

A fraction of ORF2 including region E was amplified by RT-PCR after extraction of viral RNA from FCV isolates, using the extraction kit QiAmp Viral RNA (Qiagen, Courtaboeuf, France) according to the manufacturer instructions. The RT-PCR was performed on 5 μL of viral RNA in a total volume of 25 μL with the primer set F1244 5′-GATCCCTGATGGTTGGCC-3′ (forward)/R1949 5′-ATTCCCATGTAGGAGGC-3′ (reverse) or with the primer set F832 5′-CAYCTDATGTCTGAYACTG-3′ (forward)/R1963 5′-GAATTCCCATGTAGGAGGC-3′ (reverse), using the kit PHUSION RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer instructions (proofreading Taq polymerase). The resulting 706 bp (first set of primers) or 1131 bp (second set of primers) amplification band was visualized on agarose electrophoresis with ethidium bromide straining. The RT-PCR products were directly sequenced on both strands, using the same primers (Eurofins Genomics, Anzinger, Germany). The sequences were assembled and translated (software Vector NTI, Thermo Fisher Scientific, Waltham, MA, USA) to obtain the protein sequence of the region E part of VP1.