key: cord-1022303-g2yy8e99
authors: Kitajima, Masaaki; Ahmed, Warish; Bibby, Kyle; Carducci, Annalaura; Gerba, Charles P.; Hamilton, Kerry A.; Haramoto, Eiji; Rose, Joan B.
title: SARS-CoV-2 in wastewater: State of the knowledge and research needs
date: 2020-04-30
journal: The Science of the total environment
DOI: 10.1016/j.scitotenv.2020.139076
sha: 732c31132a3d0355f17cc90b736a0a012e31f1b3
doc_id: 1022303
cord_uid: g2yy8e99

Abstract The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a Public Health Emergency of International Concern, which was officially declared by the World Health Organization. SARS-CoV-2 is a member of the family Coronaviridae that consists of a group of enveloped viruses with single-stranded RNA genome, some of which have been known to cause common colds. Although the major transmission routes of SARS-CoV-2 are inhalation from person-to-person and aerosol/droplet transmission, currently available evidence indicates that the viral RNA is present in wastewater, suggesting the need to better understand wastewater as potential sources of epidemiological data and human health risks. Here, we review the current knowledge related to the potential of wastewater surveillance to understand the epidemiology of COVID-19, methodologies for the detection and quantification of SARS-CoV-2 in wastewater, and information relevant for human health risk assessment of SARS-CoV-2. There has been growing evidence of gastrointestinal symptoms caused by SARS-CoV-2 infections and the presence of viral RNA not only in feces of COVID-19 patients but in wastewater. One of the major challenges in SARS-CoV-2 detection/quantification in wastewater samples is the lack of an optimized and standardized protocol. Currently available data are also limited for conducting a quantitative microbial risk assessment (QMRA) for SARS-CoV-2 exposure pathways. However, modeling-based approaches have a potential role to play in reducing the impact of the ongoing COVID-19 outbreak, and QMRA parameters obtained from previous studies on relevant respiratory viruses help to inform risk assessments of SARS-CoV-2. Our understanding on the potential role of wastewater in SARS-CoV-2 transmission is largely limited by knowledge gaps in its occurrence, persistence, and removal in wastewater. There is an urgent need for further research to establish methodologies for wastewater surveillance and understand the implications of the presence of SARS-CoV-2 in wastewater.

In December 2019, China reported an outbreak of pneumonia of unknown etiology occurring in Wuhan, Central China's Hubei Province to the World Health Organization (WHO) (WHO, 2020a) .

Shotgun metagenomic sequencing of bronchoalveolar lavage samples indicated that this outbreak was associated with a novel coronavirus (nCoV) . The nCoV was confirmed to have 75-80% nucleotide similarity to severe acute respiratory syndrome coronavirus (SARS-CoV) and was officially designated as SARS-CoV-2 after being provisionally named as 2019-nCoV (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020) . SARS-CoV-2 together with SARS-CoV belong to the species Severe acute respiratory syndrome-related coronavirus in the subgenus Sarbecovirus of the family Coronaviridae that consists of a group of enveloped viruses with a single-stranded, positive-sense RNA genome. SARS-CoV and SARS-CoV-2 are distantly related to Middle East respiratory syndrome coronavirus (MERS-CoV) , which belongs to the species Middle East respiratory syndrome-related coronavirus within the genus Betacoronavirus (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020). SARS-CoV-2 is also distantly related to 'classical' human CoV strains (229E, OC43, NL63, and HKU1) belonging to the genus Alphacoronavirus or Betacoronavirus that have been studied since the 1960s and are estimated to cause 15 to 30% of cases of common colds worldwide (Mesel-Lemoine et al., 2012) .

The disease caused by SARS-CoV-2 is referred to as coronavirus disease 2019 .

Symptoms of COVID-19 at the onset of illness include fever, myalgia, fatigue, and dry cough, and J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f enterovirus, rotavirus, and hepatitis A virus (Okabayashi et al., 2008; Rodríguez-Lázaro et al., 2012; Yoshida et al., 2009 ), but also for other viruses that are rarely or never reported by epidemiological surveillance systems, such as Saffold virus, cosavirus, and salivirus/klassevirus (Bonanno Ferraro et al., 2020; Kitajima et al., 2015 Kitajima et al., , 2014 Thongprachum et al., 2018) .

SARS-CoV-2 is known to cause asymptomatic or pauci-symptomatic infections (Lai et al., 2020; Mizumoto et al., 2020; Nishiura et al., 2020; A. Tang et al., 2020a) making it difficult to determine the actual degree of viral circulation in a community and in making comparisons among different countries that have different clinical diagnostic testing capabilities with even different diagnostic methods/assays (Ortiz-Ospina and Hasell, 2020) . Meanwhile, wastewater surveillance could provide an unbiased method of evaluating the spread of infection in different areas, even where resources for clinical diagnosis are limited and when reporting systems are unavailable or not feasible, such as in developing countries. Moreover, wastewater monitoring can help to detect variations in the circulating strains through phylogenetic analysis, allowing for comparisons between regions and assessment of evolution of the virus genome over time as demonstrated previously for enteric viruses (Bisseux et al., 2018; La Rosa et al., 2014; Lodder et al., 2013) , and more recently for SARS-CoV-2 (Nemudryi et al., 2020) .

The importance of wastewater surveillance is also highlighted by its ability to detect low levels of viruses; this can happen when the number of infected cases is decreasing following public health interventions, which has been successful in poliovirus eradication programs (Asghar et al., 2014) . It is also useful to determine when a new virus is introduced into a population (Savolainen-Kopra et J o u r n a l P r e -p r o o f highlighting ethical issues related to basic access to sanitation, privacy, and rights may be required.

