Global Trends in Norovirus Genotype Distribution among Children with Acute Gastroenteritis.

Noroviruses are a leading cause of acute gastroenteritis (AGE) among adults and children worldwide. NoroSurv is a global network for norovirus strain surveillance among children <5 years of age with AGE. Participants in 16 countries across 6 continents used standardized protocols for dual typing (genotype and polymerase type) and uploaded 1,325 dual-typed sequences to the NoroSurv web portal during 2016-2020. More than 50% of submitted sequences were GII.4 Sydney[P16] or GII.4 Sydney[P31] strains. Other common strains included GII.2[P16], GII.3[P12], GII.6[P7], and GI.3[P3] viruses. In total, 22 genotypes and 36 dual types, including GII.3 and GII.20 viruses with rarely reported polymerase types, were detected, reflecting high strain diversity. Surveillance data captured in NoroSurv enables the monitoring of trends in norovirus strains associated childhood AGE throughout the world on a near real-time basis.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Global Spread of Norovirus GII.17 Kawasaki 308, 2014-2016.

Analysis of complete capsid sequences of the emerging norovirus GII.17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.

Addressing heterogeneity of individual blood cancers: the need for single cell analysis.

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.

Norovirus genotype profiles associated with foodborne transmission, 1999-2012.

Worldwide, noroviruses are a leading cause of gastroenteritis. They can be transmitted from person to person directly or indirectly through contaminated food, water, or environments. To estimate the proportion of foodborne infections caused by noroviruses on a global scale, we used norovirus transmission and genotyping information from multiple international outbreak surveillance systems (Noronet, CaliciNet, EpiSurv) and from a systematic review of peer-reviewed literature. The proportion of outbreaks caused by food was determined by genotype and/or genogroup. Analysis resulted in the following final global profiles: foodborne transmission is attributed to 10% (range 9%%-11%) of all genotype GII.4 outbreaks, 27% (25%-30%) of outbreaks caused by all other single genotypes, and 37% (24%%-52%) of outbreaks caused by mixtures of GII.4 and other noroviruses. When these profiles are applied to global outbreak surveillance data, results indicate that ≈14% of all norovirus outbreaks are attributed to food.

Metagenomic analysis of viruses in feces from unsolved outbreaks of gastroenteritis in humans.

The etiology of an outbreak of gastroenteritis in humans cannot always be determined, and ∼25% of outbreaks remain unsolved in New Zealand. It is hypothesized that novel viruses may account for a proportion of unsolved cases, and new unbiased high-throughput sequencing methods hold promise for their detection. Analysis of the fecal metagenome can reveal the presence of viruses, bacteria, and parasites which may have evaded routine diagnostic testing. Thirty-one fecal samples from 26 gastroenteritis outbreaks of unknown etiology occurring in New Zealand between 2011 and 2012 were selected for de novo metagenomic analysis. A total data set of 193 million sequence reads of 150 bp in length was produced on an Illumina MiSeq. The metagenomic data set was searched for virus and parasite sequences, with no evidence of novel pathogens found. Eight viruses and one parasite were detected, each already known to be associated with gastroenteritis, including adenovirus, rotavirus, sapovirus, and Dientamoeba fragilis. In addition, we also describe the first detection of human parechovirus 3 (HPeV3) in Australasia. Metagenomics may thus provide a useful audit tool when applied retrospectively to determine where routine diagnostic processes may have failed to detect a pathogen.

Structure based design of novel 6,5 heterobicyclic mitogen-activated protein kinase kinase (MEK) inhibitors leading to the discovery of imidazo[1,5-a] pyrazine G-479.

