key: cord-0816865-2ee0p8sf
authors: McCall, Camille; Wu, Huiyun; Miyani, Brijen; Xagoraraki, Irene
title: Identification of multiple potential viral diseases in a large urban center using wastewater surveillance
date: 2020-07-07
journal: Water Res
DOI: 10.1016/j.watres.2020.116160
sha: a8e32c20291a96978f020a83d7bdd194c79da7ed
doc_id: 816865
cord_uid: 2ee0p8sf

Viruses are linked to a multitude of human illnesses and can disseminate widely in urbanized environments causing global adverse impacts on communities and healthcare infrastructures. Wastewater-based epidemiology was employed using metagenomics and quantitative polymerase chain reaction (qPCR) assays to identify enteric and non-enteric viruses collected from a large urban area for potential public health monitoring and outbreak analysis. Untreated wastewater samples were collected from November 2017 to February 2018 (n = 54) to evaluate the diversity of human viral pathogens in collected samples. Viruses were classified into virus types based on primary transmission routes and reviewed against viral associated diseases reported in the catchment area. Metagenomics detected the presence of viral pathogens that cause clinically significant diseases reported within the study area during the sampling year. Detected viruses belong to the Adenoviridae, Astroviridae, Caliciviridae, Coronaviridae, Flaviviridae, Hepeviridae, Herpesviridae, Matonaviridae, Papillomaviridae, Parvoviridae, Picornaviridae, Poxviridae, Retroviridae, and Togaviridae families. Furthermore, concentrations of adenovirus, norovirus GII, sapovirus, hepatitis A virus, human herpesvirus 6, and human herpesvirus 8 were measured in wastewater samples and compared to metagenomic findings to confirm detected viral genus. Hepatitis A virus obtained the greatest average viral load (1.86 × 10(7) genome copies/L) in wastewater samples compared to other viruses quantified using qPCR with a 100% detection rate in metagenomic samples. Average concentration of sapovirus (1.36 × 10(6) genome copies/L) was significantly greater than norovirus GII (2.94 × 10(4) genome copies/L) indicating a higher burden within the study area. Findings obtained from this study aid in evaluating the utility of wastewater-based epidemiology for identification and routine monitoring of various viruses in large communities. This methodology has the potential to improve public health responses to large scale outbreaks and viral pandemics.

To explore human virus diversity between sampling locations and dates, purified nucleic acid 144 from each biological replicate was pooled together for a total of 18 samples. These samples 145 represent genetic material from all three interceptors during each of the six sampling dates. 146 Nucleic acid from each sample was reverse transcribed and subjected to random amplification as 147 previously described (Wang et al., 2003) to evaluate both RNA and DNA viruses. HAV and SaV was quantified using a two-step RT-qPCR based on a previously described 226 methods (Jothikumar et al., 2005; Oka et al., 2006) . Briefly, viral RNA was reverse transcribed 227 using iScript RT-qPCR Supermix (Bio-Rad) according to the manufacturer's protocol. For HAV, 5 µL of cDNA, negative control, or positive control was transferred to a 15 μl reaction mix 229 containing HAV primers and TaqMan probe. Reactions were performed with the following 230 conditions: 95ºC for 15 min, followed by 45 cycles of 95ºC for 15 s, 55ºC for 20 s, and 72ºC for 231 15 s. SaV quantification was carried out in a 25 uL reaction containing each primer and probe.

Reactions were performed with the following conditions: 95ºC for 15 min, followed by 45 cycles 233 of 94ºC for 15 s, 62ºC for 1 min, and 72ºC for 15 s.

