Prevalence of Non-polio Enteroviruses in the Sewage of Guangzhou City, China, Running title: Prevalence of NPEVs in Sewage in Guangzhou, China

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1 AEM Accepts, published online ahead of print on 4 October 2013 Appl. Environ. Microbiol. doi: /aem Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Prevalence of Non-polio Enteroviruses in the Sewage of Guangzhou City, China, from 2009 to Running title: Prevalence of NPEVs in Sewage in Guangzhou, China Huanying Zheng 1*, Jing Lu 1,5*, Yong Zhang 2*, Hiromu Yoshida 3, Xue Guo 1, Leng Liu 1, Hui Li 1, Hanri Zeng 1, Ling Fang 1, Yanling Mo 1, Lina Yi 1,5, Toru Chosa 4, Wenbo Xu 2, Changwen Ke 1 1 Guangdong Provincial Center for Disease Control and Prevention. No. 160, Qunxian Road, Panyu District, Guangzhou, China. 2 WHO WPRO Regional Polio Reference Laboratory and Ministry of Health Key Laboratory for Medical Virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. No. 155, Changbai Road, Changping District, Beijing, China. 3 Department of Virology II, National Institute of Infectious Diseases, Tokyo , Japan 4 Bureau of International Cooperation, International Medical Center of Japan, Toyama, Shinjuku-ku, Tokyo 162, Japan 5 Guangdong Provincial Institution of Public Health, Guangzhou, China *:These authors contributed equally to this work. 1

2 Corresponding author: Zheng Huanying, Institute of Pathogenic Microbiology, Guangdong Provincial Center for Disease Control and Prevention, No.160, Qunxian Road, Dashi town, Panyu District, Guangzhou City, Guangdong Province, China. Phone: (86) Fax: (86) Downloaded from on April 12, 2019 by guest 2

3 Abstract: Background: The human pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted from patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. Methods: In this study, a 4-year ( ) surveillance study was conducted to detect non-polio enteroviruses (NPEVs) in the urban sewage of Guangzhou City, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. Results: During the 4-year study, 17 different NPEV serotypes were identified in sewage of Guangzhou City. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, ECHO12, Coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010, but was rarely isolated in 2011 and In contrast, CVB3 was not observed in 2009 and 2010, but was increasingly detected in 2011 and Conclusion: Our study, for the first time, provides an overview of the serotype distribution and circulation patterns of NPEVs in sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help 3

4 54 interpret the EV trends and warn of future outbreaks in this area. 55 4

5 Introduction Human enteroviruses (EVs) are members of the genus Enterovirus within the order Picornavirales, family Picornaviridae, and consist of 4 species: EV-A, EV-B, EV-C, and EV-D (11). Based on their pathogenicity in humans, EVs were initially classified into 4 subgroups: polioviruses (PV, types 1 3), Coxsackie group A viruses (CVA, types 1 22, and 24), Coxsackie group B viruses (CVB, types 1 6), and echoviruses viruses (ECHO, types 1 7, 9, 11 27, and 29 34) (15). EVs are associated with diverse clinical syndromes, ranging from mild fever, headache, herpangina, and hand-foot-and-mouth disease, to severe and potentially fatal illnesses such as aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis (3, 5, 10, 25). In recent years, outbreaks of different EV infections and related diseases have been frequently reported in China (12, 17, 32, 34); however, only limited data are available on the circulation patterns of EVs in the environment (22, 23). Although different serotypes of EVs can co-circulate, the predominant serotype is determined by spatial and temporal factors. For instance, in Beijing, China, CVA21 and EV-D68 were the predominant serotypes in patients with acute EV respiratory infections from 2006 to 2010 (29). However, in France and Spain, ECHO11 and ECHO6 are the most frequently detected agents in human EV positive adults with acute respiratory tract infections (7, 26). The high incidence of aseptic meningitis that occurred in Alberta, Canada in 2010 was caused by CVA9, and an aseptic meningitis outbreak in Korea in 2008 was mainly caused by ECHO6 and ECHO30 infections (9, 5

6 ). For this reason, determining the temporal and geographic patterns of EV circulation, especially the dynamics of EV serotype shifts are critical The presence of human pathogenic viruses in urban sewage has served as an indicator of their existence in a given population (2, 18, 19, 27). In urban populations with absent or questionable surveillance, monitoring the viruses in circulating sewage can provide valuable supplementary information, especially when persistent virus circulation or frequent re-introduction is suspected. In this study, we provide an overview of the non-polio enteroviruses (NPEV) circulating in urban sewage of Guangzhou China from 2009 to During this 4-year period, 17 co-circulated NPEVs were isolated from sewage samples and the circulation patterns of predominant NPEVs (ECHO6, ECHO7, ECHO11, ECHO12, and CVB5) were described. To the best of our knowledge, this is the longest most systematic study of EV prevalence in sewage in China, and these results will help us to better understand the changes in enteric disease trends and the potential risk of an enteric disease epidemic. 6

