Going beyond SARS-CoV-2: genomic surveillance of monkeypox in German wastewater

Going beyond SARS-CoV-2: genomic surveillance of monkeypox in German wastewater | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Going beyond SARS-CoV-2: genomic surveillance of monkeypox in German wastewater Shelesh Agrawal, Laura Orschler, Sofie Waengler, Robert Greither, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2350648/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Fear of stigma poses a challenge in tracking the 2022-outbreak of monkeypox virus (MPXV) infection. Patients shed MPXV via skin lesions, gastrointestinal route, and seminal fluids into the wastewater. Monitoring MPXV in wastewater can support tracking transmission. We developed a sensitive NGS panel “P4Mpox22”, to track MPXV in sewage. Since May 2022, we have monitored the sewage of German cities using P4Mpox22 and dPCR to quantify MPXV. Despite only 12 clinical cases reported in the respective sewersheds, we obtained up to 90% MPXV genome coverage. Sewage-derived MPXV genomes cluster with lineage B.1 and exhibit APOBEC-type hypermutations. Using dPCR, we quantified MPXV even in sewersheds with only one clinical case. We show that MPXV sewage monitoring using existing SARS-CoV-2 wastewater surveillance infrastructure could be implemented immediately. One-Sentence Summary Human monkeypox virus sequencing in wastewater enables community-wide surveillance while overcoming stigmatization challenges Biological sciences/Microbiology/Environmental microbiology/Water microbiology Health sciences/Health care/Public health/Epidemiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction A zoonotic disease caused by monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has been known to be endemic in Central and West Africa, with the first human case was detected in the Congo in 1970 1 . Starting May 17, 2022 increased human-to-human transmissions without travel history to Africa were reported from the UK, quickly followed by cases in several European countries, as well as in North America 2 . Since then, we are facing the largest known outbreak of monkeypox in the world ever, with most confirmed cases coming from the European Union (EU). By June 23, 2022, 3413 cases had been reported to the World Health Organization (WHO), including 2933 cases from the EU 3 . The WHO decided not to declare the situation a Public Health Emergency of International Concern (PHEIC) at the time. Within a month, by July 23. 2022, the number of cases had increased to 14533. Because of this rapid increase and the paucity of information on new transmission routes, the WHO decided to declare the situation a PHEIC 4 . In Germany, by August 11, 2022, 3065 cases have been reported 5 . Health authorities around the world are taking various measures to monitor and control the disease. Currently, most cases are associated with men who have sex with men (MSM) 6 . However, this association could exacerbate the stigmatization of the disease while creating barriers to assessing the true situation of its spread and thus to taking prompt countermeasures. For instance, several positive cases refused to share information about their sexual contacts with the UK health agencies, posing a major challenge for public health action 7 . In addition, the current definition of the typical route of transmission through sexual contact is increasingly being weakened. For example, community transmission is already taking place in the United Kingdom 7,8 . In this context, wastewater surveillance can help to monitor the spread of such a disease at temporal and spatial scales, as demonstrated by the SARS-CoV-2 monitoring currently underway in many countries 9 . Expanding the horizon of wastewater surveillance by leveraging currently existing wastewater-based epidemiology (WBE) infrastructures to monitor trends in the spread of monkeypox can strengthen the surveillance of this emerging disease while overcoming the challenge of information access for public health officials. Among all specimen types with positive test results used for diagnosing MPXV in Europe, urine samples account for 1.4% of total samples by August 10, 2022 10 . The presence of MPXV DNA in fecal samples (i.e. in gastrointestinal samples) has been also confirmed 11 . Discharge of MPXV during showering from the exposed skin lesions is known 12 , another plausible source of MPXV in wastewater. Thus, the probability of detecting MPXV in wastewater is relatively high, also considering relative high stability of orthopoxvirus (OPV) in environmental samples such as storm water and soil 13 . With the monkeypox outbreak, genome sequencing of clinical samples began immediately to understand the evolution and genetic diversity of the virus and to find answers to current human-to-human transmission. However, genomic analysis of MPXV in wastewater samples, similar to SARS-CoV-2, is challenging due to the presence of inhibitors, contamination with other genomic material, and especially the very low concentration of the target material 14,15 . Thus, we set out for rapid implementation of a robust wastewater surveillance system for monitoring MPXV, using dPCR and next-generation sequencing (NGS), while using a currently established SARS-CoV-2 monitoring approach. Results and Discussion Detection of MPXV in wastewater while few clinical cases are known. We developed and applied an amplicon-sequencing panel, named as “P4Mpox22” (details in Supplementary, Data S1), to detect MPXV in wastewater samples even in very low concentrations, for example when only a handful of clinical cases are reported to the public health authorities. Since early May 2022, we have analyzed wastewater samples from ten German cities (Table S1 for more information about the cities) for MPXV with digital PCR (dPCR) using the commercially available assay and our NGS amplicon panel. Overall in Germany, the first two cases were reported in week 20 2 , while in the federal state of Hessen in Germany, the first two cases were reported in week 21 (Fig. S1). We found MPXV already in wastewater samples from week 19 in two cities (i.e. Giessen: GI and Darmstadt: DA) (Fig.1). Among the 10 German cities, we have observed a quadruple increase from week 19 to week 23 in the samples from GI, whereas, in wastewater samples from Biebrich (BR) and Giresheim (GR) the MXPXV concentration increased two-fold after week 27 to week 32 (Fig.1). In Hanau (HU) samples an increase in signal from week 23 to 27 (up to 0.6 gene copies/µL), which is not detectable anymore