key: cord-1038984-or8dcz75
authors: Ahmed, Warish; Bertsch, Paul M.; Bibby, Kyle; Haramoto, Eiji; Hewitt, Joanne; Huygens, Flavia; Gyawali, Pradip; Korajkic, Asja; Riddell, Shane; Sherchan, Samendra P.; Simpson, Stuart L.; Sirikanchana, Kwanrawee; Symonds, Erin M.; Verhagen, Rory; Vasan, Seshadri S.; Kitajima, Masaaki; Bivins, Aaron
title: Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology
date: 2020-08-27
journal: Environ Res
DOI: 10.1016/j.envres.2020.110092
sha: 2d802d6a3ab16f7f207efe9a7a6f621f6e11f771
doc_id: 1038984
cord_uid: or8dcz75

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay 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, then matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T(90) (time required for 1 log(10) reduction) for RNA of SARS-CoV-2 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 T(90) for RNA of MHV at 4 to 37˚C ranged from 7.44 to 56.6 in untreated wastewater, 5.58 to 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 characteristics of SARS-CoV-2 and MHV were identified; thus, MHV is suggested as 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.

, an enveloped positive-sense RNA virus belonging to the genus Betacoronavirus decay and can impact the persistence of viruses in environmental systems. Increased decay rates are manner. For example, infectious viruses and/or the nucleic acids of somatic and F+ coliphages (Lee 111 and Sobsey, 2011; Sokolova et al., 2012) , as well as coxsackie virus B5, echovirus type 6 and 112 noroviruses all persisted longer at lower temperatures (Lo et al., 1976; Wait and Sobsey, 2001;  113 Skraber et al., 2007; Ngazoa et al., 2008) . The existing data suggest that SARS-CoV-2 surrogates, 114 such as MHV and transmissible gastroenteritis virus can remain infectious for up to 7 days when 115 seeded in pasteurized settled sewage, up to 22 days in deionised water at 25°C, and 4 weeks or 116 longer in these matrices at lower temperatures (<4°C) (Casanova et al., 2009) . Similarly, feline 117 infectious peritonitis virus (FIPV) and human coronavirus (CoV) 229E were found to also remain 118 infectious significantly longer in tap water at 4°C compared to 23°C (Gundy et al., 2009) . For SARS-

CoV, the etiologic agent of the severe acute respiratory epidemic that occurred in 2003, it was found 120 to remain infectious for 14 days in wastewater and 17 days in feces and urine at 4°C, but only two data suggest that the stability of enveloped viruses is negatively affected by elevated temperatures, 123 observations for SARS-CoV-2 RNA have not yet been published and may significantly impact 124 interpretation of WBE surveillance.

Biotic interactions, such as predation by bacterivorous protozoa, are a known cause of bacterial enveloped viruses such as SARS-CoV-2.

is derived from studies of other human CoV or viral surrogates. Gundy et al. (2009) determined the 138 survival of human CoV 229E and FIPV in water and wastewater using plaque assay or tissue culture 139 infectious dose 50% (TCID 50 ) techniques. The survival of both human CoV 229E and FIPV showed 140 similar patterns which was highly dependent on water temperature, level of organic matter, and 141 biological activity. In tap water, the T 99 (99% or 2 log 10 reduction) of both human CoV 229E and FIPV 142 were lower at 23°C (7-9 days) than 4°C (87 days). The inactivation rates of both these viruses were 143 greater in filtered tap water compared to unfiltered tap water at 23°C, suggesting increased protection 

There are critical gaps in our understanding of the persistence of enveloped viruses, and in 152 particular, SARS-CoV-2. The goal of this study was to compare the decay characteristics of SARS-

CoV-2 and MHV RNA using RT-qPCR, with attention to the effect of: (i) the water matrix (untreated 154 wastewater, autoclaved wastewater and dechlorinated tap water); (ii) environmentally relevant 155 temperatures (4, 15, 25 and 37°C); and, (iii) wastewater microbiota (comparison of untreated 156 wastewater and autoclaved wastewater) on the decay of the virus RNA. To achieve this, microcosms of MHV in the mouse fecal samples was determined using RT-qPCR assay (further details provided 169 below). Each MHV-positive fecal sample (approximately 250 mg) was suspended into 1 mL of 170 phosphate buffered saline (1 × PBS) and then the samples were pooled together to produce a 171 homogeneous fecal slurry. The slurry was centrifuged at 1,000 × g for 10 min. The pellet was 172 discarded, and the supernatant was stored at -80°C for three days. This supernatant, containing irradiated SARS-CoV-2 and the MHV suspension using the QIAamp Viral RNA Mini Kit (Qiagen,

Valencia, CA, USA) according to the manufacturer's instructions. The concentrations of gamma-qPCR assays, respectively (see below for detailed methodologies).

A sample of untreated wastewater (2 L) was collected from a WWTP in Brisbane, Australia and 181 transported to the laboratory on ice. The WWTP receives domestic wastewater from approximately 182 325,000 people, and also includes industrial and hospital wastewaters. While stormwater also enters 183 the WWTP, the study catchment did not receive any precipitation during the 24 h period prior to the 184 wastewater sample collection. The wastewater sample was stored at 4°C for 24 h before setting up the microcosm experiment. The collected wastewater sample was RT-qPCR negative for both SARS-microcosm was collected on day 0 (the day that they were prepared) and on days 3, 7, 12, 18, 25, 

The RT-qPCR assays were performed using a Bio-Rad CFX96 thermal cycler (Bio-Rad Laboratories). standard curves. Both CDC N1 and MHV standard dilutions ranged from 1 × 10 5 to 1 copy/µL. RT-227 qPCR data were generated using master standard curves reported in our recent studies (Ahmed et 228 al., 2020a; Ahmed et al., 2020b) . For each assay in this study, the assay limit of detection (ALOD),

defined as the minimum copy number with a 95% probability of detection, was determined as 

The effects of untreated and autoclaved wastewater samples on RT-qPCR amplifications of SARS-

CoV-2 and MHV were determined by comparing the Cq values obtained for wastewater samples to 237 the tap water samples (i.e., reference point). A sample was considered to have PCR inhibitors (i.e., 238 matrix interference) when Cq value was >2 compared to the reference Cq from tap water. The 

As expected, the log 10 transformed first-order decay rate increased with increasing temperature for 

CoV-2 and MHV RNA as observed at 4, 15, 25, and 37°C in each matrix. Each marker represents one 698 mean log 10 k value as observed in the current study

Temperatures (