Epoch 2

Sequenced SARS-CoV-2-positive RNA samples (n = 60; 10 per variant) were randomly selected from adult (30–60 years old) patients (men and women). In addition, 10 negative samples were used as controls. RNA concentration and purity were measured with a spectrophotometer (Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA Thermo). Because of the small amounts of RNA in nasopharyngeal samples, all samples were vacuum-lyophilized (LABCONCO, Kansas City, MO, USA) at −56 °C and 0.123 mBar, for 48 h. The samples were then resuspended in nuclease-free water for a final concentration of 60 ng/µL. The methylation assay was performed with the EpiQuik^TM m6A RNA Methylation Quantification Kit (Epigentek, Farmingdale, NY, USA), according to the manufacturer’s instructions. Absorbance at 450 nm was measured in a microplate reader (EPOCH2, BioTek, Thermo Fisher Scientific, Waltham, MA, USA). The standard curve was obtained and the relative methylation level m6A (%) was determined according to the following formula: $$m6A (\%)=\frac{\left(O{D}_{sample}-O{D}_{NC}\right)÷S}{\left(O{D}_{PC}-O{D}_{NC}\right)÷P}\times 100$$
where OD_sample is the optical density of the sample, OD_NC is the optical density of the negative control, OD_PC is the optical density of the positive control, S is the amount of RNA (ng) applied to the well, and P is the amount of RNA (ng) applied to the positive control well (Supplementary File S2).

The glucose uptake colorimetric assay was performed according to the manufacturer’s instructions (Sigma, St. Louis, MO, USA). Briefly, human VICs were seeded at 1500 cells per well in 96-well plates after appropriate stimulation and incubated in an incubator containing 5% CO2 at 37 °C overnight. VICs were then rinsed twice with sterile PBS and starved in serum-free medium overnight. After another several washes, VICs were glucose-starved in Krebs-Ringer phosphate HEPES buffer containing 2% BSA for 40 min, followed by stimulation with or without insulin for 20 min. VICs were then cultured with 1 mM of the fluorescent glucose analog 2-Deoxy-d-glucose (2-DG) for 20 min. Following incubation, the cells were washed three times with PBS and then lysed using 80 μl of extraction buffer per well. After heating at 85 °C for 40 min, the cell lysates were incubated with Reaction Mix A and Reaction Mix B on a horizontal shaker. Fluorescence was calculated using absorbance at 485 nm in the EPOCH2 microplate reader (Thermo, EPOCH2).

Detection of PDV after agroinfiltration or mechanical transmission was performed using DAS-ELISA, and RT-PCR. DAS-ELISA was performed using the PDV DAS-ELISA kit to detect the viral CP of PDV (Agdia, Elkhart, IN, USA) following the manufacturer’s specifications. Results from DAS-ELISA were analyzed using a Fisher Scientific BioTek Epoch 2 microplate reader (Thermo Fisher Scientific, Mississauga, ON, Canada). For RT-PCR total RNAs were extracted from newly emerging leaves. The extracted RNA was digested with DNAse I (Thermo Fisher Scientific, Mississauga, ON, Canada) and cDNAs were generated using the superscript III cDNA synthesis kit, utilizing random hexamer primers, following the manufacturers protocol (Thermo Fisher Scientific, Mississauga, ON, Canada). PCR was carried out with PDV specific primers which amplify a portion of the gene which encodes the CP (Supplementary Table S1). PCR reactions were comprised of 12 μL 2× Taq Froggamix (FroggaBio, Vaughan, ON, Canada), 1 μL of newly synthesized cDNAs, 1μL of each forward and reverse primer (each at a concentration of 0.2 μM), and 10 μL of sterile distilled water with a total volume of 25 μL. PCR was performed using an initial denaturation step of 3 min at 94°C, followed by 35 cycles of 30 s at 94 °C for denaturation, annealing for 30 s at 54 °C, and 30 s at 72°C for extension, followed by a 5 min extension step at 72 °C.