in week 32. A similar trend was seen for the Kassel (KA) samples, but in a lower concentration range (<0.2 gene copies/µL). And, in Fulda (FU), Niederrad (NR), Sindlingen (SL) and Wiesbaden (WI) samples MPXV was not detectable using dPCR analysis. Whole genome amplicon sequencing of MPXV in wastewater. We applied the NGS panel described in this study to successfully sequence and partially reconstruct MPXV genomes from sewage samples, limiting to samples with positive signal for dPCR analysis (Fig. S2). The performance of the P4Mpox22 Panel was checked using the MPXV DNA obtained from virus isolated from skin pustules of the first German case in Munich 2 , as positive control. The panel allowed to achieve >2M mapped reads with average read identity of >99% to our reference genome hMpxV/Belgium/UZ_Rega_1/2022 (GISAID accessionID - EPI_ISL_13052282) which was used to design the panel, and average base coverage depth of 1800 bp. Coverage overview of the control MPXV DNA is shown in Fig. S3. For wastewater samples, with an increase in reported clinical cases from week 19 to 32, the percentage of reference genome bases covered also increased from 0.14% in week 19 to 89.5% in week 32 (Fig. 2A, Fig. S4, Fig. S5). While the total reported clinical cases in Hesse increased to 9 in week 27, the genome coverage increased to around 40% in comparison to <4% genome coverage in week 23 when no cases were reported (Fig. 2A and B). As the total number of cases in Hesse increased in week 32, the DNA concentration and thus length of fragments recovered from the wastewater sequences also increased. (Fig. 2C). For week 32, we sequenced sample from two cities (i.e. BR and GR). During week 32, only one case was reported in BR district and 12 cases in GR district (Fig S4). A significant increase in fragment length was observed in the samples from week 32, the median fragment length was 1000 bp and maximum up to 7500 bp (Fig 2C). High genome coverage of samples from week 32 (Fig. 2A, Fig. S5), allowed us to construct consensus genomes which represent predominant MPXV strain circulating in the respective sewersheds at this timepoint. We looked at the alignment of the consensus genome sequences to a set of clinical MPXV sequences released in the National Center for Biotechnology Information (NCBI) before August 10, 2022. Whole-genome alignment demonstrated higher sequence similarity with the 2022 outbreak-related MPXV genome sequences (Fig. 3A). Subsequently, the FR and BR genome sequences clustered together with 2022 outbreak-related MPXV cluster (lineage B.1) 16 , tightly placed together with lineage B.1 sequences from Germany (Fig. 3B). The lineage B.1 cluster formed a divergent branch from a genome sequence from USA 2021 (lineage A.1.1) 16 descendant from 2018 MPXV cluster. Although the quality of genome sequences from other wastewater samples was very low, we looked at their placement together with BR and GR sequences from week 32. Figure 3C shows the impact of the genome quality: more fragmented genomes from samples with less MPXV DNA concentration (Fig.1) tend to cluster together and farther away from the genomes of week 32. Nevertheless, low-quality genomes also partially aligned with clinical MPXV sequences (Fig. S6). Detecting hyper-mutation signature of 2022 MPXV (lineage B.1) in wastewater. The 2022-outbreak MPXV genome sequences have been associated with a specific mode of mutation which is ascribed to the action of apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) enzymes 17 , specifically referring to deamination of cytosine to uracil (C-to-U) by APOBEC3 18 . In clinical samples, GA>AA and TC>TT hypermutations have been reported as APOBEC3-type signature mutations 17 . Therefore, we also screened the wastewater sample sequences for GA>AA and TC>TT signature mutations using MPXV-UK_P2, 2018 (GenBank accession no. MT903344.1) as a reference sequence similar to a previous study 17 , to determine whether hyper-mutation signatures of 2022 MPXV (lineage B.1) can be detected in wastewater samples. However, we report only mutations with 100% allele frequency. Figure 4A emphasizes the impact of target base coverage evidently, lack of dense cluster of GA>AA and TC>TT mutation for samples from week 19 to 27, with maximum of 26 mutations for HU_27, in comparison to sample BR and GR from week 32. As the quality of target base coverage is important for reliable GA>AA and TC>TT hypermutation analysis, only samples BR_32 and GR_32 were further analyzed. We found clusters of GA>AA and TC>TT across the entire genome in those wastewater samples (Fig.4B). Distribution of the observed mutation cluster across the genome is also seen in clinical sequences from the current MPXV outbreak 17 . Although multiple mutation clusters were spread across the genome in the wastewater samples, most mutations were found in genomic regions: 3000-5000 bp and 120000-150000 bp (Fig. 4C). In genome sequences from clinical samples in Germany, the mutations were also concentrated in multiple genomic regions, including 30000-50000 bp and 120000-150000 bp (Fig. 4D). To achieve better confidence in the wastewater samples, we compared signature motifs (which included five nucleotides before and after the mutation against reference sequence, MT903344.1, found in global clinical sequences with all 12 wastewater samples. For six out of these 12 wastewater samples, we found a match for motifs in the global clinical sequences and the maximum number of matches was observed for sample GR_32 (Fig. 4E), also emphasizing the impact of genome coverage from wastewater samples. Challenges with PCR based MPXV surveillance. The 196,858-bp MPXV genome has 190 open reading frames containing at least 60 amino acid residues each 19 . However, it seems that just 11 genes have been used as likely targets for PCR analysis. The choice of target for the specific detection of MPXV is crucial but also challenging. While highly similar sequences lead to nonspecific primer/probe binding, highly divergent or variable sequence regions pose considerable challenges for the detection 19,20 . In this study, we also observed that detecting MPXV in wastewater using dPCR, which target specific region, was less sensitive, while it was possible to detect MPXV genomes in wastewater using amplicon sequencing approach by targeting whole genome using multiple primers. In this study, we were able to detect the MPXV in the state Hesse in sewage of different city districts and counties by digital PCR as well as by NGS, while a handful of cases have been