key: cord-0853909-hu512mhz authors: Shi, Danyang; Ma, Hui; Miao, Jing; Liu, Weili; Yang, Dong; Qiu, Zhigang; Shen, Zhiqiang; Yin, Jing; Yang, Zhongwei; Wang, Huaran; Li, Haibei; Chen, Zhengshan; Li, Junwen; Jin, Min title: Levels of human Rotaviruses and Noroviruses GII in urban rivers running through the city mirror their infection prevalence in populations date: 2020-09-03 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2020.142203 sha: 829f9f92b3ff5a7bc6d19e43178f26deaceb51c7 doc_id: 853909 cord_uid: hu512mhz Enteric viruses exposed to water pose a huge threat to global public health and can lead to waterborne disease outbreaks. A sudden increase in enteric viruses in some water matrices also underpins the prevalence of corresponding waterborne diseases in communities over the same time period. However, few efforts have been focused on water matrices whose viral pollution may best reflect the clinical prevalence in communities. Here, a one-year surveillance of human enteric viruses including Enteroviruses (EnVs), Rotaviruses (HRVs), Astroviruses (AstVs), Noroviruses GII (HuNoVsGII) and Mastadenoviruses (HAdVs) in four representative water matrices: an urban river (UR) running through city, effluent from Wastewater Treatment Plant (EW), raw water for Urban Water Treatment Plant (RW), and tap water (TW) were performed by qPCR. The relationship between the virus detection frequency at each site and their prevalence in clinical PCR assay was further analyzed. We found that the detection frequencies of HRVs, HuNoVsGII, and AstVs in stools peaked in winter, while EnVs peaked in autumn. No EnVs occurred in EW, RW, or TW, but HuNoVsGII and AstVs occurred intensively in winter. For UR, all types of enteric viruses could be detected and the levels of acute gastroenteritis viruses (HRVs, HuNoVsGII, AstVs, and HAdVs) were highest in autumn or winter, whereas EnVs peaked in summer. In terms of correlation analyses, only HRVs and HuNoVsGII levels in UR showed a strong positive correlation with their prevalence in clinical stool samples. This study indicated that HRVs and HuNoVsGII levels in URs may mirror the local virus prevalence, thereby implying the possibility of revealing their local epidemiology by monitoring them in the URs. Human enteric viruses transmitted by the fecal-oral route cause millions of infections each year and result in various diseases such as gastroenteritis, meningitis, hepatitis, and encephalitis (Okoh et al., 2010) . Generally, they circulate continuously between an infected population and the environmental water. After propagation in infected individuals, they are released into the surrounding environment via excrement (up to 10 11 viral particles/g stool) (Teunis et al., 2015) . Since they cannot be removed from wastewater by traditional sewage treatment processes and they have strong resistance to unfavorable conditions, they may survive in various water matrices for an extended period once discharged into wastewater (Lopman et al., 2012; Okoh et al., increase in viral load in wastewater or environmental water that receives wastewater or treated effluent may support the prevalence of viral waterborne disease in cities over the same period. Therefore, the occurrence of enteric viruses in some water matrices may be used as an indicator to mirror the disease's prevalence among the local population. Nowadays, there is increasing evidence that human enteric viruses can be independently detected in almost all types of water, such as wastewater (Bisseux et al., 2018; Farkas et al., 2018; Jahne et al., 2019; Simhon et al., 2019) and city surface water (Goh et al., 2019; Keller et al., 2019; Masachessi et al., 2018; Pang et al., 2019; Sassi et al., 2018; Sedji et al., 2018; Tandukar et al., 2018) . Wastewater-based epidemiology can be used to capture a near real-time picture of the viral disease burden within a community (Bisseux et al., 2018; McCall et al., 2020) . However, few studies (Prevost et al., 2015) have linked the dynamics of enteric virus in other water matrices to the epidemiology of viral infections over the same period. The correlation between virus occurrence in various water matrices and clinical prevalence remains unclear. In this study, surveillance of human enteric viruses was performed from September Then, the relationship between virus occurrence in water and the prevalence in clinical samples was further analyzed to determine the water matrices whose viral contamination was epidemiologically the most closely related to the virus infection. To our knowledge, this was the first study to reveal the relationship between enteric virus levels in urban representative water matrices and the local prevalence. This study implied the applicability of monitoring enteric virus in environmental water for the aim of helping reveal the local epidemiology of waterborne disease outbreaks. The biological and physicochemical parameters of various representative water samples were assayed (Table 1 ). According to the standard membrane filter procedure (Jin et al., 2014) , heterotrophic plate counts (HPC) and total coliforms (TC) were assayed on Luria-Bertani agar and M-Endo (BD Difco, USA), respectively. The physicochemical parameters turbidity and conductivity were measured with a portable turbidity meter (HACH 1900C, USA) and a conductivity meter (HACH sension5, USA), respectively. Chemical oxygen demand (COD Mn ) and ammonium content were measured according to standard methods (APHA, 1998). 