Use of the tools of SBDD including crystallography led to the discovery of novel and potent 6,5 heterobicyclic MEKi's [J. Med. Chem.2012, 55, 4594]. The core change from a 5,6 heterobicycle to a 6,5 heterobicycle was driven by the desire for increased structural diversity and aided by the co-crystal structure of G-925 [J. Med. Chem.2012, 55, 4594]. The key design feature was the shift of the attachment of the five-membered heterocyclic ring towards the B ring while maintaining the key hydroxamate and anilino pharamcophoric elements in a remarkably similar position as in G-925. From modelling, changing the connection point of the five membered ring heterocycle placed the H-bond accepting nitrogen within a good distance and angle to the Ser212 [J. Med. Chem.2012, 55, 4594]. The resulting novel 6,5 benzoisothiazole MEKi G-155 exhibited improved potency versus aza-benzofurans G-925 and G-963 but was a potent inhibitor of cytochrome P450's 2C9 and 2C19. Lowering the logD by switching to the more polar imidazo[1,5-a] pyridine core significantly diminished 2C9/2C19 inhibition while retaining potency. The imidazo[1,5-a] pyridine G-868 exhibited increased potency versus the starting point for this work (aza-benzofuran G-925) leading to deprioritization of the azabenzofurans. The 6,5-imidazo[1,5-a] pyridine scaffold was further diversified by incorporating a nitrogen at the 7 position to give the imidazo[1,5-a] pyrazine scaffold. The introduction of the C7 nitrogen was driven by the desire to improve metabolic stability by blocking metabolism at the C7 and C8 positions (particularly the HLM stability). It was found that improving on G-868 (later renamed GDC-0623) required combining C7 nitrogen with a diol hydroxamate to give G-479. G-479 with polarity distributed throughout the molecule was improved over G-868 in many aspects.

Persistence of Infectious Human Norovirus in Estuarine Water.

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.

Study of Shellfish Growing Area During Normal Harvesting Periods and Following Wastewater Overflows in an Urban Estuary With Complex Hydrography.

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 m3), 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.

PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

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.

Molecular epidemiology of norovirus gastroenteritis outbreaks in New Zealand from 2002-2009.

Noroviruses are the most common cause of acute non-bacterial gastroenteritis outbreaks worldwide, including New Zealand. New Zealand has a population of 4.4 million, which allows for centralized outbreak surveillance and a Norovirus Reference Laboratory, which facilitates efficient diagnosis, surveillance, and tracking of norovirus outbreaks. Norovirus outbreak strains are identified, sequenced, and compared with international reference strains. Between January 2002 and December 2009, 1,206 laboratory-confirmed norovirus outbreaks were recorded. The predominant outbreak settings were healthcare institutions for the elderly and acute care patients. Other outbreak settings included catering establishments, cruise ships, homes, community events, school camps, child-related settings, and consumption of contaminated shellfish. Of the 1,206 outbreaks, 105 (8.7%) were caused by norovirus genogroup I (GI) strains, 1,085 (89.9%) were caused by genogroup II (GII) strains, and both GI and GII strains were detected in 9 (0.8%) outbreaks. The genogroup was not identified in 7 (0.6%) outbreaks. A range of norovirus genotypes, including GI genotypes 1-6, GII genotypes 2-8, and GII.12, were associated with these outbreaks. The predominant genotype was GII.4, which was identified in 825 (68.4%) outbreaks. Norovirus GII.4 variant strains, including 2002 (Farmington Hills), 2004 (Hunter), 2006a (Laurens, Yerseke), 2006b (Minerva), and 2010 (New Orleans) implicated in overseas outbreaks also occurred in New Zealand, providing evidence of global spread.

Reductions of human enteric viruses in 10 commonly used activated carbon, polypropylene and polyester household drinking-water filters.

Drinking-water treatment in non-networked rural communities relies on the use of point-of-use (PoU) household filters. Source waters treated by PoU filters are often microbially contaminated, but information about human enteric virus reductions in these filters is limited. This study evaluated human rotavirus, adenovirus and norovirus reductions in 10 commonly used, new PoU carbon, polypropylene and polyester microfilters. The viruses were spiked into chlorine-free tap water (pH 8.0, ionic strength 1.22 mM), and 3 sequential challenge tests were conducted in each filter under a constant flow rate of 1 L/min. In most of the filters investigated, the norovirus and adenovirus reductions were similar (P > 0.49). Compared with the norovirus and adenovirus reductions, the rotavirus reductions were significantly lower in the carbon filters (P ≤ 0.009), which may relate to rotavirus's higher zeta potential and lower hydrophobicity. Virus reductions appeared to be dictated by the filter media type through electrostatic and hydrophobic interactions; the effects of filter media pore sizes on virus reductions via physical size-exclusion were very limited. The virus reductions in the carbon filters were significantly greater than those in the polypropylene and polyester filters (P ≤ 0.0001), and they did not differ significantly between the polypropylene and polyester filters (P > 0.24). None of the filters met the "protective" rotavirus reduction level (≥3 log10) required for household drinking-water treatment. Our study's findings highlight a critical need for additional water treatment when using PoU microfilters, for example, water boiling or ultraviolet radiation, or the use of effective surface-modified filter media to prevent drinking-waterborne infections from enteric viruses.

