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Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Viral testing combined with hydrographic studies is considered standard good practice in determining microbiological impacts on shellfish growing areas following wastewater overflows. In this study, norovirus genogroup I and II, indicators of viral contamination (F-RNA bacteriophage genogroup II (F-RNA GII), crAssphage, pepper mild mottle virus) and Escherichia coli were monitored during periods of normal harvesting and following overflows in two commercial shellfish growing areas in Otago Harbour (Aotearoa New Zealand). Dye tracing, drogue tracking and analysis of particle tracking modelling were also undertaken to assess the dispersion, dilution and time of travel of wastewater discharged from a pump station discharge that impacts the growing areas. Norovirus was not detected in any of the 218 shellfish samples tested. PMMoV and crAssphage were more prevalent than F-RNA GII as determined by RT-qPCR. The dye study indicated long residence time of the waters (≥5 days) in the embayment impacted by the discharge. No relationships were found between the concentrations of viral indicators or E. coli and wastewater dilution, distance between the discharge and the growing areas or time since the last overflow. For the three spills studied (≤327 m 3 ), there was little evidence of microbiological impact on the growing areas. This was likely associated with a deep shipping channel that enhances water flushing in the harbour and reduces contaminant transport to the growing areas. We recommend flexibility in the approach for closure/reopening growing areas impacted by spills, particularly for small duration/volume spills and when norovirus is not present in the community., (© 2024. The Author(s).)

Norovirus is the predominant cause of viral acute gastroenteritis globally. While person-to-person is the most reported transmission route, norovirus is also associated with waterborne and foodborne illness, including from the consumption of contaminated bivalve molluscan shellfish. The main cause of shellfish contamination is via the bioaccumulation of norovirus from growing waters impacted by human wastewater. However, data on the persistence of infectious norovirus in the environment are limited due to a lack of a human norovirus culture method in the past. In this study, we applied the recently established method of norovirus replication in human intestinal enteroids to determine the persistence of norovirus in artificial estuarine water at 25 ppt for up to 21 days at 4 °C and 16 °C in the dark. Infectious norovirus was detected for up to 21 days. The relative infectivity declined from 100 to 3% at day 21, with decay rate constants of 0.07 day -1 at 4 °C and 0.17 day -1 at 16 °C. There was no decrease in norovirus titres as measured by reverse transcription-droplet digital PCR (RT-ddPCR), confirming the lack of the relationship between norovirus infectivity and direct detection by PCR. The results confirm that norovirus can remain infectious for at least 3 weeks in an estuarine water environment, presenting associated health risks., (© 2024. The Author(s).)

The BioFire FilmArray ® Gastrointestinal Panel was evaluated for the rapid detection of adenovirus, astrovirus, norovirus, rotavirus and sapovirus from influent and effluent wastewater and shellfish. The multiplex BioFire FilmArray ® Gastrointestinal Panel compared well to singleplex qPCR/RT-qPCR methods for the detection of adenovirus, astrovirus, rotavirus and sapovirus from influent and effluent wastewater samples. However, the BioFire FilmArray ® Gastrointestinal Panel showed poor performance for the detection of norovirus, significantly underestimating its presence in wastewater and shellfish samples when compared with the singleplex norovirus GI and GII RT-qPCR assays. Therefore, improvement on detection efficiency for norovirus from environmental and food samples is necessary before using results from the FilmArray ®  Gastrointestinal Panel to assess associated public health risks.

Historically in developed countries, reported hepatitis E cases were typically travellers returning from countries where hepatitis E virus (HEV) is endemic, but now there are increasing numbers of non-travel-related ("autochthonous") cases being reported. Data for HEV in New Zealand remain limited and the transmission routes unproven. We critically reviewed the scientific evidence supporting HEV transmission routes in other developed countries to inform how people in New Zealand may be exposed to this virus. A substantial body of indirect evidence shows domesticated pigs are a source of zoonotic human HEV infection, but there is an information bias towards this established reservoir. The increasing range of animals in which HEV has been detected makes it important to consider other possible animal reservoirs of HEV genotypes that can or could infect humans. Foodborne transmission of HEV from swine and deer products has been proven, and a large body of indirect evidence (e.g. food surveys, epidemiological studies and phylogenetic analyses) support pig products as vehicles of HEV infection. Scarce data from other foods suggest we are neglecting other potential sources of foodborne HEV infection. Moreover, other transmission routes are scarcely investigated in developed countries; the role of infected food handlers, person-to-person transmission via the faecal-oral route, and waterborne transmission from recreational contact or drinking untreated or inadequately treated water. People have become symptomatic after receiving transfusions of HEV-contaminated blood, but it is unclear how important this is in the overall hepatitis E disease burden. There is need for broader research efforts to support establishing risk-based controls.

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log 10 was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log 10 loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log 10 , with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.

Cancer heterogeneity is a significant factor in response to treatment and escape leading to relapse. Within an individual cancer, especially blood cancers, there exists multiple subclones as well as distinct clonal expansions unrelated to the clinically detected, dominant clone. Over time, multiple subclones and clones undergo emergence, expansion, and extinction. Although sometimes this intra-clonal and inter-clonal heterogeneity can be detected and/or quantified in tests that measure aggregate populations of cells, frequently, such heterogeneity can only be detected using single cell analysis to determine its frequency and to detect minor clones that may subsequently emerge to become drug resistant and dominant. Most genetic/genomic tests look at the pooled tumor population as a whole rather than at its individual cellular components. Yet, minor clones and cancer stem cells are unlikely to be detected against the background of expanded major clones. Because selective pressures are likely to govern much of what is seen clinically, single cell analysis allows identification of otherwise cryptic compartments of the malignancy that may ultimately mediate progression and relapse. Single cell analysis can track intra- or inter-clonal heterogeneity and provide useful clinical information, often before changes in the disease are detectable in the clinic. To a very limited extent, single cell analysis has already found roles in clinical care. Because inter- and intra-clonal heterogeneity likely occurs more frequently than can be currently appreciated on a clinical level, future use of single cell analysis is likely to have profound clinical utility.