Norovirus GII was quantified using a one-step RT-qPCR as previously described (Le Guyader et 236 al., 2009) . In short, the RT-qPCR was carried out in a 25 μL reaction mixture containing primers 237 and probe, 2 μL of iScript RT-qPCR Supermix, and 5 μL of viral RNA, negative control, or 238 positive control. Reactions were performed with the following conditions: reverse transcription 239 at 25ºC for 5 min, 46ºC for 20 min, and 95ºC for 1 min, followed by 45 cycles of 95ºC for 15 s, 240 60ºC for 1 min, 65ºC for 1 min. 241 242 DNA viruses HAdV, HHV-6, and HHV-8 were quantified according to previously established 243 methods (Gautheret-Dejean et al., 2002; Lallemand et al., 2000; Xagoraraki et al., 2007) . HAdV 244 and HHVs were qualified in 20 uL reactions containing 5 uL of DNA or standard control. influenza-like illness (ILI) to represent any disease displaying symptoms of this nature. The 259 etiological agent of the disease is unspecified, but could be of viral, bacterial, or parasitic origin. Bray-Curtis dissimilarity analysis was used to determine the similarity between samples at the 295 family taxonomic level after alignment against human viral protein sequences. According to the 296 Bray-Curtis analysis, there were more similarities within sampling dates rather than sampling 297 locations with samples collected during three consecutive sampling dates (14-Dec.,19-Jan., and The most frequently detected virus type was enteric and respiratory, followed by other, 307 bloodborne, and vector-borne ( Figure 4b , Table 2 ). Four of ten enteric viruses detected belong to 308 the Picornaviridae family, namely, hepatovirus, enterovirus, parechovirus, and cardiovirus. (Table 2) . 100%, 50%, 22%, and 0% in metagenomic samples, respectively. SaV and HHV-6 were 350 quantified in 94% and 39% of the 18 samples considered with a 28% and 83% detection in 351 sequenced samples. All select viruses except HHV-6 were detected during each sampling date 352 using qPCR or RT-qPCR ( Figure 6 ). There were significant differences in average 353 concentrations for some viruses where HAV > HAdV > NoV (p < 0.0001). Mean SaV 354 concentrations were significantly less than HAV (p < 0.0001) and greater than NoV (p < 355 0.0001). There was no significant difference between SaV and HAdV (p > 0.05). Concentrations 356 of HHV-6 and HHV-8 were significantly lower than HAV, HAdV, SaV, and NoV (p <0.0001).

There was no significant difference between mean concentrations of HHV-6 and HHV-8 (p > 358 0.05) in collected samples. 

A temporal investigation of NoV GII and SaV concentrations in wastewater samples was carried 362 out to assess the potential burden of these viruses and their contribution to gastrointestinal illnesses within the service community. Average concentrations of SaV and NoV GII were 364 1.36x10 6 and 2.94x10 4 genome copies/L, respectively. The highest average concentration for 365 both viruses occurred during the 14-Dec sampling date (Figure 7) . However, according to 366 spearman's correlation analysis, there was no significant association between the number of 367 reported GI cases and concentrations of NoV GII and SaV per sampling week (Table 3) . including SARS-CoV in 2003 (Kuiken et al., 2003 , MERS-CoV in 2012 (Chan et al., 2015 , and illnesses have also been reported in patients with BCoV infections (Lai et al., 2020) . Albeit viral Tamura, 2014) . No mandatory reporting was required for primary infections associated with the 501 above-mentioned viruses during the study period. respectively (Armstrong and Andreadis, 2013; Weaver et al., 2004) . Although EEEV infections 517 in humans are rare compared to other clinically relevant vector-borne infections, it is the 518 deadliest with a fatality rate of 35-75% (Armstrong and Andreadis, 2013) . This contrasts with 519 VEEV infections where fatalities rates of less than 1% have been reported (Weaver et al., 2004) . (Glass et al., 2009; Oka et al., 2015) . year.

• Findings reveal evidence of re-emerging vector-borne viruses.

• Frequent and rigorous wastewater sampling along with integrative sample processing 593 strategies can be employed to identify the etiological agent of non-specific diseases and 594 viruses that poses a significant burden among inhabitants.

• Results presented in this study suggests that WBE has the potential to advance the area of 

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• Optimized sequence alignment and qPCR identified the presence of important viral pathogens• Detected viral pathogens in wastewater associated with reported clinically significant diseases• Wastewater-based epidemiology can be used for identification of viral outbreaks

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: