key: cord-0899366-qhalvit2 authors: Hemalatha, M.; Tharak, A.; Kopperi, H.; Kiran, U.; Gokulan, C. G.; Mishra, R. K.; Venkata Mohan, S. title: Comprehensive and Temporal Surveillance of SARS-CoV-2 in Urban Water Bodies: Early Signal of Second Wave Onset date: 2021-05-12 journal: nan DOI: 10.1101/2021.05.08.21256881 sha: 7e1eb1f243f33cb53f56185e1d3f79925e1a7b21 doc_id: 899366 cord_uid: qhalvit2 The possible faecal-oral transmission of SARS-CoV-2 through domestic discharges has emerged as a serious public health concern. Based on persistence of the virus in environment, the wastewater-based epidemiology (WBE) enabled the surveillance of infection in a community. The water bodies connected to the anthropogenic activities have strong possibility of presence of the SARS-CoV-2 genetic material. In this work, we monitored urban, peri-urban and rural lakes in and around Hyderabad as a long-term surveillance study for presence of enteric virus SARS-CoV-2 gene fragments. The study time of seven months coincided with the first and second wave of COVID-19 infection. The study depicted differential viral RNA copies in the urban lake with high viral load observed during the peaks of wave I and wave II. Distinct variability in viral genes detection was observed amongst all five lakes which were in concordance with the human activity of the catchment area. The SARS-CoV-2 genes were not detected in peri-urban and rural lakes, whereas the urban lakes having direct functional attributes from domestic activity, in the community showed presence of viral load. The outcome of the study clearly shows that the urban water streams linked with domestic discharge will function as a proxy for wastewater epidemiological studies. The surge in viral gene load from February 2021 sample suggests the on shoot of the second wave of infection, which correlated well with the prevailing pandemic situation. Implementation of regular WBE based monitoring system for the water bodies/wastewater in the urban and semi-urban areas will help to understand the outbreak and spread of virus in the community. The (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 4 community, however, can be used as surveillance tool to understand the infection onset and spread. In this work we monitored an urban lake (UL-1) for seven months for the detection SARS-CoV-2 virus material along with less polluted urban lakes (UL-2 and UL-3), periurban (PL) and rural lake (RL). Water samples from selected lentic water bodies viz., urban, peri-urban and rural areas in and around Hyderabad, were collected by employing grab sampling protocol between 8:00 to 10 under per-urban and rural functions respectively. Long term surveillance (weekly and monthly) was performed for UL-1 with two samples (lake and lake outlet) for four weeks (Week-1 (7/10/2020); Week-4 (28/10/2020); Week-5 (04/11/2020)); Week-6 (18/11/2020)) and monthly monitoring for 7 months (October 2020 to April 2021) were collected. Week 2 (14/10/2020) and Week 3 (21/10/2020) samples were not collected due to the occurrence of multiple rainfall events. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. After collection, samples were packed in a disposable pack to avoid leakage during the transportation. Samples were brought to lab within 3 h of sample collection and processed within 24 h. Samples were initially filtered using 1 mm filter paper to remove larger debris followed by 0.2 μ m filtration to separate suspended solids. A 60 mL of filtrate was further subjected to ultrafiltration (4000 rpm; 4 °C; 10 min) using 15 mL Amicon (30 kDa Amicon® Ultra-15 (Merck Millipore)) to concentrate to 600 μ L. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 6 A 150 μ L of concentrate was used for RNA extraction using a viral RNA isolation kit (QIAamp, Qiagen) as per manufactures protocol. Sterile material devoid of DNA/RNA crosscontamination and RNase-free water was used for RNA isolation. From the C T value obtained RNA copies/L of water using the linear fit equation of E-gene (Eq. 1) and relative standard deviation (RSD) (Eq. 2) for the individual gene were calculated All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 7 Peddacheruvu-Nacharam Lake (UL-1) selected for long term viral RNA surveillance is a manmade lake spreading over 90 acres surround by a population of ~10,00,000. The catchment area of water body is surrounded by densely populated anthropogenic activities and receives majorly three-point sources viz., treated discharge of 10 MLD capacity sewage treatment plant (STP) and the other two domestic discharges with relatively low flow. The lake water samples analysed showed COD of 152 mg/L with TDS of 800 mg/L and TSS of 110 mg/L respectively. The unfiltered sample of lake showed a yellowish tinge while the lake appears dark grey in colour with eutrophication behaviour. A total of 8 samples over a period of six weeks were collected (07/10/2020 (week 1); 28/10/2020 (week 4); 04/11/2020 (week 5); 18/11/2020 (week 6)) accounting 4 samples each from Peddacheruvu lake (UL1) and its outlet point. Week 2 (14/10/2020) and Week 3 21/10/2020) were not collected due to the occurrence of multiple rainfall events. All the lake (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 8 and 25.56±3.40% respectively. The continuous decrement in C T of all the targeted genes represents the increase in viral load in the community surrounding the UL-I with time. Viral RNA copies were calculated based on the C T of E-gene using linear fit equation All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 9 Interestingly 75% of Lake Outlet samples showed positive signals for the viral genetic material presence. E-genes was detected in all the outlet samples, while N-gene was detected in Week 1, 4, and 5 but ORF1 gene was detected in week 1 and 2 samples only. The trend of C T values showed relatively lower viral load in outlets samples when compared to lake samples. Comparatively lower RNA copies (1,823/L) were observed in Week 1 while Week 5 depicted higher RNA copies (99,914/L) (Fig. 2) . 