2.3. Virus recovery from water samples with electropositive granule media (EGM) filter Fifty liters of water collected from each sampling site was quickly transported to the lab under refrigerated conditions for further virus concentration tests. EGM filters were prepared to concentrate the virus from water samples as described previously (Jin et al., 2014) . Briefly, after each 50 L water sample was flowed through an EGM filter, the filter was eluted with 3 L of elution buffer (2% sodium hydroxide, 0.375% glycine, 1.5% sodium chloride, 3% tryptone, and 1.5% beef powder). Then, 0.1 mol/L HCl was J o u r n a l P r e -p r o o f added to adjust the pH of the eluate to 7.0±0.2 immediately after collection and 10% polyethylene glycol (PEG) was added to the eluate before overnight incubation and centrifugation (15,000 rpm for 30 min at 4°C). Then, the pellets were resuspended in 40 mL PBS and stored at −70 °C for further analysis. To evaluate the virus recovery efficiency, 10 5 PFU of bacteriophage MS2 cultivated by confluent lysis on its host strain E. coli (ATCC 15597) was added to water samples as an indicator and detected using the double-layer plaque assay (Hornstra et al., 2011) . Virus recovery, all of which were demonstrated above 90%, was calculated using the following eq. (1): Where A is the number of MS 2 added into the water samples, B is the number of Handbook, Qiagen, Hilden, Germany). Quantification of viruses by (RT-)qPCR was performed as previously described (Miao et al., 2018) . Briefly, virus RNA was first reverse-transcribed using a cDNA first-strand synthesis system (Thermo Fisher Scientific, Waltham, MA). According to the instructions, the RNA template was first mixed with Random Hexamer primer, incubated at 65°C for five minutes, and then chilled on ice. Then, the reaction mixture was added to the samples, and the reaction was performed in a thermocycler (Applied Biosystems, USA) to synthesize cDNA. The qPCR reaction was performed in an ABI 7300 sequence detection system (Applied Biosystems, USA). Two microliters of DNA or cDNA samples were added to a 20 μL reaction mixture contending 10 μL PCR SuperMix-UDG (Platinum PCR SuperMix-UDG, Invitrogen, USA), 0.5 μL primer (10 μmol/L), 0.5 μL Taqman probe (5 μmol/L), and 6.5 μL nuclease-free water. The reaction conditions were 95°C for 30 s followed by 40 cycles of 95°C for 30 s and 60°C for 1 min. All qPCR analyses were performed in triplicate with positive controls for each target and DEPC-treated water as the negative controls. Table S1 shows all primers and probes (Jin et al., 2014; Kageyama et al., 2003; Le Cann et al., 2004; Miao et al., 2018; Xagoraraki et al., 2007) labeled with FAM detector dyes and TAMRA quencher dyes (Invitrogen, Shanghai, China). Table S2 shows the standard curves for J o u r n a l P r e -p r o o f the quantification of virus. To avoid inhibition during RT-qPCR, HCV RNA IC was added to 2 μL of nucleic acids extracted from the samples (diluted 10-fold or 50-fold or undiluted) or DEPC water (blank control) at a concentration of 10 5 genome copies (GCs) per reaction before RT-qPCR. If the threshold cycle value (Ct) of the HCV RNA IC detected in the blank control was one cycle fewer than that in the sample nucleic acid extracts, the reaction's inhibition had occurred and the nucleic acid extracts were diluted before RT-qPCR until no inhibition was observed. As background control, all samples were verified as being free of HCV using RT-qPCR prior to inhibition testing. The quantification of HCV RNA IC was carried out using the same RT-qPCR conditions as virus detection with the Primers and TaqMan probe sequences listed in Table S1 . The equation for calculating the sample inhibition is: (2) Where A is the GCs of IC per reaction in the blank control and B is the measured GCs of IC per reaction mixed with the nucleic acid extracts in the tested water samples. The standard curves for the quantification of HRVs, HuNoVsGⅡ, AstVs, EnVs, and HAdVs were obtained by analyzing 10-fold serial dilutions of viral RNA or DNA J o u r n a l P r e -p r o o f standards (Jin et al., 2014) . Virus concentrations in all water samples were calculated using the Equation (3) HRVs, HuNoVsGⅡ, AstVs, EnVs, and HAdVs using PCR as previously described (Ouyang et al., 2012) . The primers used to detect above viruses are listed in Table S3 . Kruskal-Wallis test, and students' T-test was used to analyze the differences in viral concentrations between seasons. The Pearson test was calculated using R Studio to measure the associations of enteric virus concentrations between different water matrices. This was followed by a Student-Newman-Keuls-q test to analyze the correlation between these variables. Table 2 shows