reported. This demonstrates the successful and rapid adaption of an established virus detection and NGS based sequencing pipeline to a new approach and underlines the benefits/necessity of including wastewater-based monitoring into the national surveillance strategy of public health relevant pathogens. Wastewater surveillance comes of age, starting from poliovirus early warning systems to SARS-CoV-2 monitoring at present. SARS-CoV-2 and monkeypox are very dissimilar viruses, not only by their biological properties: one is a never-seen-before RNA virus and the other one is a DNA virus known for decades with zoonotic transmission endemic to Sub-Saharan Africa. Yet, there are more unknowns than knowns during the early days of worldwide outbreaks ( 21 ). WBE has proven a viable tool during the COVID-19 pandemic to monitor viral loads and complement other surveillance systems. Furthermore, it can be used in the deceleration as well as the interpandemic phase with sampling strategies prioritized to transmission nodes or sentinel sites ( 22 ). With special regards to new emerging viral pathogens such as MPXV or currently re-emerging poliovirus in already polio-free regions ( 23 ), we emphasize continuing to use and improve wastewater-based monitoring of public health relevant infectious agents. Methods Sampling and Extraction 200 ml of a 24h-proportional influent wastewater samples were collected every first Tuesday of the month from May till August from 10 wastewater treatment plants (S.Table1). The samples were concentrated by ultrafiltration in 100 kDa Centricon® Plus-70 centrifugal ultrafilters (Merck) to 5 ml of concentrate and DNA was extracted from the concentrate using the Ultra Microbiome kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. dPCR analysis For quantification of the monkeypox virus (MPXV) in wastewater samples, we used commercially available assay from Thermo Fisher Scientific. The analysis was performed using AbsoluteQ (Thermo Fisher Scientific). 9µl of total master mix was prepared for each sample and was loaded in the QuantStudio Absolute Q MAP16 Digital PCR Plate (Thermo Fisher Scientific). 9µl of total master mix consist of 1.8µl Absolute Q DNA Digital PCR Master Mix (5X); 0.45µl of target assay; 1.75µl of NFW; and 5 µl of DNA template. The thermal profile used for the quantification was 96°C for 5 min.; 45 cycles of 96°C for 5 sec followed by 62°C for 30 sec. Next-generation sequencing (NGS) panel design and analysis We designed AmpliSeq™ custom panel- named as “P4Mpox22 Panel”. This panel was developed using the 2022 outbreak clinical MPXV genome sequence (GISAID accessionID - EPI_ISL_13052282). This panel consist of 958 amplicons, with amplicon size ranging 125 - 275 bp. To achieve higher specificity and reduction in primer interferences, primers were divided into two-pool system. Pool 1 consist of 480 primes, and pool 2 has 478 primers. The design BED file, as an auxillary file (Data S1), is also provided for people looking for synthesizing the NGS panel. We performed library preparation, using the P4Mpox22 panel, on Ion Chef (Thermo Fisher Scientific) using the DL8 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The library preparation allowed barcoded amplicons for each sample. Libraries were multiplexed and sequenced using an Ion Torrent 550 chip on an Ion S5 sequencer (Thermo Fisher Scientific). The sequences were mapped against reference genome sequence (GISAID accessionID - EPI_ISL_13052282) and reads with minimum 100 bp length and at least 50 bp alignment to the reference sequence were retained for further analysis. Variant caller, consensus genome construction and Hypermutation analysis For SNP calling, we used mpileup (24) and VarScan (25) at default setting. Consensus genome sequences were called using the ivar consensus (26) using parameters: Minimum quality score threshold to count base – 20, minimum read depth - 10. For hypermutation analysis, we screened filtered wastewater sample sequences for GA>AA and TC>TT signature mutations using MPXV-UK_ P2, 2018 (GenBank accession no. MT903344.1) as a reference sequence, similar to a previous study (17) . The graphs were constructed in RStudio Server (build 554) in R environment (v 4.2.1) using ggplot2_3.3.6, forcats_0.5., GenomicRanges_1.48.0, ggpubr_0.4.0, and hrbrthemes_0.8.0 packages. Phylogenetic reconstruction and placement of sewage-derived consensus sequences We generated a multiple sequence alignment via MAFFT v7.455 (27) and default parameters for a selection of input sequences comprising a) genome sequences from former outbreaks, b) sequences from clinical sampling of the 2022 hMPXV outbreak from different countries with a focus on Germany, and c) the reconstructed consensus sequences for the sewage samples obtained in week 29 (GR_32, BR_32). We only used sewage sequences from week 29 in this initial alignment to reduce alignment bias and potential long branch attractions in the final phylogeny caused by the highly fragmented consensus genomes especially derived from sewage samples with low DNA concentrations at earlier sampling dates. We plotted an overview figure of this initial MSA via CIAlign v1.0.18 (28) using default parameters and reconstructed a phylogenetic tree with IQ-TREE v2.2.0.3 (29) and 1000 ultra-fast bootstraps and the genome sequence from Nigeria 1971 as defined outgroup. We used Newick Utilities v1.6 (^30) to visualize the tree. We additionally calculated an MSA and tree using the same commands focusing only on the sewage-derived consensus sequences. Declarations Acknowledgments: We gratefully acknowledge the contribution from the originating laboratories responsible for obtaining the specimens and the submitting laboratories where genetic sequence data were generated and shared via the GISAID Initiative (https://www.gisaid.org). We thank all WWTP operators for providing wastewater samples. Funding: JJB Medical Biological Defense Research Program of the Bundeswehr Medical Service Author contributions: Conceptualization: SA, LO Methodology: SA, LO, RG, MH, SW Investigation: SA, MH, LO, RG, SW Visualization: SA, MH, LO Funding acquisition: SL, SA Project administration: SL, LO, SA Supervision: LO, SA Writing – original draft: SA, LO, MH Writing – review & editing: MH, SA, SL, SB, JJB, AN, AB Competing interests: Authors declare that they have no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. The NGS panel design Bed file is provided as an auxillary file “Data S1”. Raw metagenomic sequence data are available from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under Submission ID SUB11975131, BioProject number PRJNA874069. References Ladnyj, I. D., Ziegler, P. & Kima, E. