SARS-CoV-2 wastewater variant surveillance: pandemic response leveraging FDA's GenomeTrakr network.

Wastewater surveillance has emerged as a crucial public health tool for population-level pathogen surveillance. Supported by funding from the American Rescue Plan Act of 2021, the FDA's genomic epidemiology program, GenomeTrakr, was leveraged to sequence SARS-CoV-2 from wastewater sites across the United States. This initiative required the evaluation, optimization, development, and publication of new methods and analytical tools spanning sample collection through variant analyses. Version-controlled protocols for each step of the process were developed and published on protocols.io. A custom data analysis tool and a publicly accessible dashboard were built to facilitate real-time visualization of the collected data, focusing on the relative abundance of SARS-CoV-2 variants and sub-lineages across different samples and sites throughout the project. From September 2021 through June 2023, a total of 3,389 wastewater samples were collected, with 2,517 undergoing sequencing and submission to NCBI under the umbrella BioProject, PRJNA757291. Sequence data were released with explicit quality control (QC) tags on all sequence records, communicating our confidence in the quality of data. Variant analysis revealed wide circulation of Delta in the fall of 2021 and captured the sweep of Omicron and subsequent diversification of this lineage through the end of the sampling period. This project successfully achieved two important goals for the FDA's GenomeTrakr program: first, contributing timely genomic data for the SARS-CoV-2 pandemic response, and second, establishing both capacity and best practices for culture-independent, population-level environmental surveillance for other pathogens of interest to the FDA. IMPORTANCE: This paper serves two primary objectives. First, it summarizes the genomic and contextual data collected during a Covid-19 pandemic response project, which utilized the FDA's laboratory network, traditionally employed for sequencing foodborne pathogens, for sequencing SARS-CoV-2 from wastewater samples. Second, it outlines best practices for gathering and organizing population-level next generation sequencing (NGS) data collected for culture-free, surveillance of pathogens sourced from environmental samples.

Risk Assessment of Norovirus Illness from Consumption of Raw Oysters in the United States and in Canada.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Bivalve molluscan shellfish is one commodity commonly identified as being a vector of NoV. Bivalve molluscan shellfish are grown in waters that may be affected by contamination events, tend to bioaccumulate viruses, and are frequently eaten raw. In an effort to better assess the elements that contribute to potential risk of NoV infection and illness from consumption of bivalve molluscan shellfish, the U.S. Department of Health and Human Services/Food and Drug Administration (FDA), Health Canada (HC), the Canadian Food Inspection Agency (CFIA), and Environment and Climate Change Canada (ECCC) collaborated to conduct a quantitative risk assessment for NoV in bivalve molluscan shellfish, notably oysters. This study describes the model and scenarios developed and results obtained to assess the risk of NoV infection and illness from consumption of raw oysters harvested from a quasi-steady-state situation. Among the many factors that influence the risk of NoV illness for raw oyster consumers, the concentrations of NoV in the influent (raw, untreated) and effluent (treated) of wastewater treatment plants (WWTP) were identified to be the most important. Thus, mitigation and control strategies that limit the influence from human waste (WWTP outfalls) in oyster growing areas have a major influence on the risk of illness from consumption of those oysters.

Determination of buildup and dilution of wastewater effluent in shellfish growing waters through a modified application of super-position.

Since 1925, dilution analysis has been used to minimize pathogenic impacts to bivalve molluscan shellfish growing areas from treated wastewater effluent in the National Shellfish Sanitation Program (NSSP). For over twenty five years, the U.S. Food and Drug Administration (FDA) has recommended a minimum of 1000:1 dilution of effluent within prohibited closure zones established around wastewater treatment plant (WWTP) discharges. During May 2010, using recent technologies, a hydrographic dye study was conducted in conjunction with a pathogen bioaccumulation study in shellfish adjacent to a WWTP discharge in Yarmouth, ME. For the first time an improved method of the super-position principle was used to determine the buildup of dye tagged sewage effluent and steady state dilution in tidal waters. Results of the improved method of dilution analysis illustrate an economical, reliable and more accurate and manageable approach for estimating the buildup and steady state pollutant conditions in coastal and estuarine waters.

Detection and molecular characterization of norovirus from oysters implicated in outbreaks in the US.

Human noroviruses are the leading cause of non-bacterial shellfish associated gastroenteritis. Here we report on the detection and characterization of norovirus (NoV) in shellfish associated outbreaks. Requests were received from state and federal officials for technical assistance in the analysis of shellfish for NoV and male specific coliphage (MSC; an enteric virus surrogate) during the years 2009 thru 2014. In outbreaks where NoV was detected, genogroup II (GII) levels ranged from 2.4 to 82.0 RT-qPCR U/g of digestive diverticula (DD) while NoV genogroup I (GI) levels ranged from 1.5 to 29.8 RT-qPCR U/g of DD. Murine norovirus extraction efficiencies ranged between 50 and 85%. MSC levels ranged from <6 to 80 PFU/100 g. Phylogenetic analysis of the outbreak sequences revealed strains clustering with GI.8, GI.4, GII.3, GII.4, GII.7, and GII.21. There was 100% homology between the shellfish and clinical strains occurring in 2 of 8 outbreaks. Known shellfish consumption data demonstrated probable infectious particles ingested as low as 12. These investigations demonstrate effective detection, quantification, and characterization of NoV in shellfish associated with illness.