7 Materials and Methods Sewage sample collection Raw sewage samples were collected monthly from January 2009 to December 2012 from the primary sedimentation tanks at the Liede wastewater treatment plant (WWTP) in Guangzhou City, China. This WWTP is located in the northern part of Guangzhou City and serves a population of about 2,150,000. Four samples (1 L each) were obtained from the inlets of the primary sedimentation tanks on a routine basis each month. The samples were immediately transported to the laboratory and sample treatment was started within 2 hours after the samples arrived at the laboratory. Viral concentration Viruses in the sewage samples were concentrated using a previously described improved negative charge filter membrane absorption and sonication method (6). Briefly, the collected sewage samples were centrifuged at 3,000 rpm for 30 min. Then, MgCl 2 was added to the supernatant at the final concentration of 0.05 M, and the ph was adjusted to with HCl. The samples were then slowly passed through a negatively charged membrane filter (mixed cellulose ester membrane filter; Advantec Co. Ltd., Tokyo, Japan) under gentle positive pressure. To elute the viruses, the filter with adsorbed viruses was cut into pieces and sonicated for 1 min in 10 ml of a 3% beef extract solution (PH 9.6) followed centrifugation at 1,940 g for 30 min twice to yield 2 eluents (first and second eluents). Finally, the eluents were passed through a 0.22-μm syringe filter to remove bacteria and fungi. 7

8 Viral isolation Human rhabdomyosarcoma (RD) and human laryngeal epidermoid carcinoma (HEp-2) cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and were used for viral isolation. For each cell line and concentrated eluent, six parallel cell tubes were set up with four for the first eluents and two for the second eluents. Total 200 µl of each concentrated eluent was used for inoculating the standard monolayer of cells. Each tube was examined microscopically for the appearance of cytopathic effect (CPE) daily for up to 7 days. The tube in which cells developed CPE was recorded and culture supernatant was collected separately as a positive isolate and performed molecular typing independently. RNA extraction and molecular typing To type the NPEV isolates, nucleic acid was extracted from the collected cultures with a QIAamp Viral RNA mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer s instructions. Viral RNA was eluted by adding 60 μl of sterile nuclease-free water and stored at 80 C until use. RT-PCR was performed according to the method developed by Nix et al. (13). Primers sequences used in this study were listed in Supplement Table 1. Briefly, cdna was synthesized by using QIAGEN OneStep RT-PCR Kit (final volume, 20 μl) with 1 μm each cdna primer (primers AN32, AN33, AN34, and AN35). Following incubation at 37 C for 60 min, 2.5 μl cdna was then used in the first PCR (PCR1 8

9 final volume, 25 μl) by using Taq PCR Master Mix Kit (QIAGEN) with 0.5 μm each of primers 224 and 222 targeting highly conserved motif in VP3 and VP1 region respectively. After 40 cycles of amplification (95 C for 30 s, 42 C for 30 s, 72 C for 45 s), 2.5 μl PCR1 products was used as a template in the second round PCR with 0.5 μm each of primers AN88 and AN89 targeting partial VP1 region. After 40 cycles of amplification (95 C for 30 s, 60 C for 30 s, 72 C for 45 s). The PCR products were analyzed on 1.2% agarose gels, and the positive products (~350 to 400 nt) were purified using the QIAquick PCR purification kit (QIAGEN) and sent for sequencing by using primer AN88 or AN89. The sequences were analyzed with the Basic Local Alignment Search Tool (BLAST) server at the National Center for Biotechnology Information (NCBI) and the serotype of each isolate was determined according to a previously described molecular typing method (14). In general, a pending EV was classified as the same serotype as the prototype strain if it had >75% nucleotide identity and >85% amino acid sequence identity in the VP1 coding region; the pending EVs were classified into different serotypes if they had <70% nucleotide identity and <85% amino acid sequence identity. 9