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull World Health Organ 46 , 593–597 (1972). Noe, S. et al. Clinical and virological features of first human monkeypox cases in Germany. Infection (2022) doi:10.1007/s15010-022-01874-z. World Health Organization. Meeting of the International Health Regulations (2005) Emergency Committee regarding the multi-country monkeypox outbreak. https://www.who.int/news/item/25-06-2022-meeting-of-the-international-health-regulations-(2005)-emergency-committee--regarding-the-multi-country-monkeypox-outbreak. World Health Organization. Second meeting of the International Health Regulations (2005) (IHR) Emergency Committee regarding the multi-country outbreak of monkeypox. https://www.who.int/news/item/23-07-2022-second-meeting-of-the-international-health-regulations-(2005)-(ihr)-emergency-committee-regarding-the-multi-country-outbreak-of-monkeypox. Robert Koch Institute. RKI - Infektionskrankheiten A-Z - Internationaler Affenpocken-Ausbruch: Fallzahlen und Einschätzung der Situation in Deutschland. https://www.rki.de/DE/Content/InfAZ/A/Affenpocken/Ausbruch-2022-Situation-Deutschland.html;jsessionid=E13EBFC92D61B1E07ED381EA0530D5EC.internet062?nn=2386228. European Centre for Disease Prevention and Control. Monkeypox multi-country outbreak . 22 (2022). Vivancos, R. et al. Community transmission of monkeypox in the United Kingdom, April to May 2022. Eurosurveillance 27 , (2022). Pan, D. et al. Monkeypox in the UK: arguments for a broader case definition. The Lancet 399 , 2345–2346 (2022). Medema, G., Been, F., Heijnen, L. & Petterson, S. Implementation of environmental surveillance for SARS-CoV-2 virus to support public health decisions: Opportunities and challenges. Curr Opin Environ Sci Health 17 , 49–71 (2020). European Centre for Disease Prevention and Control/WHO Regional Office for Europe. Joint ECDC-WHO Regional Office for Europe Monkeypox Surveillance Bulletin . https://monkeypoxreport.ecdc.europa.eu (2022). Peiró-Mestres, A. et al. Frequent detection of monkeypox virus DNA in saliva, semen, and other clinical samples from 12 patients, Barcelona, Spain, May to June 2022. Eurosurveillance 27 , (2022). CDC. Monkeypox in the U.S. Centers for Disease Control and Prevention https://t.cdc.gov/K6XB9 (2022). Essbauer, S., Meyer, H., Porsch-Özcürümez, M. & Pfeffer, M. Long-Lasting Stability of Vaccinia Virus (Orthopoxvirus) in Food and Environmental Samples. Zoonoses Public Health 54 , 118–124 (2007). Agrawal, S. et al. Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities. Water Research 118162 (2022) doi:10.1016/j.watres.2022.118162. Larsen, D. A. & Wigginton, K. R. Tracking COVID-19 with wastewater. Nature Biotechnology 38 , 1151–1153 (2020). Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus - Monkeypox. Virological https://virological.org/t/urgent-need-for-a-non-discriminatory-and-non-stigmatizing-nomenclature-for-monkeypox-virus/853 (2022). Isidro, J. et al. Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med (2022) doi:10.1038/s41591-022-01907-y. Pecori, R., Di Giorgio, S., Paulo Lorenzo, J. & Nina Papavasiliou, F. Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination. Nat Rev Genet 23 , 505–518 (2022). Shchelkunov, S. N. et al. Analysis of the Monkeypox Virus Genome. Virology 297 , 172–194 (2002). Hammarlund, E. et al. Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox. Nat Med 11 , 1005–1011 (2005). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterialsFinal.docx Supplementary Text Figs. S1 to S6 Tables S1 DataS1.txt Dataset 1 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2350648","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":169935150,"identity":"cdd88bf0-e8b3-4649-9998-e6dea1561aaf","order_by":0,"name":"Shelesh Agrawal","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9365-5951","institution":"Technical University of Darmstadt","correspondingAuthor":true,"prefix":"","firstName":"Shelesh","middleName":"","lastName":"Agrawal","suffix":""},{"id":169935151,"identity":"26193054-e3dc-4031-ac8f-99c904e20388","order_by":1,"name":"Laura Orschler","email":"","orcid":"","institution":"Technical University of Darmstadt","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Orschler","suffix":""},{"id":169935152,"identity":"82d43f18-9189-4336-b3c8-381fc25eae9b","order_by":2,"name":"Sofie Waengler","email":"","orcid":"","institution":"Technical University of Darmstadt","correspondingAuthor":false,"prefix":"","firstName":"Sofie","middleName":"","lastName":"Waengler","suffix":""},{"id":169935153,"identity":"db21f129-82a2-4648-ab66-076fd035fb40","order_by":3,"name":"Robert Greither","email":"","orcid":"","institution":"Life Technologies","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Greither","suffix":""},{"id":169935154,"identity":"0aa7b557-14fb-4109-8612-8274eec2b3cc","order_by":4,"name":"Sindy Boettchers","email":"","orcid":"","institution":"Robert Koch Institute","correspondingAuthor":false,"prefix":"","firstName":"Sindy","middleName":"","lastName":"Boettchers","suffix":""},{"id":169935155,"identity":"8a66f81f-f679-4ac5-ab5c-6de45934bfb1","order_by":5,"name":"Annika Brinkmann","email":"","orcid":"","institution":"Robert Koch Institute","correspondingAuthor":false,"prefix":"","firstName":"Annika","middleName":"","lastName":"Brinkmann","suffix":""},{"id":169935156,"identity":"faf0d98c-f434-4363-a327-41a701c7951f","order_by":6,"name":"Andreas Nitsche","email":"","orcid":"","institution":"Robert Koch Institute","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Nitsche","suffix":""},{"id":169935157,"identity":"c80009e0-ae11-4f27-9f7f-be5fc36ef030","order_by":7,"name":"Joachim Bugert","email":"","orcid":"","institution":"Bundeswehr Institute of Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Joachim","middleName":"","lastName":"Bugert","suffix":""},{"id":169935158,"identity":"758dfc2a-fbaf-42e7-82cb-0e648ed0616c","order_by":8,"name":"Martin Hölzer","email":"","orcid":"","institution":"Methodology and Research Infrastructure, Bioinformatics, Robert Koch Institute","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Hölzer","suffix":""},{"id":169935159,"identity":"d6ff1272-5ae7-4d15-8578-f4d413cdd44d","order_by":9,"name":"Susanne Lackner","email":"","orcid":"","institution":"Technical University of Darmstadt","correspondingAuthor":false,"prefix":"","firstName":"Susanne","middleName":"","lastName":"Lackner","suffix":""}],"badges":[],"createdAt":"2022-12-06 15:36:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2350648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2350648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84921246,"identity":"cb646307-53c9-4960-a804-3c1206bdad54","added_by":"auto","created_at":"2025-06-18 19:47:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe concentration of MPXV measured in wastewater based on dPCR analysis. \u003c/strong\u003eThe concentration of MPXV DNA was measured in wastewater samples collected from ten wastewater treatment plants (WWTPs), using a commercially a 5 vailable TaqMan assay.