It should be widely understood that one of the advantages of WBE is that this approach provides epidemiological information on disease prevalence in a community by circumventing individual stigmatization, which often results from clinical diagnosis in the ongoing COVID-19 outbreak (Murakami et al., 2020) .

Although viral loads in feces of COVID-19 patients are variable (Table 1) , SARS-CoV-2 RNA can be sometimes detected with comparable concentrations to many enteric viruses (~10 8 viruses per gram of feces) (Bosch, 1998; Prüss et al., 2002; Wyn-Jones and Sellwood, 2001) . Nevertheless, it will likely be necessary to perform a virus concentration step(s) prior to subsequent detection of SARS-CoV-2, even in untreated wastewater, as conducted previously Lodder and de Roda Husman, 2020; Medema et al., 2020; Nemudryi et al., 2020; Wurtzer et al., 2020) (Table 2) .

Numerous types of methods have been developed for concentrating viruses in wastewater; however, most of those studies aimed to establish concentration methods for nonenveloped enteric viruses such as norovirus, enterovirus, adenovirus, and hepatitis A virus, using culturable viruses and/or bacteriophages as model viruses (Haramoto et al., 2018) . Electropositive or electronegative membranes have been widely used to concentrate enteric viruses in untreated and treated wastewater samples (Cashdollar and Wymer, 2013; Haramoto et al., 2018; Ikner et al., 2012) . These methods were developed based on electrostatic interactions between filters and viruses, utilizing the fact that a majority of enteric viruses have a net negative electrostatic charge in environmental water near neutral pH. In this method, negatively charged virus particles directly adsorb onto electropositive J o u r n a l P r e -p r o o f (Lewis and Metcalf, 1988), ultracentrifugation (Fumian et al., 2010) , and skimmed-milk flocculation (Calgua et al., 2013) have also been used for concentrating viruses from wastewater samples.

The effectiveness of these virus concentration methods has been well demonstrated by successful detection of various types of indigenous enteric viruses which were not used as a model virus during the method development (Fong and Lipp, 2005; Haramoto et al., 2018) . However, limited knowledge is available on recovery efficiencies of enveloped viruses, including CoVs, with the existing virus concentration methods. Ye et al. (2016) reported greater adsorption of enveloped viruses (mouse hepatitis virus [MHV] and Pseudomonas phage Φ6) to the solid fraction of wastewater compared to nonenveloped viruses. Haramoto et al. (2009) reported that enveloped koi herpesvirus showed high adsorption efficiency to an electronegative filter. Taken together, these results suggest that virus concentration methods using filters may potentially be used to recover SARS-CoV-2 from water and wastewater and requires further investigation. Even within enteric viruses, recovery efficiencies of viruses can vary greatly depending on virus and water types (Haramoto et al., 2018) . Therefore, little scientific evidence is available to inform judgments of the usefulness of these existing virus concentration methods for enveloped SARS-CoV-2, which has quite different characteristics in structural and physical properties from enteric viruses. For example, Wang et al. (Xin Wei Wang et al., 2005b) reported that recovery of SARS-CoV from wastewater was only 1% using an electropositive membrane filter method, a significant decrease in performance compared to that observed for many types of enteroviruses (Li et al., 1998) .

Nevertheless, virus concentration will likely be necessary to increase the chance of detection of SARS-CoV-2 in wastewater and research is needed to evaluate the recovery efficiency. Meanwhile, efforts are needed to evaluate the applicability of these existing methods to concentrating SARS-CoV-2. For method evaluation and development, low-pathogenic CoV strains (such as MHV and classical human CoVs) and/or Pseudomonas phage Φ6 may be used as models of SARS-CoV-2 for J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Nalla et al. (2020) evaluated the performance of seven RT-qPCR assays targeting RdRp, E, and N genes (CDC, 2020a; Corman et al., 2020) , where clinical respiratory and swab samples including SARS-CoV-2 positive samples were tested (Nalla et al., 2020) . Based on the results of experiments using dilutions of a SARS-CoV-2-positive sample, the authors found that the N gene-(N2) and E gene-RT-qPCR assays developed by CDC (2020a) and Corman et al. (2020) , respectively, showed the highest sensitivity of ~6.3 genomic equivalents per reaction. However, since the limited number of SARS-CoV-2-positive samples collected from a certain region were used in this study, further studies using samples from various locations worldwide are needed to establish a 'gold standard' assay.

Unlike clinical samples, a lower ALOD value is required when SARS-CoV-2 is tested in a wastewater sample with low virus concentration due to dilution and low prevalence of COVID-19.

Unfortunately, data regarding ALOD are not available for many of the existing RT-qPCR assays, most likely because these methods were designed for application to rapidly screening clinical samples. It is likely that RT-qPCR assays showing ALOD of <10 copies per reaction could be useful for screening of wastewater samples for SARS-CoV-2 Shirato et al., 2020) . A SYBR Green based qPCR targeting spike (S) protein gene has been also developed, although no ALOD data are provided . Digital RT-PCR may enable more sensitive and accurate detection/quantification of SARS-CoV-2 RNA in wastewater samples than RT-qPCR as suggested recently for clinical samples Suo et al., 2020) .

When detecting SARS-CoV-2 RNA in wastewater by qPCR, confirmation of positive qPCR signals by sequencing analysis is highly recommended until the assay specificities have been validated against environmental samples, because the currently available RT-qPCR assays were developed for clinical diagnosis, which may be quite different from environmental applications.

