Long-Range Reverse Transcription as a Useful Tool to Assess the Genomic Integrity of Norovirus

Abstract

The effects of heat (72°C) and UV-light (254 nm) on the infectivity of murine norovirus-1 (MNV-1) and human norovirus (NoV) are reported. The infectivity of MNV-1 was measured using standard cell-culture plaque assays and was compared with a specific duplex RT-qPCR for MNV-1, which targets two fragments of the MNV genome, one 142 bp (short-range, sRT-qPCR) and a second 4.6 kb (long-range, loRT-qPCR) upstream from the reverse transcription (RT) priming site (poly-A tail). After the heat treatment of MNV neither the sRT- nor loRT-qPCR correlated with decreased infectivity as assessed by the plaque assay. However, after UV-exposure, the lo-qPCR showed decreased qPCR amplification, which became more pronounced with increased UV-exposure time, and reflected decreased infectivity as shown in the plaque assay. The sRT-qPCR amplification was not affected by UV-exposure. Similar results were obtained when challenging non-culturable human GI and GII NoV with UV-light. Using previously published RT-qPCR assays for GI and GII NoV, no decrease in RT-qPCR amplification was seen when using the respective reverse primers of the RT-qPCR assay in the RT reaction, whereas a significant decrease in qPCR amplification occurred when the RT reaction was primed at the poly-A tail about 2.3 kb from the qPCR amplification site. The results indicate that, in contrast to RT-qPCR with a long-range RT, RT-qPCR with a short-range RT does not reflect genomic integrity and therefore viral infectivity. Furthermore, human NoV appears to be highly sensitive to UV-irradiation which could lead to more efficient decontamination procedures.