the detection frequencies of enteric viruses in the samples from representative water matrices. It presents the diversity of enteric viruses in different water matrices with the exception of HAdVs, which were found in all tested water samples with a 100% detection rate. The UR, including the Haihe and Jinhe Rivers, had abundant virus types, and all five tested human enteric viruses were detected in them; all detection rates were >75%. In addition, the UR showed the highest detection frequencies-83.33% for both HuNoVsGⅡ and AstVs and 91.67% for EnVs-among all observed water matrices. However, for EW, RW, and TW, four types of acute gastroenteritis viruses (HAdVs, HRVs, HuNoVs GⅡ, and AstVs) could be detected positively but no EnVs were detected throughout the whole year. Above all, HRVs were the only viruses to show a higher detection rate there than in the Haihe and Jinhe J o u r n a l P r e -p r o o f Rivers. The detection rate of HRVs in EW reached 100%, followed by RW and TW, both of which were 91.76%. In addition, the detection rates of HuNoVsGⅡ and AstVs in RW and TW were <35%, significantly lower than those in EW and UR (P < 0.05). Based on further analysis of the detection frequency's seasonal distribution (Table S4) , EnVs in UR presented the lowest frequency in winter but always occurred in the water samples from other seasons. HRVs, HuNoVs GⅡ, and AstVs could be found in all samples from UR and EW collected in spring and winter, but AstVs only appeared in the RW and TW samples collected in winter. No inhibition to qPCR was found in the concentrates of EGM filter from all the observed water matrices. Figure 2 shows the average concentration of human enteric virus throughout the year in each representative water sample. For samples collected from the Haihe and Jinhe Rivers, EnVs showed the highest average concentration, reaching 1.39×10 6 GC/L and 4.34×10 5 GC/L, respectively, followed by AstVs with 4.11×10 5 GC/L and 4.20×10 5 GC/L. However, in other water matrices, including EW, RW, and TW, AstVs were always the richest, reaching 1.25-6.62×10 5 GC/L. Among all the water matrices, the maximal concentrations of HuNoVs GⅡ and AstVs, in the range 3.56-6.62×10 5 GC/L, occurred in the EW, while HAdVs, HRVs, and EnVs had the highest detection levels in UR. RW and TW presented lesser virus abundance and all target viruses remained at a lower level than that of the other water matrices (P < 0.05). Furthermore, in comparison with RW, there was significant declination in the concentration of all viruses in TW after chlorination (P < 0.05). Enteric viral infections were found in 775 of the 1,906 cases (40.66%) with HRVs showing the highest detection rate (19.88%), followed by HuNoVsGⅡ (11.91%). HAdVs was only positive in 2.36% of stool samples. Therefore, HRVs and HuNoVsGⅡ were the main epidemic EnVs in Tianjin in September 2014-August 2015 (Table S5) . Currently, wastewater-based epidemiology is considered a powerful tool to understand the actual incidence of human viruses in a community, such as enteric viruses and Aichi virus (Cuevas-Ferrando et al., 2019; Lodder et al., 2013; McCall et al., 2020; Rimoldi et al., 2020) . However, due to its poor biosafety and inconvenient collection of sewage samples that entered WWTP through the fully closed pipeline, it was not a wise approach to monitor the virus occurrence in the raw sewage. Previous J o u r n a l P r e -p r o o f studies detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in secondary-treated wastewater when the cases peaked in the community (Haramoto et al., 2020; Randazzo et al., 2020) . However, in this study, we did not find the correlation Previous studies limitedly revealed the existence of a close relationship between inhabitants' health status and the viral contamination of WWTP effluents. However, our study is the first to demonstrate that viral abundance in WWTP effluents has no J o u r n a l P r e -p r o o f correlation with local prevalence, but the concentration of HRVs and HuNoVsGⅡ in UR running through a city significant positively correlates with their occurrence in clinical prevalence. Considering HRVs and HuNoVs are leading etiologic agents of gastroenteritis outbreaks worldwide, our findings gave an indication that the analysis of HRVs and HuNoVs GⅡ in UR could be used as an indicator to monitor their prevalence in local populations, which would help reveal the viral epidemic in a timely manner and take measures to control disease outbreaks. 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Weili Liu: Methodology, Investigation Methodology, Investigation, Zhigang Qiu: Methodology, Investigation, Supervision Zhiqiang Shen: Methodology, Investigation Zhongwei Yang: Methodology, Investigation Methodology, Investigation Methodology, Investigation Zhengshan Chen: Investigation, Project administration, Visualization Conceptualization, Methodology, Validation, Investigation, Supervision, Funding acquisition This study was supported by grants from National Key R&D Program of China (2018YFC1603500) and Tianjin Science and Technology Support Program (16YFZCSF00340).