Two nested RT-PCR assays targeting open reading frame 1a (ORF1a) and S protein genes are also available (Shirato et al., 2020) , which could be used to elucidate the genetic diversity of SARS-CoV-2 circulating in human populations. As a thermal cycler is essential for PCR, novel assays which do not require any thermal cycler, such as loop-mediated isothermal amplification (LAMP) method, are expected to be developed, which will enable detection of SARS-CoV-2 RNA more easily and rapidly, especially in situations where sufficient laboratory equipment is not available.

Additional efforts may be made to assess viral infectivity in wastewater using an engineered cell line with high susceptibility to SARS-CoV-2 (Matsuyama et al., 2020) and/or to detect infectious viral particles selectively by utilizing viability qPCR, such as ethidium bromide monoazide (EMA) or propidium monoazide (PMA) treatment followed by RT-qPCR, or integrated cell culture (ICC)-RT-PCR/qPCR (Farkas et al., 2020) .

A critical issue in the application of molecular-based methods including RT-qPCR to wastewater samples is PCR inhibition during the detection process. It has been recommended that a process control(s) should be included in the analysis to monitor the levels of loss of targets and/or inhibition from the sample concentration to the detection steps (Haramoto et al., 2018) . Three types of process controls are proposed, depending on the points of their inoculation: (i) whole process controls, to be inoculated into a water sample before virus concentration; (ii) molecular process controls, to be inoculated into a viral concentrate before nucleic acid extraction; and (iii) RT-qPCR controls, to be inoculated before RT-qPCR. At least one of these process controls is recommended to be included to avoid false-negative results and for concentration methods with low virus recovery efficiencies. Based on the results, samples may need to be reanalyzed (Haramoto et al., 2018) . For a reliable process control, it is appropriate to select a virus which is genetically closely related to and/or has similar physical characteristics as the target virus and is expected not to be present in the J o u r n a l P r e -p r o o f

The magnitude of human health risks varies depending on the decay of pathogens, including SARS-CoV-2 in water environments. Understanding the decay of SARS-CoV-2 and its RNA will ultimately improve control measures and wastewater treatment requirements, but little has been documented on the persistence of CoVs in water and wastewater matrices. Wang et al. (2005) investigated the persistence of SARS-CoV, Escherichia coli and f 2 phage in hospital wastewater, domestic sewage, tap water, phosphate buffered saline, feces, urine, water, and wastewater with high various concentrations (5, 10, 20 and 40 mg/L) of chlorine and chlorine dioxide. They also investigated the effect of contact time on inactivation of SARS-CoV in wastewater with low (10 mg/L chlorine and chlorine dioxide) and high (20 mg/L of chlorine and 40 mg/L of chlorine dioxide) concentrations. Results indicated that coronavirus persisted longer (inoculated titer of 10 5 TCID 50 ; detectable with RT-PCR for 14 days) at 4°C compared to 20°C (3 days) in hospital wastewater, domestic sewage, and dechlorinated tap water. At 20°C, SARS-CoV persisted in three fecal samples for 3 days and two urine samples for 17 days (inoculated titer of 10 5 TCID 50 ). SARS-CoV was more vulnerable to disinfectants compared to E. coli and f 2 phage. Free chlorine was more effective in inactivating SARS-CoV than chlorine dioxide. Free residue chlorine of >0.5 mg/L or chlorine J o u r n a l P r e -p r o o f faster at 23°C (7-9 days) than 4°C (>87 days). The inactivation rates of both CoVs were faster in filtered tap water compared to unfiltered tap water at 23°C, suggesting increased protection and survival in the presence of organic matter and suspended solids. CoVs were inactivated rapidly in wastewater, with T 99 values of <3 days (Gundy et al., 2009 ). Casanova et al. (2009) determined the persistence of two surrogate CoVs, transmissible gastroenteritis virus (TGEV), and MHV in reagent grade water, lake water, and pasteurized settled sewage in North Carolina, USA using quantal assays for cytopathic effect (CPE). In general, both the surrogate viruses persisted for significantly shorter durations at 25°C compared to 4°C for all water types. For reagent grade water, TGEV and MHV persisted for shorter durations (T 99 = 22 and 16 days, respectively) at 25°C than at 4°C (>220 days for both viruses). For lake water, TGEV and MHV T 99 values were 13 and 10 days, respectively, over a 14-day experiment. However, at 4°C, one log 10 reduction was observed at day 14 for TGEV, while no reduction was observed for MHV up to day 14 at 4°C. Both viruses persisted shorter in pasteurized settled sewage samples, and T 99 reduction times were nine days for TGEV, and seven days for MHV. At 4°C, T 99 values of TGEV and MHV were 49 and 70 days, respectively, suggesting surrogate CoVs can remain infectious for long periods in water and pasteurized settled sewage at a lower temperature (Casanova et al., 2009) .