10 Results In total, 947 positive isolates were collected including 916 NPEV and 31 non-typable viruses. 17 NPEV serotypes were identified based on the molecular typing of a 340-bp fragment sequence in the VP1 region, and the number of EV serotypes ranged from 10 to 16 during the period from 2009 to The distribution of serotypes identified EVs is presented in table 1. Overall, the 6 most commonly identified EVs were ECHO11 (26.4% of all isolates), ECHO6 (24.1%), ECHO7 (13.4%), ECHO12 (13.3%), CVB5 (9.5%) and CVB3 (3.1%). To investigate the circulating NPEVs according to season, the number of NPEV isolates detected each month was determined. As shown in figure 1, during , the number of NPEVs typically increased from February and generally peaked in July, except in 2010 when a large number (35 isolates) of NPEVs was isolated in June and only a few (9 isolates) were detected in July. However, some variations in the seasonal prevalence of NPEVs were observed. In 2009, there was another peak in April and 36 (16.7%) NPEVs were isolated. In 2010 and 2011, numerous NPEVs were isolated in October (32 isolates) and November (26 isolates), and a similar pattern was not observed in Interestingly, our results differ from those of other studies (8, 20), in which the number of NPEVs in Guangzhou sewage samples did not remain high during the summer and fall, and a sharp decrease in the number of NPEVs was observed in June, July, and August of 2009, 2010, and 2011, respectively. The circulation pattern of each NPEV serotype also differed. As shown 10

11 in figure 2, a large number of ECHO6 and ECHO12 viruses were detected in June, whereas most ECHO7 and ECHO11 viruses were isolated around October The distribution pattern of NPEV serotypes also varied over the years, even though the total numbers of NPEVs isolated from sewage each year were similar. Among these NPEV serotypes, 10 were detected every year, and another 2 serotypes (ECHO1 and CVB4) were detected in only a single year. The predominant serotypes also differed each year. As shown in table 1 and figure 2, ECHO6 and ECHO11 were the most prevalent serotypes during the study period, whereas ECHO7 and CVB5 were prevalent only in 2009 and 2010, respectively. ECHO12 was increasingly detected starting in 2011 and became most common serotype in (Fig. 2d). While CVB5, the virus frequently isolated in 2009 and 2010 (15.7% and 14.8% of total isolates, respectively), it was rarely detected in 2011 and 2012 (Fig. 2e). Although only a single CVB3 isolate was detected in 2009 and 2010, an increasing number was detected in the subsequent years, and 21 isolates (10% of total isolates in 2012) were identified in 2012 (Fig. 2f). In this study, different cell lines also exhibited different sensitivity to NPEVs. Our results indicated that RD cells were more sensitive to echoviruses with 83.1% of echoviruses were isolated from RD cells while only 16.9% were isolated from HEp-2 cells. Conversely, HEp-2 cells seemed to be better for CVB detection since 59.8% of CVBs were isolated from HEp-2 cells, whereas 40.2% were isolated from RD cells

12 Discussion Circulating EVs in sewage is a proven indicator of their presence in a given community. Therefore, sewage surveillance is regarded as a complementary approach to determine the prevalence and duration of epidemic EVs in a human population (6, 20-22). For instance, in a serotype-based surveillance study performed by Sedmak et al., clinical isolates were compared with sewage isolates in Wisconsin from August 1994 to December 2002 (20). This study showed that the most commonly detected EV serotypes in sewage were similar to the most commonly detected EV serotypes in clinical samples. Also, the annual peaks in sewage EV titers were accompanied by peaks in clinical cases, which occurred in late summer or early fall. Moreover, the high sequence similarity between EVs from sewage and clinical samples provide substantial evidences at molecular level. In a study conducted by Iwai et al. from 2002 to 2003 demonstrated that the nucleotide sequences of ECHO13 isolated from sewage water were closely related to those isolated from patients with aseptic meningitis in Toyama Prefecture, Japan (6). In this study, we reported an overview of NPEV prevalence in the sewage of Guangzhou City, the capital city of Guangdong Province China. During the 4-year surveillance study, the 10 common circulating NPEV serotypes were identified in Guangzhou, which were (in descending order) ECHO11, ECHO6, ECHO7, ECHO12, CVB5, CVB3, ECHO3, ECHO13, ECHO24, and ECHO19. In contrast, ECHO1, ECHO20, ECHO29, ECHO30, CVB1, CVB2, and CVB4 were only occasionally 12