A Framework for Public Health Authorities to Evaluate Health Determinants for Wastewater-Based Epidemiology.

BACKGROUND: Wastewater-based epidemiology (WBE) is rapidly developing as a powerful public health tool. It can provide information about a wide range of health determinants (HDs), including community exposure to environmental hazards, trends in consumption of licit and illicit substances, spread of infectious diseases, and general community health. As such, the list of possible candidate HDs for WBE is almost limitless. Consequently, a means to evaluate and prioritize suitable candidates for WBE is useful, particularly for public health authorities, who often face resource constraints. OBJECTIVES: We have developed a framework to assist public health authorities to decide what HDs may be appropriate for WBE and what biomarkers could be used. This commentary reflects the experience of the authors, who work at the interface of research and public health implementation. DISCUSSION: To be suitable for WBE, a candidate HD should address a public health or scientific issue that would benefit from better understanding at the population level. For HDs where information on individual exposures or stratification by population subgroups is required, WBE is less suitable. Where other methodologies are already used to monitor the candidate HD, consideration must be given to whether WBE could provide better or complementary information to the current approach. An essential requirement of WBE is a biomarker specific for the candidate HD. A biomarker in this context refers to any human-excreted chemical or biological that could act as an indicator of consumption or exposure to an environmental hazard or of the human health state. Suitable biomarkers should meet several criteria outlined in this commentary, which requires background knowledge for both the biomarker and the HD. An evaluation tree summarizing key considerations for public health authorities when assessing the suitability of candidate HDs for WBE and an example evaluation are presented. https://doi.org/10.1289/EHP11115.

Canadian perspectives in multiple myeloma on the use of steroids in clinical practice based on patient and healthcare provider interviews.

Corticosteroid (steroid) medications are associated with challenging adverse effects that can negatively impact patient quality of life. However, owing to a long legacy of effective use in treatment protocols, they remain a cornerstone of multiple myeloma (MM) care. We conducted a roundtable with Canadian healthcare providers (HCPs) with diverse healthcare backgrounds and involvement in MM care as well as with patients with MM. Our goal was to develop clear guidance for steroid management aimed at improving patient quality of life, taking into account patient perspective and experiences with managing the disease. Our recommendations, which are based on the insights acquired from this discussion, can be categorized to the following areas: steroid prescribing, dosing, and modifications; managing adverse effects; and patient-HCP communication. These recommendations can be used by the entire multi-disciplinary hematology team to improve patient quality of life while being treated with steroid medication for multiple myeloma.

Sensitivity of wastewater-based epidemiology for detection of SARS-CoV-2 RNA in a low prevalence setting.