A technical brief from WHO suggested that there is no evidence about the survival of SARS-CoV-2 in wastewater or drinking water. It is likely that enveloped CoVs are less stable in the environment and is more susceptible to chlorine, pH, and temperature than most of nonenveloped enteric viruses (WHO, 2020d). Therefore, conventional wastewater treatment processes should inactivate SARS-CoV-2, and multiple barriers used in drinking water treatment plants should suffice to remove SARS-CoV-2 to levels of non-detect and low risks (<10 -4 annual risk). However, limited J o u r n a l P r e -p r o o f viruses such as low-pathogenic human CoV (e.g., 229E or OC43), feline CoV, MHV, or Pseudomonas phage Φ6 can be used. Ye et al. (2016) compared the persistence and partitioning behavior of two model enveloped viruses, MHV and Pseudomonas phage Φ6 in raw and pasteurized wastewater samples using cell culture and plaque assays. MHV and Φ6 were seeded into unpasteurized and pasteurized wastewater and incubated at 10 and 25°C to mimic typical winter and summer temperatures of wastewater. The

T 90 values of MHV and Φ6 in unpasteurized wastewater at 25°C were 13 and 7 h, respectively. In contrast, the T 90 values of MHV and Φ6 were slower in unpasteurized wastewater at 10°C with T 90 values of 36 and 28 h, respectively. Both viruses persisted relatively longer in pasteurized wastewater than unpasteurized wastewater. Based on the results, the authors concluded that although MHV and Φ6 were inactivated rapidly in wastewater, their persistence could still be of concern for wastewater treatment facilities, stormwater overflows, and wastewater intrusion in drinking water (Ye et al., 2016) . Their results on comparative viral persistence in pasteurized and unpasteurized wastewater implied that enhancement of favoring competition and predation contributed by indigenous microbial communities in wastewater could be a potential medium-term strategy to fight against the ongoing and future viral disease outbreaks.

Similarly, Aquino de Carvalho et al. (2017) evaluated the persistence of Φ6 in a variety of matrices, including water and wastewater. The T 90 of Φ6 under these conditions was highly variable, from 24 min to 117 days. Significant factors included temperature, biological activity, and the composition of the test media. Beyond direct study findings, the authors reported that the aqueous stability of enveloped viruses in water matrices was highly variable, and a single surrogate was insufficient to capture the behavior of all enveloped viruses (Aquino de Carvalho et al., 2017) .

Given the limited available data on SARS-CoV-2 in water matrices, it may also be informative to consider recent reports of viral persistence on surfaces. van Doremalen et al. (2020) evaluated the surface stability of SARS-CoV-2 compared to SARS-CoV. The half-life of SARS-CoV-2 varied from 0.8 hours on copper to 6.8 hours on plastic. The authors also identified comparable environmental persistence between the two viruses (van Doremalen et al., 2020) . Chin et al. (2020) J o u r n a l P r e -p r o o f also reported on the surface persistence and disinfection of SARS-CoV-2. The authors identified a high temperature dependence on the inactivation kinetics, and rapid removal of the virus using bleach, ethanol, benzylalkonium chloride, povidone-iodine, and chloroxylenol (Chin et al., 2020) .

Overall, these limited results suggest that previous data on CoVs are likely to be useful for informing the environmental persistence of SARS-CoV-2, and that SARS-CoV-2 is likely rapidly inactivated under increased temperature and by major disinfectants. In fact, a number of the existing disinfectant products have been approved by the United States Environmental Protection Agency (USEPA) for use against SARS-CoV-2 (USEPA, 2020).

In the field of environmental virology, the focus on waterborne transmission has been primarily on enteric viruses. However, respiratory viruses including adenoviruses, coxsackieviruses, and indeed CoVs have been known to occur in wastewater (Sinclair et al., 2008; Xin Wei Wang et al., 2005a ) and wastewater-polluted waters (Wigginton et al., 2015) . Going back to early descriptions, it has been known that these viruses cause diarrheal as well as respiratory diseases (Britton, 1980) There is no doubt that swimming in sewage-contaminated waters is associated with respiratory disease; however, the etiological agent is not frequently identified (Wade et al., 2010) . Studies on the Great Lakes suggest this could be due to adenoviruses . Respiratory disease as an occupational risk for sewage workers has also been studied with mixed results. Four key studies were reviewed as shown in Table 5 . In Switzerland, no health impacts were found in garbage collectors and wastewater treatment plant (WWTP) workers (Tschopp et al., 2011) . However, in J o u r n a l P r e -p r o o f three other investigations, gastrointestinal effects were observed, and two of the three studies noted respiratory health impacts (Khuder et al., 1998; Lee et al., 2009; Smit et al., 2005) .

Environmental engineering and science, and in particular QMRA have major roles to play in reducing the impact of the current COVID-19 outbreak (Haas, 2020; Wigginton and Boehm, 2020) .

The process of QMRA involves relating an environmental concentration of an infectious agent to an exposure dose and subsequently a probability of developing an infection or illness (Haas et al., 2014) .

Gaps needed to fill include characterizing persistence, fate and transport (including airborne transport and deposition, for example), and exposure to be able to define the risk. In addition to basic questions like "what is the risk" (potentially in relation to some baseline) for a particular context, QMRA can be used to address questions for SARS-CoV-2 such as: (i) What ventilation/air exchange rate is recommended for different settings (e.g. workplace, healthcare facility) to prevent transmission consistent with a risk target?; (ii) Is a 6-ft / 2m "social distance" protective enough?;

(iii) What should surface disinfection targets be for different settings and what are the best technologies or disinfectants for achieving these targets (e.g. UV light)?; (iv) What wastewater treatment disinfection targets might be needed? SARS-CoV-2 transmission is known to occur via hand-to-face (nasal-pharyngeal: eyes, nose, and mouth) contact with contaminated fomites, and inhalation of aqueous aerosols including coughs.

The fecal-oral route or aspiration have been postulated as potential exposure routes, although no cases of transmission via the fecal-oral route have been reported to date (CDC, 2020b; Y. Wu et al., 2020; Yeo et al., 2020) . There are some preliminary data to suggest that the virus is shed longer from the digestive tract than the respiratory tract .