\u003cstrong\u003e \u003c/strong\u003eThe samples were collected on the first Tuesday of each month. BR – Wiesbaden Biebrich, DA – Darmstadt, FU – Fulda, GI – Giessen, GR – Griesheim, HU – Hanau, KA – Kassel, NR – Niederrad, SL – Sindlingen, and WI – Wiesbaden.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/a6569aa800b0799efb193085.png"},{"id":84920675,"identity":"c5a5b1b0-bc4d-4144-a922-2a1ab6882b05","added_by":"auto","created_at":"2025-06-18 19:39:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe quality of MPXV genome from wastewater samples. \u003c/strong\u003e(A) Boxplot showing the target base coverage achieved for the wastewater samples. (B) Time distribution of clinical cases in Hessen. (C) Boxplot showing the length of c 5 ontinuous fragments achieved from overlapping amplicons.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/97105f59a424f71b3a8b616b.png"},{"id":84920678,"identity":"902b3635-fa8a-45de-89c9-589d2b19de9d","added_by":"auto","created_at":"2025-06-18 19:39:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple sequence alignment and phylogenetic placement of wastewater-based MPXV consensus sequences against a collection of MPXV genomes from clinical sampling. (A) \u003c/strong\u003eFirst, the reconstructed sewage genomes were aligned against MPXV sequences from clinical sampling. The multiple sequence alignment 5 comprises all sequences shown in\u003cstrong\u003e \u003c/strong\u003e(B)\u003cstrong\u003e \u003c/strong\u003eand includes two sewage MPXV genomes of higher quality (GR_32, BR_32); cyan color, bottom rows).\u003cstrong\u003e (B)\u003c/strong\u003e An MPXV global phylogeny based on the alignment shown in (A). The BR and GR week 32 genome sequences cluster with the 2022 outbreak (lineage B.1). \u003cstrong\u003e(C)\u003c/strong\u003e Phylogenetic tree only focusing on the diversity among the genomes recovered from the wastewater samples. 10 Wastewater samples of week 19 – SL_19, FU_19; week 23 – GI_23, KA_23,HU_23; week 27 – KA_27,GR_27,Da_27,HU_27, BR_27; week 32 – GR_32, BR_32.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/8e3e8240984b051792474cc2.png"},{"id":84920679,"identity":"4daa20de-d765-47a6-8bc2-bc97437e942a","added_by":"auto","created_at":"2025-06-18 19:39:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNP profiles characteristic for the 2022-MPXV outbreak.\u003c/strong\u003e (A) Mutational profile of the wastewater samples, showing the mutations GA \u0026gt; AA and TC\u0026gt; TT exhibiting 100% allele frequency. (B) Showing the GA \u0026gt; AA and TC\u0026gt; TT mutation clusters for BR_32 and GR_32, together with (C) the density of the GA 5 \u0026gt; AA and TC\u0026gt; TT mutations found across the wastewater samples throughout the MPXV genome. (D) Showing the density of the GA \u0026gt; AA and TC\u0026gt; TT mutations found across the German clinical samples throughout the MPXV genome. (E) Plot showing the common sequence motifs between the wastewater samples and global clinical samples (submitted in GISAID), along with their respective mutations. 10 Wastewater samples of week 19 – SL_19, FU_19; week 23 – GI_23,KA_23,HU_23; week 27 – KA_27,GR_27,Da_27,HU_27, BR_27; week 32 – GR_32, BR_32.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/557a13d3e23eb4a0f5b231b4.png"},{"id":84921247,"identity":"8032cb1e-1b27-461e-a31e-54dbe303af35","added_by":"auto","created_at":"2025-06-18 19:47:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":933084,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/dd6ad4b4-1ed5-4fc9-96ce-99657e426a4a.pdf"},{"id":84920680,"identity":"5faa0c54-2c3a-4824-9f90-2b454aa53cb8","added_by":"auto","created_at":"2025-06-18 19:39:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2666519,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Text\u003c/p\u003e\n\u003cp\u003eFigs. S1 to S6\u003c/p\u003e\n\u003cp\u003eTables S1\u003c/p\u003e","description":"","filename":"SupplementaryMaterialsFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/c690b9113c3cccb0f7704b37.docx"},{"id":84920677,"identity":"5ee7f00d-f469-4e37-a083-a27434709e18","added_by":"auto","created_at":"2025-06-18 19:39:40","extension":"txt","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":120809,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 1\u003c/p\u003e","description":"","filename":"DataS1.txt","url":"https://assets-eu.researchsquare.com/files/rs-2350648/v1/e2053950651339392a889f9e.txt"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Going beyond SARS-CoV-2: genomic surveillance of monkeypox in German wastewater","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA zoonotic disease caused by monkeypox virus (MPXV), a member of the \u003cem\u003eOrthopoxvirus\u003c/em\u003e genus, has been known to be endemic in Central and West Africa, with the first human case was detected in the Congo in 1970 \u003csup\u003e1\u003c/sup\u003e. Starting May 17, 2022 increased human-to-human transmissions without travel history to Africa were reported from the UK, quickly followed by cases in several European countries, as well as in North America \u003csup\u003e2\u003c/sup\u003e. Since then, we are facing the largest known outbreak of monkeypox in the world ever, with most confirmed cases coming from the European Union (EU). By June 23, 2022, 3413 cases had been reported to the World Health Organization (WHO), including 2933 cases from the EU \u003csup\u003e3\u003c/sup\u003e. The WHO decided not to declare the situation a Public Health Emergency of International Concern (PHEIC) at the time. Within a month, by July 23. 