Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.