03±2.09%) ). For ORF1ab, the decrease in C T was observed from October 2020 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint RNA copies of lake outlet samples were also followed the same trend as highest copies were All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 1 1 observed in November 2020 (99.914 RNA copies/L) followed by December 2020 (6, 090 RNA copies/L) (Fig. 3) . Whereas ORF1ab C T values sowed marginal decrement from December 2020 to January All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 1 2 2021. RNA copies were not calculated as the sample showed no detection of E-gene for all the samples. The lake outlet samples did not detected for the three targeted genes. Relatively young lake, non-signs of eutrophication and self-regeneration capacity might be the probable reasons for not detection all the genes in the lake water samples even though untreated domestic sewage discharge was there. Also, the composition of domestic discharges specifically with surfactants will also influence the viral genetic material presence in lake water body. Hussain Sagar Lake (UL-3), a heart of Hyderabad is an important man-made lake with a Both the samples showed the amplification of two (E-gene and N-gene). The detected samples showed higher E-gene with C T of 30.82±0.06% and 33.44±0.12% for July 2020 and April 2021 respectively. Whereas the N-gene C T of 31.18±0.11% (July 2020) and 31.96±0.16% (April 2021) was observed. RNA copies of 4,160/L and 664 RNA copies/L were observed in July 2020 and April 2021 respectively. This might be possible as the lake receives majority of treated water as discharge and also due to dilution effect apart from the ecosystem nature. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 1 3 The Peri-urban lake (Edulabad Lake, PL) is located near to Ghatkesar covers about 1236 acres of area. The lake catchment area is covered by villages and agricultural fields. It gets both non- A comprehensive long-term monitoring of different types of lentic water bodies located in urban, semi-urban and rural locations showed the presence of RNA genetic material of virus attributing to the associated functional activities of the lake catchment area. Urban lakes encompassing the domestic activities showed the prevalence of viral load and may be consider as proxy for WBE studies to assess the community infection rate. The receiving of domestic discharge from the population residing around the catchment area forms a major basis for this. UL-1 samples showed positive signal for all the three genes, UL-2 and UL-3 showed positive signal for two genes out of three (UL-2 (N-gene and ORF1ab) and UL-3 (Egene and N-gene)). The referral lakes PL and RL did not show detection for the targeted genes. From the results it is clear that urban lakes are impacted probably due to non-treated sewage discharge resulted in high viral load. The trend of RNA copies curve (with reference to UL-1) is correlating with the dynamics of COVID cases matching with Wave I and Wave II scenario in India. This observation indicates a clear-cut function of the water bodies to act All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 1 4 as a proxy for surveillance studies to predict the epidemic/pandemics as early warning signal (as part of WBE studies), to assess the infection rates during the ongoing pandemic and to understand the dynamics of viral load pertains to the community in the catchment area. The SARS-CoV-2 gene fragments have been detected were clearly in the urban lakes which are surrounded by the anthropogenic activities. Also, the dynamic of viral load showed the proximity to understand the infection rates as well as also served as early warning signal. The surge in February 2021 sample showed the on shoot of the second wave of infection which correlated well with the prevailing pandemic situation. The reference water bodies (peri-urban and rural lakes) did not detected SARS-CoV-2 fragments. The functional attributes of lake to anthropogenic activity will be the guiding factor for the viral presence. The presence of viral fragments and its load can be used as surveillance tool to understand the infection spread. This study depicted the need for regular monitoring of the water bodies/wastewater as part of WBE studies in the urban and semi-urban areas to understand the outbreak and to assess spread of viral as well. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint 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. 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No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 12, 2021. ; https://doi.org/10.1101/2021.05.08.21256881 doi: medRxiv preprint