2022, the number of cases had increased to 14533. Because of this rapid increase and the paucity of information on new transmission routes, the WHO decided to declare the situation a PHEIC \u003csup\u003e4\u003c/sup\u003e. In Germany, by August 11, 2022, 3065 cases have been reported \u003csup\u003e5\u003c/sup\u003e. Health authorities around the world are taking various measures to monitor and control the disease. Currently, most cases are associated with men who have sex with men (MSM) \u003csup\u003e6\u003c/sup\u003e. However, this association could exacerbate the stigmatization of the disease while creating barriers to assessing the true situation of its spread and thus to taking prompt countermeasures. For instance, several positive cases refused to share information about their sexual contacts with the UK health agencies, posing a major challenge for public health action \u003csup\u003e7\u003c/sup\u003e. In addition, the current definition of the typical route of transmission through sexual contact is increasingly being weakened. For example, community transmission is already taking place in the United Kingdom \u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this context, wastewater surveillance can help to monitor the spread of such a disease at temporal and spatial scales, as demonstrated by the SARS-CoV-2 monitoring currently underway in many countries \u003csup\u003e9\u003c/sup\u003e. Expanding the horizon of wastewater surveillance by leveraging currently existing wastewater-based epidemiology (WBE) infrastructures to monitor trends in the spread of monkeypox can strengthen the surveillance of this emerging disease while overcoming the challenge of information access for public health officials. Among all specimen types with positive test results used for diagnosing MPXV in Europe, urine samples account for 1.4% of total samples by August 10, 2022 \u003csup\u003e10\u003c/sup\u003e. The presence of MPXV DNA in fecal samples (i.e. in gastrointestinal samples) has been also confirmed \u003csup\u003e11\u003c/sup\u003e. Discharge of MPXV during showering from the exposed skin lesions is known \u003csup\u003e12\u003c/sup\u003e, another plausible source of MPXV in wastewater. \u0026nbsp;Thus, the probability of detecting MPXV in wastewater is relatively high, also considering relative high stability of orthopoxvirus (OPV) in environmental samples such as storm water and soil \u0026nbsp;\u003csup\u003e13\u003c/sup\u003e. With the monkeypox outbreak, genome sequencing of clinical samples began immediately to understand the evolution and genetic diversity of the virus and to find answers to current human-to-human transmission. However, genomic analysis of MPXV in wastewater samples, similar to SARS-CoV-2, is challenging due to the presence of inhibitors, contamination with other genomic material, and especially the very low concentration of the target material \u003csup\u003e14,15\u003c/sup\u003e. Thus, we set out for rapid implementation of a robust wastewater surveillance system for monitoring MPXV, using dPCR and next-generation sequencing (NGS), while using a currently established SARS-CoV-2 monitoring approach.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eDetection of MPXV in wastewater while few clinical cases are known.\u003c/strong\u003e We developed and applied an amplicon-sequencing panel, named as “P4Mpox22” (details in Supplementary, Data S1), to detect MPXV in wastewater samples even in very low concentrations, for example when only a handful of clinical cases are reported to the public health authorities. Since early May 2022, we have analyzed wastewater samples from ten German cities (Table S1 for more information about the cities) for MPXV with digital PCR (dPCR) using the commercially available assay and our NGS amplicon panel. Overall in Germany, the first two cases were reported in week 20 \u003csup\u003e2\u003c/sup\u003e, while in the federal state of Hessen in Germany, the first two cases were reported in week 21 (Fig. S1). We found MPXV already in wastewater samples from week 19 in two cities (i.e. Giessen: GI and Darmstadt: DA) (Fig.1). Among the 10 German cities, we have observed a quadruple increase from week 19 to week 23 in the samples from GI, whereas, in wastewater samples from Biebrich (BR) and Giresheim (GR) the MXPXV concentration increased two-fold after week 27 to week 32 (Fig.1). In Hanau (HU) samples an increase in signal from week 23 to 27 (up to 0.6 gene copies/µL), which is not detectable anymore in week 32. A similar trend was seen for the Kassel (KA) samples, but in a lower concentration range (\u0026lt;0.2 gene copies/µL). And, in Fulda (FU), Niederrad (NR), Sindlingen (SL) and Wiesbaden (WI) samples MPXV was not detectable using dPCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome amplicon sequencing of MPXV in wastewater.\u0026nbsp;\u003c/strong\u003eWe applied the NGS panel described in this study to successfully sequence and partially reconstruct MPXV genomes from sewage samples, limiting to samples with positive signal for dPCR analysis (Fig. S2). The performance of the P4Mpox22 Panel was checked using the MPXV DNA obtained from virus isolated from skin pustules of the first German case in Munich \u003csup\u003e2\u003c/sup\u003e, as positive control. \u0026nbsp;The panel allowed to achieve \u0026gt;2M mapped reads with average read identity of \u0026gt;99% to our reference genome hMpxV/Belgium/UZ_Rega_1/2022 (GISAID accessionID - EPI_ISL_13052282) which was used to design the panel, and average base coverage depth of 1800 bp. Coverage overview of the control MPXV DNA is shown in Fig. S3. For wastewater samples, with an increase in reported clinical cases from week 19 to 32, the percentage of reference genome bases covered also increased from 0.14% in week 19 to 89.5% in week 32 (Fig. 2A, Fig. S4, Fig. S5). While the total reported clinical cases in Hesse increased to 9 in week 27, the genome coverage increased to around 40% in comparison to \u0026lt;4% genome coverage in week 23 when no cases were reported (Fig. 2A and B). \u0026nbsp;As the total number of cases in Hesse increased in week 32, the DNA concentration and thus length of fragments recovered from the wastewater sequences also increased. (Fig. 2C). For week 32, we sequenced sample from two cities (i.e. BR and GR). During week 32, only one case was reported in BR district and 12 cases in GR district (Fig S4). \u0026nbsp;A significant increase in fragment length was observed in the samples from week 32, the median fragment length was 1000 bp and maximum up to 7500 bp (Fig 2C).