J o u r n a l P r e -p r o o f occupational populations (e.g. wastewater workers and nurses). Many QMRAs have also focused on wastewater biosolids applications, addressing adenovirus, astrovirus, coxsackievirus, echovirus, enterovirus, hepatitis A virus, hepatitis E virus, norovirus, and rotavirus, both during the actual period of application as well as at various exposures from farm to fork (Hamilton et al., 2020) .

A key component of bioaerosol QMRA is modeling the dispersion of aerosols and/or transfer of microorganisms from water to air. A key study of stormwater reuse for inhalation-ingestion of adenovirus and norovirus provides an example of considering the aerosol size profile of a particular activity in order to calculate the number of viruses aerosolized and the subsequent deposited dose (Lim et al., 2015) . Other methods utilized for other microbial risk studies are the use of a water-toair transfer coefficient or computational fluid dynamics approaches (Hamilton and Haas, 2016) . The studies of viral aerosols emerging from wastewater facilities have often focused on coliphage as an indicator for human pathogenic viruses, but most studies have not simultaneously sampled the wastewater and the aerosols produced, or identified how the viruses are distributed in aerosols with respect to the aerosol size profile (Table 6) . Fannin et al. (1985) were not able to detect animal viruses in the aerosols (Fannin et al., 1985) . Adenoviruses are known to be quite stable in air and high concentrations were found by qPCR, thus viable viruses were not addressed (Masclaux et al., 2014) . The phage data suggest a 10,000-fold level of dilution and inactivation (Brenner et al., 1988 ).

There has been no published study to date testing SARS-CoV-2 in aerosols from wastewater facilities, but a recent laboratory-scale study on persistence of coronaviruses in aerosols revealed that SARS-CoV-2 could maintain its infectivity in aerosols for up to 16 hours (Fears et al., 2020) , suggesting potential human health risks if wastewater aerosols contain viable SARS-CoV-2. Further investigations are needed to elucidate the presence of SARS-CoV-2 and its viability in wastewater bioaerosols and associated public health risks.

A recent QMRA study for enteric viruses via exposure to wastewater bioaerosols focused on adenoviruses as the hazards (Carducci et al., 2018) . The analysis highlighted the following: (i) workers at highest risk were related to exposures at the influent and biological oxidation tanks for more than 3 min; (ii) adenovirus concentrations drove the risk; (iii) risks of 10 -2 , 10 -3 , 10 -4 , and 10 -5 J o u r n a l P r e -p r o o f 21 were related to levels of 565, 170, 54 and 6 copies/m 3 in the bioaerosol, respectively; (iv) this relates to an estimated level of approximately 10 4 to 10 6 copies/L in the oxidation tank. Similarly, a recent study focusing on rotavirus and norovirus bioaerosol exposures to WWTP workers noted risks that exceeded common public health risk benchmarks of 10 -4 infections or 10 -6 disability adjusted life years (DALY) per person per year for airborne concentrations above the aeration tank of 27 and 3,099 viruses/m 3 -h, for rotavirus and norovirus, respectively (Pasalari et al., 2019) . Taken together, these data help to provide a comparison of relative risks and put concentrations of viruses into perspective.

To date, few QMRAs for CoVs have been conducted including one for MERS-CoV in a hospital setting (Adhikari et al., 2019) and another for a residential bathroom exposure (Watanabe et al., 2010) . Adhikari et al. (2019) where 99 cases of SARS were reported to arrive at estimates of 16−49 plaqueforming units (PFU) on the first floor, 63−160 PFU for residents on a middle floor, and 42−117 PFU for residents on upper floors, with higher attack rates on higher floors likely due to air flow in the building. Tabulating attack rates for SARS-CoV-2 could provide similarly useful information for estimating doses and/or risks in retrospect, however such an exercise requires information on an environmental measurement coupled with the total number of persons exposed in addition to the number of individuals infected and/or ill. Given the parameters developed in the existing QMRA J o u r n a l P r e -p r o o f models, a summary of parameters for potential use in QMRA models and data gaps are summarized in Table 7 .

To conduct a QMRA, a dose-response model is required providing the relationship between exposure and health outcome or endpoint. To date, no quantitative dose-response model is available for SARS-CoV-2. This is partially due to the absence of an appropriate animal model for pathogen dosing (Gralinski and Menachery, 2020) . Contributing factors to the lack of appropriate animal model include the inability to cause disease without passaging the virus through a mouse host and milder disease in primates compared to humans (Gralinski and Menachery, 2020) . The search for an appropriate animal model for various applications (treatments and vaccines) is underway and several models are being explored, placing strains on the supply of transgenic laboratory mice (Boodman, 2020; Callaway, 2020; Warren, 2020) .

(macaques, cynomolgus monkeys, African green monkeys, rhesus macaques, and common marmosets), hamsters, ferrets, and transgenic mice (Gretebeck and Subbarao, 2015) . A recent study by Rockx et al. (2020) indicated that SARS-CoV-2 results in a severity of infection that is intermediate between that of SARS-CoV and MERS-CoV based on a direct comparison of the three viruses in a combined intratracheal and intranasal dosing study of female adult cynomolgus macaques with a dose of 10 6 TCID 50 for all viruses. Shedding varied depending on the age of the animal, with higher levels detected in nasal swabs of aged animals compared to younger animals (Rockx et al., 2020) .