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log10 reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log10 gc/liter; 95% credible interval [CI], 3.5, 4.3 log10 gc/liter) is larger than the value for NoV GI (1.5 log10 gc/liter; 95% CI, 0.4, 2.4 log10 gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log10 reductions were -2.4 log10 gc/liter (95% CI, -3.9, -1.1 log10 gc/liter) for NoV GI, -2.7 log10 gc/liter (95% CI, -3.6, -1.9 log10 gc/liter) for NoV GII, and -2.9 log10 PFU per liter (95% CI, -3.4, -2.4 log10 PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were -1.4 log10 gc/liter (95% CI, -3.3, 0.5 log10 gc/liter) for NoV GI, -1.7 log10 gc/liter (95% CI, -3.1, -0.3 log10 gc/liter) for NoV GII, and -3.6 log10 PFU per liter (95% CI, -4.8, -2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log10 reduction in NoV GII and the mean log10 reduction in MSCs.

Susceptibility of murine norovirus and hepatitis A virus to electron beam irradiation in oysters and quantifying the reduction in potential infection risks.

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D(10) value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10(5) PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10(2) PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.

Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assay provides a tool to help identify the origin of fecal contamination. Primers and probes were designed using complete genomic sequences from 29 FRNA phages. The final selection of primer/probe sets were based on (i) ability to amplify a single, specific product, (ii) genogroup specificity, (iii) lack of cross-reactivity, and (iv) experimental reproducibility and sensitivity over a range of target concentrations. Assay time was reduced by using heat-released viral RNA rather than purified RNA. For quality assurance, a custom RNA molecule was employed as an internal, non-competitive control. The usefulness of this method to identify sources of fecal contamination was tested on a total of 49 FRNA phages isolated from various warm-blooded animals, sewage and combined sewage overflow. FRNA phages from animal wastes were genotyped as 86% I, 4% III Q-like and 9% IV. Two sewage isolates typed to genogroup I and combined sewage overflow isolates genotyped as 40% II and 52% III. Primer specificity designed from this comprehensive sequence database may better discriminate FRNA from different sources.

Bacterial and viral pathogens in live oysters: 2007 United States market survey.

Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic Vibrio parahaemolyticus and V. vulnificus numbers and for the presence of toxigenic V. cholerae, Salmonella spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN), Escherichia coli (MPN), male-specific bacteriophage, and aerobic plate counts, were also determined. V. parahaemolyticus and V. vulnificus levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic V. parahaemolyticus were typically several logs lower than total V. parahaemolyticus levels regardless of season or region. Pathogenic V. parahaemolyticus levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic V. cholerae, 0%; Salmonella, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.

Use of N stable isotope and microbial analyses to define wastewater influence in Mobile Bay, AL.

We assessed short-term ecological and potential human health effects of wastewater treatment plant (WTP) effluent by measuring delta 15N per thousand and microbial concentrations in oysters and suspended particulate matter (SPM). We also tested male-specific bacteriophage (MSB) as an alternative to fecal coliforms, to assess potential influence of wastewater contamination on shellfish. WTP effluent did not affect oyster growth or survival, but SPM and oysters acquired wastewater-specific delta 15N per thousand. delta 15N values were depleted near the WTP, typical of low-level processed wastewater. Fecal coliform and MSB concentrations were higher in samples taken closest to the WTP, and MSB values were significantly correlated with delta 15N per thousand in oyster tissues. Overall, oysters demonstrated relatively rapid integration and accumulation of wastewater-specific delta 15N per thousand and indicator microorganisms compared to water samples. These data suggest oysters were superior sentinels compared to water, and MSB was a more reliable indicator of wastewater influence on shellfish than fecal coliforms.

Inactivation of a norovirus by high-pressure processing.

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20 degrees C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log(10) PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5 degrees C; a 5-min pressure treatment of 350 MPa at 30 degrees C inactivated 1.15 log(10) PFU of virus, while the same treatment at 5 degrees C resulted in a reduction of 5.56 log(10) PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5 degrees C and 20 degrees C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5 degrees C was sufficient to inactivate 4.05 log(10) PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.

High-pressure inactivation of hepatitis A virus within oysters.

Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log(10) were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3 degrees C. Bioconcentration of nearly 6 log(10) PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.

Molecular surveillance of enterovirus and norwalk-like virus in oysters relocated to a municipal-sewage-impacted gulf estuary.

An 18-month survey was conducted to examine the prevalence of enteric viruses and their relationship to indicators in environmentally polluted shellfish. Groups of oysters, one group per 4 weeks, were relocated to a coastal water area in the Gulf of Mexico that is impacted by municipal sewage and were analyzed for enteroviruses, Norwalk-like viruses (NLV), and indicator microorganisms (fecal coliform, Escherichia coli, and male-specific coliphages). The levels of indicator microorganisms were consistent with the expected continuous pollution of the area. Fourteen of the 18 oyster samples were found by reverse transcription (RT)-PCR to harbor NLV and/or enterovirus sequences. Of the four virus-negative oysters, three had exposure to water temperatures of >29 degrees C. Concomitant with these findings, two of these four oysters also accumulated the lowest levels of coliphages. PCR primers targeting pan-enteroviruses and the NLV 95/96-US common subset were utilized; NLV sequences were detected more frequently than those of enteroviruses. Within the 12-month sampling period, NLV and enterovirus sequences were detected in 58 and 42%, respectively, of the oysters (67% of the oysters tested were positive for at least one virus) from a prohibited shellfish-growing area approximately 30 m away from a sewage discharge site. Eight (4.6%) of the 175 NLV capsid nucleotide sequences were heterogeneous among the clones derived from naturally polluted oysters. Overall, enteric viral sequences were found in the contaminated oysters throughout all seasons except hot summer, with a higher prevalence of NLV than enterovirus. Although a high percentage of the oysters harbored enteric viruses, the virus levels were usually less than or equal to 2 logs of RT-PCR-detectable units per gram of oyster meat.