key: cord-0838584-6o7xiqk2 authors: Bar-Or, Itay; Weil, Merav; Indenbaum, Victoria; Bucris, Efrat; Bar-Ilan, Dana; Elul, Michal; Levi, Nofar; Aguvaev, Irina; Cohen, Zvi; Shirazi, Rachel; Erster, Oran; Brown, Alin Sela; Sofer, Danit; Mor, Orna; Mendelson, Ella; Zuckerman, Neta S. title: Detection of SARS-CoV-2 variants by genomic analysis of wastewater samples in Israel date: 2021-05-24 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2021.148002 sha: b76a86e9c0f076c259fed9893a8c3a17d8d7fdea doc_id: 838584 cord_uid: 6o7xiqk2 Investigation of SARS-CoV-2 spread and identification of variants in sewers has been demonstrated to accurately detect prevalence of viral strains and is advantageous to clinical sampling in population catchment size. Herein, we utilized an established nationwide system of wastewater sampling and viral concentration approaches to perform large-scale surveillance of SARS-CoV-2 variants in nine different locations across Israel that were sampled from August 2020 to February 2021 and sequenced (n=58). Viral sequences obtained from the wastewater samples had high coverages of the genome, and mutation analyses successfully identified the penetration of the B.1.1.7 variant into Israel in December 2020 in the central and north regions, and its spread into additional regions in January and February 2021, corresponding with clinical sampling results. Moreover, the wastewater analysis identified the B.1.1.7 variant in December 2020 in regions in which non-sufficient clinical sampling was available. Other variants of concern examined, including P.1 (Brazil/Manaus), B.1.429 (USA/California), B.1.526 (USA/New York), A.23.1 (Uganda) and B.1.525 (Unknown origin), did not show consistently elevated frequencies. This study exemplifies that surveillance by sewage is a robust approach which allows to monitor the diversity of SARS-CoV-2 strains circulating in the community. Most importantly, this approach can pre-identify the emergence of epidemiologically or clinically relevant mutations/variants, aiding in public health decision making. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of an ongoing global pandemic of corona virus disease [1] , has affected at least 223 countries/territories with ~114 million confirmed cases and almost 2.6 million deaths reported worldwide as of beginning of March 2021 [2] . While the majority of infections result in no apparent symptoms or mild ones, some progress to acute respiratory disease, multi-organ failure, and death [3] . Community transmission of the virus, as well as anti-viral treatments, can engender novel mutations in the viral genome, potentially resulting in more virulent strains with higher mortality rates or emergence of strains resistant to treatment [4] . Recent increased sequencing efforts worldwide contributed to the discovery of SARS-CoV-2 variants of concern (VOC) with rapid spread and increased transmissibility, including B.1.1.7 (UK) [5] and B.1.351 (South Africa) [6] . The impact of these variants' mutations on the effectiveness of vaccines is being explored, and travel bans are implemented on passengers arriving from afflicted countries. In Israel as well, entry of foreigners has been minimized and returning citizens are quarantined in specialized facilities or at home. Systematic tracking of demographic and clinical patient information, and especially of the viral genome, has been shown to be necessary to effectively combat transmission of the disease [7]. Numerous countries have recently increased their viral genome sequencing capacity in aim to identify and control known and novel viral variants in a timely manner [8] . However, estimating viral diversity from clinical samples may be challenging in terms of representative sampling and funds. Wastewater-based epidemiology (WBE) has been shown to be a useful tool to track virus circulation and variants such as Poliovirus [9] . Indeed, wastewater sampling and viral concentration is established in Israel and routinely used for polio surveillance, including molecular characterization of Poliovirus strains [10] . J o u r n a l P r e -p r o o f Journal Pre-proof SARS-CoV-2 is primarily detected in respiratory tract tissues, however it has been shown that the virus can also be detected in the gastrointestinal tract, with viral RNA shedding in feces of around 50% of the infected patients, often for longer periods than for nasal swabs [11] [12] . Accordingly, since the start of the COVID-19 pandemic, wastewater real-time quantitative PCR (RT-qPCR) has been used to quantify the amount of SARS-CoV-2 RNA in sewage across many different regions globally, where generally a correlation is found between the RNA levels and the numbers of COVID-19 confirmed cases [13] [14] [15] [16] [17] . Few recent studies investigating the spread of SARS-CoV-2 variants in the community via wastewater showed accurate identification and prevalence of viral strains known to exist in the region sampled, and in addition demonstrated evidence for recent introductions of viral lineages before they are detected by local clinical tests [18] [19] [20] [21] [22] [23] . In this study, whole SARS-CoV-2 genomes were sequenced from RNA extracted from Detection of the SARS-CoV-2 RNA presence in wastewater was initially performed using RT-qPCR targeting the SARS-CoV-2 E gene as described by Corman et al. [24] . This reaction was combined in a multiplex that contained two additional reactions: hCoV OC-43 for preextraction spike control, and MS-2 phage for post extraction spike control. The protocol was adjusted for the use with the SensiFast reaction mix Fasq files underwent quality control using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC [26] , and low-quality sequences were filtered using trimmomatic [27] . Mapping was performed to the SARS-CoV-2 reference genome (NC_045512.2) using the Burrows-Wheeler aligner (BWA) [28] , followed by filtering unmapped reads, sorting and indexing of the resulting BAM files using the SAMtools suite [29] . BAM files were imported to R and the frequency of each nucleotide or gap in each position along the genome was obtained for each sample using the Rsamtools Mutations associated with known worldwide VOC were examined in each region/time ( Figure 2 A pneumonia outbreak associated with a new coronavirus of probable bat origin Coronavirus Disease (COVID-19) Pandemic Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Mechanisms of viral mutation Emergence of a new SARS-CoV-2 variant in the UK Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa Genomic sequencing effort for SARS-CoV-2 by country during the pandemic Environmental Surveillance for Polioviruses in the Global Polio Eradication Initiative Molecular Characterization of Polio from Environmental Samples: ISSP, The Israeli Sewage Surveillance Protocol Evidence for Gastrointestinal Infection of SARS-CoV-2 Prolonged presence of SARS-CoV-2 viral RNA in faecal samples SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases SARS-CoV-2 titers in wastewater foreshadow dynamics and clinical 10 Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater Implementation of environmental surveillance for SARS-CoV-2 virus to support public health decisions: Opportunities and challenges Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater Tracking SARS-CoV-2 in Sewage: Evidence of Changes in Virus Variant Predominance during COVID-19 Pandemic Monitoring SARS-CoV-2 circulation and diversity through community wastewater sequencing Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants Detection of SARS-CoV-2 variants in Switzerland by genomic analysis of wastewater samples Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Artic network, SARS-CoV-2 MultiQC: Summarize analysis results for multiple tools and samples in a single report Trimmomatic: a flexible trimmer for Illumina sequence data Fast and accurate short read alignment with Burrows-Wheeler transform The Sequence Alignment/Map format and SAMtools Rsamtools: Binary alignment (BAM), FASTA, variant call (BCF), and tabix file import Emergence of a Novel SARS-CoV-2 Variant in Southern California A SARS-CoV-2 lineage A variant (A.23.1) with altered spike has emerged and is dominating the current Uganda epidemic Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence PANGO lineages SARS-CoV-2 lineage B.1.526 emerging in the New York region detected by software utility created to query the spike mutational landscape ggplot2: Elegant Graphics for Data Analysis ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Neta zuckernan