\u003c/p\u003e\n\u003cp\u003eHigh genome coverage of samples from week 32 (Fig. 2A, Fig. S5), allowed us to construct consensus genomes which represent predominant MPXV strain circulating in the respective sewersheds at this timepoint. \u0026nbsp;We looked at the alignment of the consensus genome sequences to a set of clinical MPXV sequences released in the National Center for Biotechnology Information (NCBI) before August 10, 2022. Whole-genome alignment demonstrated higher sequence similarity with the 2022 outbreak-related MPXV genome sequences (Fig. 3A). Subsequently, the FR and BR genome sequences clustered together with 2022 outbreak-related MPXV cluster (lineage B.1) \u003csup\u003e16\u003c/sup\u003e, tightly placed together with lineage B.1 sequences from Germany (Fig. 3B). The lineage B.1 cluster formed a divergent branch from a genome sequence from USA 2021 (lineage A.1.1) \u003csup\u003e16\u003c/sup\u003e descendant from 2018 MPXV cluster. Although the quality of genome sequences from other wastewater samples was very low, we looked at their placement together with BR and GR sequences from week 32. Figure 3C shows the impact of the genome quality: more fragmented genomes from samples with less MPXV DNA concentration (Fig.1) tend to cluster together and farther away from the genomes of week 32. Nevertheless, low-quality genomes also partially aligned with clinical MPXV sequences (Fig. S6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetecting hyper-mutation signature of 2022 MPXV (lineage B.1) in wastewater.\u003c/strong\u003e The 2022-outbreak MPXV genome sequences have been associated with a specific mode of mutation which is ascribed to the action of apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) enzymes\u003csup\u003e17\u003c/sup\u003e, specifically referring to deamination of cytosine to uracil (C-to-U) by APOBEC3 \u003csup\u003e18\u003c/sup\u003e. In clinical samples, GA\u0026gt;AA and TC\u0026gt;TT hypermutations have been reported as APOBEC3-type signature mutations \u003csup\u003e17\u003c/sup\u003e. Therefore, we also screened the wastewater sample sequences for GA\u0026gt;AA and TC\u0026gt;TT signature mutations using MPXV-UK_P2, 2018 (GenBank accession no. MT903344.1) as a reference sequence similar to a previous study \u003csup\u003e17\u003c/sup\u003e, to determine whether hyper-mutation signatures of 2022 MPXV (lineage B.1) can be detected in wastewater samples. However, we report only mutations with 100% allele frequency. Figure 4A emphasizes the impact of target base coverage evidently, lack of dense cluster of GA\u0026gt;AA and TC\u0026gt;TT mutation for samples from week 19 to 27, with maximum of 26 mutations for HU_27, in comparison to sample BR and GR from week 32. As the quality of target base coverage is important for reliable GA\u0026gt;AA and TC\u0026gt;TT hypermutation analysis, only samples BR_32 and GR_32 were further analyzed. We found clusters of GA\u0026gt;AA and TC\u0026gt;TT across the entire genome in those wastewater samples (Fig.4B). Distribution of the observed mutation cluster across the genome is also seen in clinical sequences from the current MPXV outbreak \u003csup\u003e17\u003c/sup\u003e. Although multiple mutation clusters were spread across the genome in the wastewater samples, most mutations were found in genomic regions: 3000-5000 bp and 120000-150000 bp (Fig. 4C). In genome sequences from clinical samples in Germany, the mutations were also concentrated in multiple genomic regions, including 30000-50000 bp and 120000-150000 bp (Fig. 4D). To achieve better confidence in the wastewater samples, we compared signature motifs (which included five nucleotides before and after the mutation against reference sequence, MT903344.1, found in global clinical sequences\u0026nbsp;with all 12 wastewater samples. For six out of these 12 wastewater samples, we found a match for motifs in the global clinical sequences and the maximum number of matches was observed for sample GR_32 (Fig. 4E), also emphasizing the impact of genome coverage from \u0026nbsp;wastewater samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChallenges with PCR based MPXV surveillance.\u003c/strong\u003e The 196,858-bp MPXV genome has 190 open reading frames containing at least 60 amino acid residues each \u003csup\u003e19\u003c/sup\u003e. However, it seems that just 11 genes have been used as likely targets for PCR analysis. The choice of target for the specific detection of MPXV is crucial but also challenging. While highly similar sequences lead to nonspecific primer/probe binding, highly divergent or variable sequence regions pose considerable challenges for the detection \u003csup\u003e19,20\u003c/sup\u003e. \u0026nbsp;In this study, we also observed that detecting MPXV in wastewater using dPCR, which target specific region, was less sensitive, while it was possible to detect MPXV genomes in wastewater using amplicon sequencing approach by targeting whole genome using multiple primers.\u003c/p\u003e\n\u003cp\u003eIn this study, we were able to detect the MPXV in the state Hesse in sewage of different city districts and counties by digital PCR as well as by NGS, while a handful of cases have been reported. This demonstrates the successful and rapid adaption of an established virus detection and NGS based sequencing pipeline to a new approach and underlines the benefits/necessity of including wastewater-based monitoring into the national surveillance strategy of public health relevant pathogens. Wastewater surveillance comes of age, starting from poliovirus early warning systems to SARS-CoV-2 monitoring at present. SARS-CoV-2 and monkeypox are very dissimilar viruses, not only by their biological properties: one is a never-seen-before RNA virus and the other one is a DNA virus known for decades with zoonotic transmission endemic to Sub-Saharan Africa. Yet, there are more unknowns than knowns during the early days of worldwide outbreaks (\u003cem\u003e21\u003c/em\u003e). WBE has proven a viable tool during the COVID-19 pandemic to monitor viral loads and complement other surveillance systems. Furthermore, it can be used in the deceleration as well as the interpandemic phase with sampling strategies prioritized to transmission nodes or sentinel sites (\u003cem\u003e22\u003c/em\u003e). With special regards to new emerging viral pathogens such as MPXV or currently re-emerging poliovirus in already polio-free regions (\u003cem\u003e23\u003c/em\u003e), we emphasize continuing to use and improve wastewater-based monitoring of public health relevant infectious agents.