13 223 detected in sewage samples Currently, none of surveillance system is set up for monitoring NPEV clinical infection in China. Because most people infected with enterovirus does not appear clinical symptoms or only mild symptoms, clinical data on NPEV infection is very limited and mainly from testing of patients with meningitis or meningoencephalitis and Hand, foot and mouth disease (HFMD). ECHO6 as a predominant NPEV in sewage of Guangzhou was also isolated in other region of China including Yunnan, Shandong and Henan province of China (22, 24, 28, 35). Correspondingly, the outbreaks of ECHO6 associated aseptic meningitis and HFMD was reported in Anhui and Shandong Province in 2005 and 2011 (12, 33). CVB5 which was commonly detected in 2009 and 2010 in sewage of Guangzhou was reported as the etiologic agent for aseptic meningitis outbreak in Shandong Province, China in 2009 (4). More direct evidence was provided by ECHO30. In this surveillance, ECHO30 was not detected until Although the number of positive isolates was few, the continuous identification of ECHO30 from 2010 to 2012 suggested its circulation in environments of Guangzhou. Consistently, an outbreak of aseptic meningitis occurred in Luoding city which adjoins Guangzhou city in Our most recent study revealed that the newly emerged ECHO30 was the most common isolated EV serotype in cerebrospinal fluid samples of the patients (30, 31). These coincidences of NEPV in sewage and clinical samples provide the added value of environmental surveillance for enteroviruses. 13

14 The results of this study also suggest that the circulation patterns of individual EVs change along with temporal and spatial factors. Due to the different features of each EV, the seasonal pattern of the EVs differs over time and the circulation patterns for the different serotypes might vary. In Guangzhou City, ECHO11 and ECHO6 were the most common serotypes in sewage during our 4-year surveillance study. Meanwhile, the months during which the largest number of ECHO6 and ECHO11 viruses was detected also differed. Comparing our surveillance data with those of similar reports, we found that the NPEVs detected in this study were also identified in sewage surveillance studies from other areas such as Shandong Province China, Iran, France, and the United States, whereas the predominant NPEV serotypes in these different geographical regions varied (1, 8, 20, 22). These findings demonstrate the value of environmental surveillance of EVs in this area. In the present study, the detection results using 2 cell lines with different sensitivity to the different EV serotypes are more convincing (20). Therefore, it is reasonable to assume that our sewage testing is a reflection of local EV activity. However, it should be also noted that some EVs, especially EV-As such as EV-A71, might have been missed in our study because their growth in cells lower than that of EV-Bs. Molecular typing of EVs is better for identifying strains that might have been classified as untypeable by the conventional neutralization method. Moreover, the obtained sequences allowed us to further analyze the evolution of the circulating NPEVs 14

15 267 through phylogenetic assays In the absence of a systematic EV disease surveillance system in China, our study on the prevalence of sewage-borne NPEVs has at least two advantages. First, our study provides relevant area-specific epidemiological data on potential waterborne pathogenic viruses that will help public health practitioners determine the long-term circulation patterns of individual EVs and health-based targets (i.e., water or food quality targets for pathogens). Second, since NPEVs are occasionally related to serious diseases such as myocarditis and aseptic meningitis, our description of the predominant NPEVs in sewage provides an epidemic background of the circulating EVs that can be used to interpret the trends in EV prevalence and provide a warning of possible enteroviral disease outbreaks. Acknowledgement This project was funded by Sasagawa Medical Awards in Aid for the Japan-China Cooperation Project, a grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan, and Bill & Melinda Gates Foundation (Project No. OPP ). Competing interests The authors declare that they have no competing interests

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19 Table 1. Non-poliovirus EV (NPEV) serotypes detected in the sewage of Guangzhou City, China each year from 2009 to 2012 Serotype Total (%) ECHO (0.1%) ECHO (2.5%) ECHO (24.1%) ECHO (13.4%) ECHO (26.4%) ECHO (13.3%) ECHO (1.7%) ECHO (1.3%) ECHO (0.4%) ECHO (1.5%) ECHO (0.2%) ECHO (0.4%) CVB (1.1%) CVB (0.4%) CVB (3.1%) CVB (0.3%) CVB (9.5%) Total

20 Most ECHO11 (27.6%) ECHO6 (34.8%) ECHO11 (29.4%) ECHO11 (22.7%) common ECHO7 (18.4%) ECHO11 (24.5%) ECHO12 (22.9%) ECHO6 (17.1%) serotypes ECHO6 (17.5%) CVB5 (14.8%) ECHO6 (20.9%) ECHO12 (15.0%) Downloaded from on April 12, 2019 by guest 20

21 Figure 1. Number of NPEV isolates detected each month via virus isolation from 2009 to 2012 Figure 2. Seasonal patterns of 6 predominant circulating NPEVs in Guangzhou from 2009 to (a). ECHO6; (b). ECHO7; (c). ECHO11; (d). ECHO12; (e). CVB5; (f) CVB3. Downloaded from on April 12, 2019 by guest 21

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