Persistence and Free Chlorine Disinfection of Human Coronaviruses and Their Surrogates in Water (preprint)

The COVID-19 pandemic illustrates the importance of understanding the behavior and control of human pathogenic viruses in the environment. Exposure via water (drinking, bathing, and recreation) is a known route of transmission of viruses to humans, but the literature is relatively void of studies on the persistence of many viruses, especially coronaviruses, in water and their susceptibility to chlorine disinfection. To fill that knowledge gap, we evaluated the persistence and free chlorine disinfection of human coronavirus OC43 (HCoV-OC43) and its surrogates, murine hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV), in drinking water and laboratory buffer using cell culture methods. The decay rate constants of human coronavirus and its surrogates in water varied depending on virus and water matrix. In drinking water prior to disinfectant addition, MHV showed the largest decay rate constant (2.25 day-1) followed by HCoV-OC43 (0.99 day-1) and TGEV (0.65 day-1); while in phosphate buffer, HCoV-OC43 (0.51 day-1) had a larger decay rate constant than MHV (0.28 day-1) and TGEV (0.24 day-1). Upon free chlorine disinfection, the inactivation rates of coronaviruses were independent of free chlorine concentration and not affected by water matrix, though they still varied between viruses. TGEV showed the highest susceptibility to free chlorine disinfection with the inactivation rate constant of 113.50 mg-1 min-1 L, followed by MHV (81.33 mg-1 min-1 L) and HCoV-OC43 (59.42 mg-1 min-1 L). ImportanceThis study addresses an important knowledge gap on enveloped virus persistence and disinfection in water. Results have immediate practical applications for shaping evidence-based water policies, particularly in the development of disinfection strategies for pathogenic virus control.

Detection of SARS-CoV-2 variant Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in wastewater settled solids using mutation-specific assays is associated with regional detection of variants in clinical samples (preprint)

Changes in the circulation of SARS-CoV-2 variants of concern (VOCs) may require changes in public health response to the COVID-19 pandemic, as they have the potential to evade vaccines and pharmaceutical interventions and may be more transmissive relative to other SARS-CoV-2 variants. As such, it is essential to track and prevent their spread in susceptible communities. We developed digital RT-PCR assays for mutations characteristic of VOCs and used them to quantify those mutations in wastewater settled solids samples collected from a publicly owned treatment works (POTW) during different phases of the COVID-19 pandemic. Wastewater concentrations of single mutations characteristic to each VOC, normalized by the concentration of a conserved SARS-CoV-2 N gene, correlate to regional estimates of the proportion of clinical infections caused by each VOC. These results suggest targeted RT-PCR assays can be used to detect variants circulating in communities and inform public health response to the pandemic.

Effect of storage conditions on SARS-CoV-2 RNA quantification in wastewater solids (preprint)

SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 incidence in sewersheds and therefore, there is a strong interest in using these measurements to augment traditional disease surveillance methods. A wastewater surveillance program should provide rapid turn around for sample measurements (ideally within 24 hours), but storage of samples is necessary for a variety of reasons including biobanking. Here we investigate how storage of wastewater solids at 4{degrees}C, -20{degrees}C, and -80{degrees}C affects measured concentrations of SARS-CoV-2 RNA. We find that short term (7-8 d) storage of raw solids at 4{degrees}C has little effect on measured concentrations of SARS-CoV-2 RNA, whereas longer term storage at 4{degrees}C (35-122 d) or freezing reduces measurements by 60%, on average. We show that normalizing SARS-CoV-2 RNA concentrations by concentrations of pepper mild mottle virus (PMMoV) RNA, an endogenous wastewater virus, can correct for changes during storage as storage can have a similar effect on PMMoV RNA as on SARS-CoV-2 RNA. The reductions in SARS-CoV-2 RNA in solids during freeze thaws is less than those reported for the same target in liquid influent by several authors.

Wastewater-based estimation of the effective reproductive number of SARS-CoV-2 (preprint)

The effective reproductive number, Re, is a critical indicator to monitor disease dynamics, inform regional and national policies, and estimate the effectiveness of interventions. It describes the average number of new infections caused by a single infectious person through time. To date, Re estimates are based on clinical data such as observed cases, hospitalizations, and/or deaths. Here we show that the dynamics of SARS-CoV-2 RNA in wastewater can be used to estimate Re in near real-time, independent of clinical data and without associated biases stemming from clinical testing and reporting strategies. The method to estimate Re from wastewater is robust and applicable to data from different countries and wastewater matrices. The resulting estimates are as similar to the Re estimates from case report data as Re estimates based on observed cases, hospitalizations, and deaths are among each other. We further provide details on the effect of sampling frequency and the shedding load distribution on the ability to infer Re. To our knowledge, this is the first time Re has been estimated from wastewater. This method provides a low cost, rapid, and independent way to inform SARS-CoV-2 monitoring during the ongoing pandemic and is applicable to future wastewater-based epidemiology targeting other pathogens.