Abstract
Human noroviruses (HuNoV) are the major cause of viral gastroenteritis worldwide. Similar to other positive-sense single-stranded RNA viruses, norovirus RNA replication requires the formation of a negative strand RNA intermediate. Methods for detecting and quantifying the viral positive or negative sense RNA in infected cells and tissues can be used as important tools in dissecting virus replication.In this study, we have establisheda sensitive and strand-specificTaqman-basedquantitative polymerase chain reaction (qPCR) assay for both genogroups GI and GII HuNoV. This assay shows good reproducibility, has a broad dynamic range and is able to detect a diverse range of isolates.We used tagged primers containing a non-viral sequence for the reverse transcription (RT) reaction and targeted this tag in the succeeding qPCR reaction to achieve strand specificity. The specificity of the assay was confirmed by the detection of specific viral RNA strands in the presence of high levels of the opposing strands, in both RT and qPCR reactions. Finally, we further validated the assay in norovirus replicon-bearing cell lines and norovirus-infected human small intestinal organoids, in the presence or absence of small-molecule inhibitors. Overall, we have established a strand-specific qPCR assay that can be used as a reliable methodto understand the molecular details ofthe human norovirus life cycle.
Highlights
Human noroviruses (HuNoVs) are the leading cause of both sporadic and outbreak cases of acute gastroenteritis worldwide[1]
We have previously developed the strandspecific quantitative polymerase chain reaction (qPCR) assay for murine norovirus (MNV) to study aspects of norovirus replication and to provide an additional tool to indicate that active replication is occurring[25]
A 517 basepair region of GI and a 466 base-pair region of GII HuNoV at the ORF1-ORF2 junction, previously described as being highly conserved[40,41], were amplified using standard PCR with the primers shown in Table 1 and Table 2
Summary
Human noroviruses (HuNoVs) are the leading cause of both sporadic and outbreak cases of acute gastroenteritis worldwide[1]. The HuNoV replicon system[8,9] and recently established infection models including the B-cell culture system[10], stem cell-derived organoids[11] and zebrafish larvae[12], are all valuable norovirus experimental systems. I think this figure would benefit from showing the whole workflow for both positive and negative sense RNAs, from the PCR and in vitro transcription steps (as already shown) plus the RT and qPCR steps. This would be helpful for showing each of the primer binding sites from Tables 1 and 2 and the different orientations of the +ssRNA and -ssRNA workflow at each step.
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