To assist public health responses to COVID-19, wastewater-based epidemiology (WBE) is being utilised internationally to monitor SARS-CoV-2 infections at the community level. However, questions remain regarding the sensitivity of WBE and its use in low prevalence settings. In this study, we estimated the total number of COVID-19 cases required for detection of SARS-CoV-2 RNA in wastewater. To do this, we leveraged a unique situation where, over a 4-month period, all symptomatic and asymptomatic cases, in a population of approximately 120,000, were precisely known and mainly located in a single managed isolation and quarantine facility (MIQF) building. From 9 July to 6 November 2020, 24-hr composite wastewater samples (n = 113) were collected daily from the sewer outside the MIQF, and from the municipal wastewater treatment plant (WWTP) located 5 km downstream. New daily COVID-19 cases at the MIQF ranged from 0 to 17, and for most of the study period there were no cases outside the MIQF identified. SARS-CoV-2 RNA was detected in 54.0% (61/113) at the WWTP, compared to 95.6% (108/113) at the MIQF. We used logistic regression to estimate the shedding of SARS-CoV-2 RNA into wastewater based on four infectious shedding models. With a total of 5 and 10 COVID-19 infectious cases per 100,000 population (0.005% and 0.01% prevalence) the predicated probability of SARS-CoV-2 RNA detection at the WWTP was estimated to be 28 and 41%, respectively. When a proportional shedding model was used, this increased to 58% and 87% for 5 and 10 cases, respectively. In other words, when 10 individuals were actively shedding SARS-CoV-2 RNA in a catchment of 100,000 individuals, there was a high likelihood of detecting viral RNA in wastewater. SARS-CoV-2 RNA detections at the WWTP were associated with increasing COVID-19 cases. Our results show that WBE provides a reliable and sensitive platform for detecting infections at the community scale, even when case prevalence is low, and can be of use as an early warning system for community outbreaks.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Application of crAssphage, F-RNA phage and pepper mild mottle virus as indicators of human faecal and norovirus contamination in shellfish.

Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100% (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2-3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%-100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%-99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.

Attenuation and transport of human enteric viruses and bacteriophage MS2 in alluvial sand and gravel aquifer media-laboratory studies.

Potable groundwater contamination by human enteric viruses poses serious health risks. Our understanding of virus subsurface transport has largely depended on studying bacteriophages as surrogates. Few studies have compared the transport behaviour of enteric viruses, especially norovirus, with phage surrogates. We conducted laboratory column experiments to investigate norovirus and bacteriophage MS2 (MS2) filtration in alluvial sand, and rotavirus, adenovirus and MS2 filtration in alluvial gravel aquifer media in 2 mM NaCl (pH 6.6-6.9) with pore velocities of 4.6-5.4 m/day. The data were analysed using colloid filtration theory and HYDRUS-1D 2-site attachment-detachment modelling. Norovirus removal was somewhat lower than MS2 removal in alluvial sand. The removal of rotavirus and adenovirus was markedly greater than MS2 removal in alluvial gravel. These findings concurred with the log10 reduction values, mass recoveries, attachment efficiencies and irreversible deposition rate constants. The modelling results suggested that the MS2 detachment rates were in the same order of magnitude as norovirus, but they were 1 order of magnitude faster than those of rotavirus and adenovirus. The attachment of viruses and MS2 was largely reversible with faster detachment than attachment rates, favouring free virus transport. These findings highlight the risk associated with continual virus transport through subsurface media if viruses are not inactivated and remobilising previously attached viruses could trigger contamination events. Thus, virus attachment reversibility should be considered in virus transport predictions in subsurface media. Further research is needed to compare surrogates with enteric viruses, especially norovirus, regarding their transport behaviours under different experimental conditions.

A multi-platform metabolomics approach to identify possible biomarkers for human faecal contamination in Greenshell™ mussels (Perna canaliculus).

Bivalve molluscs have the potential to bioaccumulate microbial pathogens including noroviruses from aquatic environments and as such, there is a need for a rapid and cheap in-situ method for their detection. Here, we characterise the tissue-specific response of New Zealand Greenshell™ mussels (Perna canaliculus) to faecal contamination from two different sources (municipal sewage and human faeces). This is done with the view to identify potential biomarkers that could be further developed into low cost, rapid and sensitive in-situ biosensors for human faecal contamination detection of mussels in growing areas. Tissue-specific metabolic profiles from gills, haemolymph and digestive glands were analysed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Clear differentiation of metabolic profiles was observed among treatments in each tissue type. Overall, energy pathways such as glycolysis, citrate cycle and oxidative phosphorylation were downregulated across the three mussel tissues studied following simulated contamination events. Conversely, considerable sterol upregulation in the gills was observed after exposure to contamination. Additionally, free pools of nucleotide phosphates and the antioxidant glutathione declined considerably post-exposure to contamination in gills. These results provide important insights into the tissue-specific metabolic effects of human faecal contamination in mussels. This study demonstrates the utility of metabolomics as a tool for identifying potential biomarkers in mussels.