Design, development, and validation of a strand-specific RT-qPCR assay for GI and GII human Noroviruses

Figures

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  Figure 1.. Denaturing RNA-PAGE confirms the RNA integrity of both positive and negative strands of GI and GII human norovirus. A) A schematic representation of the human norovirus genome showing the non-structural (NS1/2 to NS7) and structural (VP1 and VP2) proteins. The arrows indicate the position of the primer binding sites on the viral genome. GI was amplified from prototype Norwalk virus genome (Accession number M87661) whereas GII.4 was amplified from cDNA clone containing the accession number DQ658413. B) A schematic representation of the PCR and the in vitro transcriptions used to generate the strand-specific control RNAs used as standards in the strand-specific assay. C) The purified strand-specific RNA standards were analyzed by denaturing PAGE to confirm their integrity and size. M represents the RNA size marker in bases.

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  Figure 2.. Evaluation of the real-time strand-specific qPCR assay for HuNoV Genogroup I. The left-hand column represents positive strand and the right-hand column represents negative strand. A, B) Strand specific qPCR amplification plots for positive and negative sense GI viral RNA plotted against the cycle number. The standard curve represents 8 or 7-point ten-fold dilutions from 10 9 copy numbers. C, D) Confirmation of strand-specific qPCR products and no template control (NTC) by agarose gel electrophoresis. E, F) Strand specific qPCR assay was performed and plotted in presence or absence of a fixed amount of the opposite strand. Solid circle in line ( ) represents linear regression of the standard curve; square symbol in dotted line ( ) indicates the linear regression in the presence of opposite strand (+opp). Slope (y) and regression coefficient (R 2) of the standard curve were shown. G, H) To mimic an infection condition, the RT reactions were performed in the presence of high copy number of the opposite strand. Solid circle ( ) represents 10 7, 10 5 and 10 3 copies of the standard RNA with 10 6 copies of the opposite strand (+opp) in square symbol ( ). Samples were analyzed in three…

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  Figure 4.. HuNoV inhibitors 2'-C-methylcytidine (2-CMC) and rupintrivir reduce both the positive and the negative strand of GI HuNoV. The impact of 2-CMC and rupintrivir on HuNoV replication was examined in HGT-NV cells. Cells treated with and without 2-CMC or rupintrivir were harvested after 3 days. Extracted RNAs were quantified and analyzed by strand-specific RT-qPCR. A, C) The levels of positive (+ss) and negative (-ss) RNA strands in the absence or presence of 2-CMC ( A) and rupintrivir ( C) were measured as genome equivalents/well. B, D) The effect of treatment on virus replication was plotted as percentage of untreated control. The mean ± SEM from triplicate samples analyzed in technical duplicates is plotted. Statistically significant values are represented as: ***=p≤0.001, ****=p≤0.0001.

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  Figure 5.. Innate immune suppressors ruxolitinib (Rux) and triptolide (TPL) increase both the positive and the negative viral RNA strands in GII HuNoV infected organoid-derived culture system. Intestinal epithelial cells-derived from human duodenal organoids were inoculated with GII.3 HuNoV and ileal organoids with GII.4 HuNoV then, infected cells were subsequently treated with either DMSO, Rux, TPL or 2-CMC, as relevant control. The effects of innate immune inhibitors on HuNoV replication were examined after 2 days. The RNA extracts collected on day 0 (D0) and day 2 (D2) post infection were quantified and analyzed by strand-specific RT-qPCR. ( A, C) The levels of positive (+ss) and negative (-ss) RNA strands in the absence or presence of the drug/s were measured as genome equivalents/well. ( B, D) The effect of treatments on virus replication was plotted as fold change relative to D0. The mean ± SEM from triplicate samples analyzed in technical duplicates is plotted. Statistically significant values are represented as: ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001.