The doses used in current animal models underway for SARS-CoV-2 are 10 4 PFU−10 5.5 TCID 50 in ferrets (Blanco-Melo et al., 2020; Kim et al., 2020) , 10 2 −10 5 TCID 50 in mice (Bao et al., 2020b; S. J o u r n a l P r e -p r o o f designate a median infectious dose (ID 50 ) or median lethal dose (LD 50 ). Xia et al. (2020a) reported 100% mortality in 12 newborn mice challenged intranasally with 10 2 TCID 50 (J. Xia et al., 2020) . reported that macaques infected with 10 6 via an ocular (2/3) or intratracheal (1/3) route of exposure had a positive viral load in nose and throat swabs from 1 to 7 days post inoculation, supporting reports of ocular transmission reported for a healthcare worker infected with SARS-CoV-2 while working with a patient without eye protection . Previously infected rhesus macaques challenged intratracheally with SARS-CoV-2 at 10 6 TCID 50 did not display reinfection characteristics when challenged again with the same dose, indicating some immunity conferred from an initial infection (Bao et al., 2020a) . Sufficient data are not available for modelling or pooling dose groups from multiple studies at this time that meets the criteria of (i) more than 3 unique dose groups and (ii) at least three unique responses. A lack of a dose-response model for SARS-CoV-2 is a critical gap for conducting QMRA for this pathogen. Emerging areas for dose-response testing include dosing of organoids to represent aspects of specific pathogenesis processes such as liver damage ; however, these approaches have not been reconciled with existing quantitative dose-response modelling calculations as they do not fully encapsulate the ability to represent host immune processes (Haas, 2015) . J o u r n a l P r e -p r o o f environmental water for tropical, sub-tropical and temperate climatic zones as the persistence may be highly variable in different temperatures, as demonstrated in a recent study (Hart and Halden, 2020) . Moreover, the persistence of SARS-CoV-2 in wastewater and receiving waters and inactivation mechanisms, such as predation, UV, sunlight, and disinfection should be investigated.

Data on SARS-CoV-2 removal and/or inactivation by wastewater and water treatment processes, such as activated sludge, membrane filtration, coagulation-sedimentation, and disinfection (chlorine, chloramine, UV, ozone, etc.) is scarce. If it is difficult to determine log 10 reduction values of SARS-CoV-2 itself due to availability of the virus and/or biosafety restrictions, model enveloped virus such as human CoVs, MHV, or Pseudomonas phage Φ6 can be used for laboratory-or pilot-scale experiments.

Currently RT-qPCR assays developed for clinical specimen testing are being used for SARS-CoV-2 RNA detection in environmental water samples. Recent environmental studies reported that different assays might produce conflicting results Medema et al., 2020) .

Moreover, the false-negative rates (due to improperly designed primers/probe or virus mutation in the targeted genome region) of these assays need to be assessed by multiple laboratories. For environmental application, the sensitivities of these RT-qPCR methods need to be evaluated. The major limitation of qPCR is that it does not provide information on viability. When viability needs to be assessed, cell culture infectivity assay, EMA/PMA-RT-qPCR, and ICC-RT-qPCR may provide useful information. A standardized protocol to recover and detect SARS-CoV-2 from environmental water samples, including concentration method, qPCR assay, and process controls, should be established.

Wastewater surveillance is critical as WBE may provide valuable information on the prevalence of infections in the community. Continuous and systematic monitoring of wastewater may provide early warning signs and will potentially identify undiagnosed or successive disease at the population level, thus alerting public health officials on the ongoing or future viral disease outbreaks.

Nationwide and international wastewater surveillance campaigns should be carried out to better J o u r n a l P r e -p r o o f 26 understand temporal and spatial dynamics of disease prevalence, molecular epidemiology and evolution of the virus, and efficacy of public health interventions.

QMRA has a potential role to play in reducing the impact of the ongoing COVID-19 outbreak.

However, currently available QMRA parameters for SARS-CoV-2 are limited, although previous studies on relevant respiratory viruses (SARS-CoV, MERS-CoV, and influenza viruses) help to assess the likely risks of SARS-CoV-2. Our understanding on the potential role of wastewater in SARS-CoV-2 transmission is largely limited by knowledge gaps in its occurrence and survival in wastewater and environmental waters and removal by wastewater treatment processes. There is an urgent need for collecting these pieces of information to understand and mitigate the human health risks associated with exposure to wastewater and environmental waters potentially contaminated with SARS-CoV-2. Corman, V.M., Hartmann, W., Scheible, G., Sack, S., Guggemos, W., Kallies, R., Muth, D., Junglen, S., Müller, M.A., Haas, W., Guberina, H., Röhnisch, T., Schmid-Wendtner, M., Aldabbagh, S., Dittmer, U., Gold, H., Graf, P., Bonin, F., Rambaut, A., Wendtner, C.M., 2013. Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection. Lancet Infect. Dis. 13, 745-751. https://doi.org/10.1016/S1473-3099(13)70154-3 Fannin, K.F., Vana, S.C., Jakubowski, W., 1985. Effect of an activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses. Appl. Environ. Microbiol. 49, 1191 -1196 . https://doi.org/10.1128 /aem.49.5.1191 -1196 .1985 The WWTP workers had significantly higher gastroenteritis, abdominal pain, and headaches. No significant differences were reported for respiratory and other symptoms. (Khuder et al., 1998) WWTP workers in 67 plants in the Netherlands were evaluated via questionnaire for a 12-month study; no controls; personal endotoxin exposure was assessed (8 hr measurements: n = 460).

Dose-response relationships were found with endotoxin levels for: "lower respiratory and skin symptoms", "flu-like and systemic symptoms", and "upper respiratory symptoms". (Smit et al., 2005) WWTP workers in Iowa were evaluated via questionnaire for a 3-year study; controls were workers at water treatment plant (WTP) workers; endotoxins sampled as an exposure indicator.

