Estimated Infectivity of Human Norovirus in Environmental Water Samples by an In Situ Capture RT-qPCR Method

Introduction: Human Norovirus (HuNoV) genomic signals have been detected in environmental samples using RT-PCR. However, it does not necessarily suggest the presence of infectious viruses. Histo-blood group antigens (HBGAs) have been recognized as receptors for HuNoVs. We have, previously, demonstrated that porcine gastric mucin (PGM) contains human HBGAs, and could be bound by multiple strains of HuNoVs. Refinement of prior viral binding/sequestration techniques have led to our current version, which utilizes PGM-coated hybrid binding/amplification containers. These hybrid binding/amplification containers serve as the medium for the binding/sequestration of HBGA-binding viruses, which is, immediately, followed by in situ amplification of the captured viral genomes by RT-qPCR (ISC-RT-qPCR).

Purpose: This study was conducted to estimate the potential infectivity of HuNoV in environmental water, using the ISC-RT-qPCR method.

Methods:  Tulane virus (TV) was incorporated into samples as an internal control for the indication and quantitation of RT-qPCR inhibition. The ISC-RT-qPCR method was, first, validated using heat-released viral RNA. Samples were tested for both TV and HuNoV, using ISC-RT-qPCR, and compared against the results of RT-qPCR and RNAse protection assays.

Results: We demonstrated that heat-released TV and HuNoV viral RNAs could not be captured and amplified using the ISC-RT-qPCR method. From 72 samples, positive for GI HuNoV by RT-qPCR, 20 samples (27.8%) tested positive by ISC-RT-qPC; suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. Similar results were observed with the RNase protection assay, as only 14 (19.4%) of RT-qPCR-positive samples were resistant to RNase. Of 16 samples, positive for GII HuNoV by RT-qPCR, only one tested positive by ISC-RT-qPCR; suggesting that 93% of RT-qPCR-positive samples were unlikely to be infectious. However, five samples that had initially tested negative by RT-qPCR, tested positive for GII HuNoV by ISC-RT-qPCR. 

Significance: Overall, the ISC-RT-qPCR assay is a promising alternative for the estimation of HuNoV infectivity in environmental samples.