Rox reference dye

The primer pair and probe COG2R/QNIF2d/QNIFS (36 (link)) were used for GII genotypes, and the primer pair and probe NIFG1F/V1LCR/NIFG1P (37 (link)) were used for GI.1 using qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences). Reactions were performed on an Applied Biosystems StepOnePlus thermocycler using the following cycling conditions: 50°C (15 min) and 95°C (5 min), followed by 40 cycles of 95°C (15 s) and 60°C (35 s). A standard curve based on a recombinant HuNoV GII.4 (Houston virus [HOV]) or GI.1 (Norwalk virus) RNA transcript was used to quantitate viral genome equivalents (GEs) in RNA samples (38 (link), 39 (link)). A 0.5-log₁₀ increase in GEs after 24 hpi relative to the amount of genomic RNA detected at 1 hpi (after removal of the virus inoculum and two washes of the monolayers to remove unbound virus) was set as a threshold to indicate successful viral replication. A sample was considered to have failed replication if two independent experiments with 5 μl of undiluted stool filtrate in the presence of GCDCA failed to show a 0.5-log₁₀ increase in GEs after 24 hpi relative to GEs detected at 1 hpi.

Northern blot analysis was performed as described previously (19 (link)). Quantification of signal intensities was performed using ImageJ (78 (link)). For real-time quantitative PCR (qRT-PCR) experiments, 1 μg of RNase-free DNase-treated RNA was reverse transcribed with random hexamer primers using a RevertAid first-strand cDNA synthesis kit (Thermo Scientific). All qRT-PCRs were performed in triplicate in a 20-μL mixture containing cDNA (5 μL of 1:20 dilution), PerfeCTa SYBR green FastMix containing ROX reference dye (Quanta Biosciences), and 18 pmol of primer (Table S2). Amplification and detection of PCR products were performed with a StepOne Plus qRT-PCR system (Applied Biosystems) using the following procedure: 95°C for 10 min and then 40 cycles of 95°C for 15 s and 60°C for 60 s, followed by dissociation curve analysis. The relative expression levels of the genes studied were normalized to the 5S rRNA gene. Data were analyzed using the ΔΔC_T method (79 (link)). If not stated otherwise, at least three qRT-PCR experiments were performed in triplicate with cDNA that was reverse transcribed from independent RNA preparations.

Total RNA was extracted from each infected well using the KingFisher Flex purification system and MagMAX-96 Viral RNA isolation kit. RNA extracted at 1 hpi was used as a baseline to determine the amount of input virus that remained associated with cells after washing the infected cultures to remove unbound virus. Replication of the virus was determined by quantifying RNA levels from samples extracted at 24 hpi. For RT-qPCR, the primer pair and probe COG2R/QNIF2d/QNIFS (41 (link)) were used for GII genotypes and the primer pair and probe NIFG1F/V1LCR/NIFG1P (42 (link)) were used for GI.1 using qScript XLT One-Step RT-qPCR ToughMix reagent with ROX reference dye (Quanta Biosciences). Reactions were performed in duplicates on an Applied Biosystems StepOnePlus thermocycler using the following cycling conditions: 50°C (15 min), 95°C (5 min), followed by 40 cycles of 95°C (15 s) and 60°C (35 s). Standard curves based on recombinant GII and GI HuNoV RNA transcripts were used to quantitate viral genome equivalents (GEs) in RNA samples. The limit of detection of the RT-qPCR assay was 20 and 100 when using recombinant GII and GI RNA transcripts, respectively.