Odds ratios were statistically higher for respiratory, ocular and skin irritation, neurology, and gastrointestinal symptoms in WWTP workers. Tasks related to sludge handling were identified as high-risk. (Lee et al., 2009) A 5-year study conducted in Switzerland; controls were gardeners, waterway maintenance, public transport, and forestry workers. 

A case study evaluating the risk of infection from Middle Eastern respiratory syndrome coronavirus (MERS-CoV) in a hospital setting through bioaerosols

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community

Evaluation of Phi6 persistence and suitability as an enveloped virus surrogate

Environmental surveillance for polioviruses in the global polio eradication initiative

Reinfection could not occur in SARS-CoV-2 infected rhesus macaques

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

COVID-19: towards controlling of tenOever, B.R., 2020. SARS-CoV-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems

Glass wool concentration optimization for the detection of enveloped and non-enveloped waterborne viruses

Risk assessment of novel coronavirus COVID-19 outbreaks outside

Evidence of Saffold virus circulation

From ferrets to mice and marmosets, labs scramble to find right animals for coronavirus studies

Human enteric viruses in the water environment: A minireview

Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site

Introduction to environmental virology

Active monitoring of persons exposed to patients with confirmed COVID-19 -United States

New methods for the concentration of viruses from urban sewage using quantitative PCR

Labs rush to study coronavirus in transgenic animals--some are in short supply

Inactivation of an enveloped surrogate virus in human sewage

Methods for primary concentration of viruses from water samples: A review and meta-analysis of recent studies

CDC, 2020a. 2019-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Panel Primers and Probes

How COVID-19 spreads

Severe Acute Respiratory Syndrome (SARS) [WWW Document

Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility

Seasonal influenza a virus in feces of hospitalized adults

SARS-CoV-2-positive sputum and feces after conversion of pharyngeal samples in patients with COVID-19

Epidemiological and clinical characteristics of 99 cases of

Specific primers and probes for detection 2019 novel coronavirus

Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia

Modeling influenza epidemics and pandemics: Insights into the future of swine flu (H1N1)

Viral shedding and antibody response in 37 patients with Middle East respiratory syndrome coronavirus infection

Detection of 2019 novel coronavirus (2019-nCoV) by realtime RT-PCR

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Clinical characteristics of coronavirus disease 2019 in China

Survival of coronaviruses in water and wastewater

Quantitative microbial risk assessment

Coronavirus and environmental engineering science

Microbial dose response modeling: Past, present, and future

Comparing microbial risks from multiple sustainable waste streams applied for agricultural use: Biosolids, manure, and diverted urine

Critical review of mathematical approaches for quantitative microbial risk assessment (QMRA) of Legionella in engineered water systems: Research gaps and a new framework

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

A review on recent progress in the detection methods and prevalence of human enteric viruses in water

Development of virus concentration methods for detection of koi herpesvirus in water

Computational analysis of SARS-CoV-2/COVID-19 surveillance by

Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples

Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants

First case of 2019 novel coronavirus in the United States

SARS-CoV-2 may persist in digestive tract longer than respiratory tract

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

A modified dose-response model that describes the relationship between haemagglutination inhibition titre and protection against influenza infection

Viral loads in clinical specimens and SARS manifestations

Concentration and recovery of viruses from water: A comprehensive review

Protocol: Real-time RT-PCR assays for the detection of SARS-CoV-2

Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Facts and myths

Viral loads and duration of viral shedding in adult patients hospitalized with influenza

Increasing the translation of mouse models of MERS coronavirus pathogenesis through kinetic hematological analysis

Clinical and virological data of the first cases of COVID-19 in Europe: a case series

Incubation periods of acute respiratory viral infections: a systematic review

Enteric involvement of severe acute respiratory syndrome -Associated coronavirus infection

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples

Finding the real case-fatality rate of H5N1 avian influenza

A new and simple method for concentration of enteric viruses from water

Multi-zone modeling of probable SARS virus transmission by airflow between flats in Block E

Assessment of public health risk associated with viral contamination in harvested urban stormwater for domestic applications

Incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: A statistical analysis of publicly available case data

Transmission dynamics and control of severe acute respiratory syndrome

The reproductive number of COVID-19 is higher compared to SARS coronavirus

Evaluation of SARS-CoV-2 RNA shedding in clinical specimens and clinical characteristics of 10 patients with COVID-19 in Macau

SARS-CoV-2 in wastewater: potential health risk, but also data source

Aichi virus in sewage and surface water, the Netherlands

2019-nCoV transmission through the ocular surface must not be ignored

Dose-response and transmission: The nexus between reservoir hosts, environment and recipient hosts

Assessment of airborne Gardens

Presence of SARS-Coronavirus-2 in sewage

Middle East respiratory syndrome

A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes

Isoelectric points of viruses

Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship

Respiratory disease and virus shedding in rhesus macaques inoculated with SARS-CoV-2

Wastewater-based epidemiology can overcome representativeness and stigma issues related to COVID-19

Comparative performance of SARS-CoV-2 detection assays using seven different primer/probe sets and one assay kit

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater

High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage

Occurrence of norovirus infections unrelated to norovirus outbreaks in an asymptomatic food handler population

How many tests for COVID-19 are being performed around the world?

Viral load of SARS-CoV-2 in clinical samples

MERS transmission and risk factors: A systematic review

Assessment of airborne enteric viruses emitted from wastewater treatment plant: Atmospheric dispersion model, quantitative microbial risk assessment, disease burden

Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR [WWW Document

Canine coronavirus inactivation with physical and chemical agents

Large scale survey of enteric viruses in river and waste water underlines the health status of the local population

Estimating the burden of disease from water, sanitation, and hygiene at a global level

Global prevalence of asymptomatic norovirus infection: A meta-analysis

SARS in Hong Kong: Impact of public health interventions. Science (80-. )

Comparative pathogenesis of COVID-19, MERS and SARS in a non-human primate model

Virus hazards from food, water and other contaminated environments

A large Finnish echovirus 30 outbreak was preceded by silent circulation of the same genotype

Assessment of an enterovirus sewage surveillance system by comparison of clinical isolates with sewage isolates from

Development of genetic diagnostic methods for novel coronavirus 2019 (nCoV-2019) in Japan

Pathogen surveillance through monitoring of sewer systems

Endotoxin exposure and symptoms in wastewater treatment workers

Detection of novel coronavirus by RT-PCR in stool specimen from asymptomatic child

Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child

Estimation of the transmission risk of the 2019-nCoV and its implication for public health interventions

Detection of nineteen enteric viruses in raw sewage in Japan

Consistent detection of 2019 novel coronavirus in saliva

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2 : an observational cohort study

Exposure to bioaerosols, respiratory health and lung-specific proteins: A prospective study in garbage and wastewater workers

Estimates of the severity of coronavirus disease 2019: a model-based analysis

Rapidly measured indicators of recreational water quality and swimming-associated illness at marine beaches: a prospective cohort study

Different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures

A novel coronavirus outbreak of global health concern

Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan

Detection of SARS-CoV-2 in different types of clinical specimens

Detection of SARS-associated coronavirus in throat wash and saliva in early diagnosis

Study on the resistance of severe acute respiratory syndrome-associated coronavirus

Texas baboon troop enlisted in humankind's war on coronavirus

Dose-response assessment for influenza A virus based on data sets of infection with its live attenuated reassortants

Development of a dose-response model for SARS coronavirus

Statement on the second meeting of the International Health Regulations (2005) Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV)

Coronavirus disease (COVID-19) pandemic [WWW Document

Water, sanitation, hygiene and waste management for COVID-19

Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV) [WWW Document

Influenza (seasonal) [WWW Document

FAQs: H5N1 influenza [WWW Document

Consensus document on the epidemiology of severe acute respiratory syndrome (SARS) [WWW Document

Environmental engineers and scientists have important roles to play in stemming outbreaks and pandemics caused by enveloped viruses

Emerging investigators series: the source and fate of pandemic viruses in the urban water cycle

Virological assessment of hospitalized cases of coronavirus disease 2019

Case fatality risk of influenza a (H1N1pdm09): A systematic review

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker

Detection and characterization of human pathogenic viruses circulating in community wastewater using multi target microarrays and polymerase chain reaction

Development of a quantitative assay for SARS coronavirus and correlation or GAPDH mRNA with SARS coronavirus in clinical specimens

SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples

Time course quantitative detection of SARS-CoV-2 in Parisian wastewaters correlates with COVID-19 confirmed cases

secretions of patients with SARS-CoV-2 infection

Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Evidence for gastrointestinal infection of SARS-CoV-2. medRxiv

High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa

Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding

Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2

An animal model of mers produced by infection of rhesus macaques with MERS coronavirus

Survivability, Partitioning, and Recovery of Enveloped Viruses in Untreated Municipal Wastewater

Enteric involvement of coronaviruses: is faecal-oral transmission of SARS-CoV-2 possible?

Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes

Isolation of 2019-nCoV from a stool specimen of a laboratory-confirmed case of the coronavirus disease 2019 (COVID-19)

Recapitulation of infection and cholangiocyte damage with human liver organoids

Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus

Immunogenicity, safety, and protective efficacy of an inactivated SARSassociated coronavirus vaccine in rhesus monkeys

Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin

A novel coronavirus from patients with pneumonia in China

SARS-CoV-2 viral load in upper respiratory specimens of infected patients

This work was partly supported by the Japan Society for the Promotion of Science ( 

J o u r n a l P r e -p r o o f 46 Zhang et al., 2020) qPCR 9/17 (53%) Day 0-11; 550 -1.21 × 10 5 copies/mL qPCR 8/10 (80%) Pediatric patients; Positive for a mean of 21 (range: 5-28) days (Y. (Adhikari et al., 2019; Sueki et al., 2016; Wang et al., 2004) Feces (copies/gfeces)Up to 10 8 copies/swab 5.1×10 1 -10 7 Up to 10 3 10 3.7 -10 6 (Chan et al., 2011; Cheng et al., 2004; Drosten et al., 2013; Hung et al., 2004; Isakbaeva et al., 2004; Wigginton and Boehm, 2020; Wölfel et al., 2020) Lee et al., 2009; Ngaosuwankul et al., 2010; Wong et al., 2005; Zou et al., 2020) Attack rate (%) Up to 80 <1-100 depending on scenario 0.42-15.8 5-30 (Burke et al., 2020; Park et al., 2018; Verity et al., 2020; WHO, 2019 WHO, , 2011 Case fatality rate ( Existing dosing experiments designed to infect all animals ranged from 10 2 TCID 50 (mice)-10 6 TCID 50 (macaques) (Bao et al., 2020b (Bao et al., , 2020a Blanco-Melo et al., 2020; Chan et al., 2020; Kim et al., 2020; Munster et al., 2020; Rockx et al., 2020 Authors developed a relationship between HI titer and protection against influenza virus