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eSampling and Extraction\u003c/p\u003e\n\u003cp\u003e200 ml of a 24h-proportional influent wastewater samples were collected every first Tuesday of the month from May till August from 10 wastewater treatment plants (S.Table1). The samples were concentrated by ultrafiltration in 100 kDa Centricon® Plus-70 centrifugal ultrafilters (Merck) to 5 ml of concentrate and DNA was extracted from the concentrate using the Ultra Microbiome kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003edPCR analysis\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eFor quantification of the monkeypox virus (MPXV) in wastewater samples, we used commercially available assay from Thermo Fisher Scientific. The analysis was performed using AbsoluteQ (Thermo Fisher Scientific). 9µl of total master mix was prepared for each sample and was loaded in the QuantStudio Absolute Q MAP16 Digital PCR Plate (Thermo Fisher Scientific). 9µl of total master mix consist of 1.8µl Absolute Q DNA Digital PCR Master Mix (5X); 0.45µl of target assay; 1.75µl of NFW; and 5 µl of DNA template. The thermal profile used for the quantification was 96°C for 5 min.; 45 cycles of 96°C for 5 sec followed by 62°C for 30 sec.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eNext-generation sequencing (NGS) panel design and analysis\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWe designed AmpliSeq™ custom panel- named as “P4Mpox22 Panel”. This panel was developed using the 2022 outbreak clinical MPXV genome sequence (GISAID accessionID - EPI_ISL_13052282). This panel consist of 958 amplicons, with amplicon size ranging 125 - 275 bp. To achieve higher specificity and reduction in primer interferences, primers were divided into two-pool system. Pool 1 consist of 480 primes, and pool 2 has 478 primers. The design BED file, as an auxillary file (Data S1), is also provided for people looking for synthesizing the NGS panel.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe performed library preparation, using the P4Mpox22 panel, on Ion Chef (Thermo Fisher Scientific) using the DL8 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The library preparation allowed barcoded amplicons for each sample. Libraries were multiplexed and sequenced using an Ion Torrent 550 chip on an Ion S5 sequencer (Thermo Fisher Scientific). The sequences were mapped against reference genome sequence (GISAID accessionID - EPI_ISL_13052282) and reads with minimum 100 bp length and at least 50 bp alignment to the reference sequence were retained for further analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eVariant caller, consensus genome construction and Hypermutation analysis\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eFor SNP calling, we used mpileup \u003cem\u003e(24)\u003c/em\u003e and VarScan \u003cem\u003e(25)\u003c/em\u003e at default setting. Consensus genome sequences were called using the ivar consensus \u003cem\u003e(26)\u003c/em\u003e using parameters: Minimum quality score threshold to count base – 20, minimum read depth - 10.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor hypermutation analysis, we screened filtered wastewater sample sequences for GA\u0026gt;AA and TC\u0026gt;TT signature mutations using MPXV-UK_ P2, 2018 (GenBank accession no. MT903344.1) as a reference sequence, similar to a previous study \u003cem\u003e(17)\u003c/em\u003e. \u0026nbsp;The graphs were constructed in RStudio Server (build 554) in R environment (v 4.2.1) using ggplot2_3.3.6, forcats_0.5., GenomicRanges_1.48.0, ggpubr_0.4.0, and hrbrthemes_0.8.0 packages.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePhylogenetic reconstruction and placement of sewage-derived consensus sequences\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWe generated a multiple sequence alignment via MAFFT v7.455 \u003cem\u003e(27)\u0026nbsp;\u003c/em\u003eand default parameters for a selection of input sequences comprising a) genome sequences from former outbreaks, b) sequences from clinical sampling of the 2022 hMPXV outbreak from different countries with a focus on Germany, and c) the reconstructed consensus sequences for the sewage samples obtained in week 29 (GR_32, BR_32). We only used sewage sequences from week 29 in this initial alignment to reduce alignment bias and potential long branch attractions in the final phylogeny caused by the highly fragmented consensus genomes especially derived from sewage samples with low DNA concentrations at earlier sampling dates. We plotted an overview figure of this initial MSA via CIAlign v1.0.18 \u003cem\u003e(28)\u003c/em\u003e using default parameters and reconstructed a phylogenetic tree with IQ-TREE v2.2.0.3 \u003cem\u003e(29)\u003c/em\u003e and 1000 ultra-fast bootstraps and the genome sequence from Nigeria 1971 as defined outgroup. We used Newick Utilities v1.6 \u003cem\u003e(^30)\u003c/em\u003e to visualize the tree. We additionally calculated an MSA and tree using the same commands focusing only on the sewage-derived consensus sequences.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We gratefully acknowledge the contribution from the originating laboratories responsible for obtaining the specimens and the submitting laboratories where genetic sequence data were generated and shared via the GISAID Initiative (https://www.gisaid.org). We thank all WWTP operators for providing wastewater samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJJB Medical Biological Defense Research Program of the Bundeswehr Medical Service\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: SA, LO\u003c/p\u003e\n\u003cp\u003eMethodology: SA, LO, RG, MH, SW\u003c/p\u003e\n\u003cp\u003eInvestigation: SA, MH, LO, RG, SW\u003c/p\u003e\n\u003cp\u003eVisualization: SA, MH, LO\u003c/p\u003e\n\u003cp\u003eFunding acquisition: SL, SA\u003c/p\u003e\n\u003cp\u003eProject administration: SL, LO, SA\u003c/p\u003e\n\u003cp\u003eSupervision: LO, SA\u003c/p\u003e\n\u003cp\u003eWriting – original draft: SA, LO, MH\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing: MH, SA, SL, SB, JJB, AN, AB\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e All data are available in the main text or the supplementary materials. The NGS panel design Bed file is provided as an auxillary file “Data S1”. Raw metagenomic sequence data are available from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under Submission ID SUB11975131, BioProject number PRJNA874069.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLadnyj, I. D., Ziegler, P. \u0026amp; Kima, E. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. \u003cem\u003eBull World Health Organ\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 593\u0026ndash;597 (1972).\u003c/li\u003e\n\u003cli\u003eNoe, S. \u003cem\u003eet al.\u003c/em\u003e Clinical and virological features of first human monkeypox cases in Germany. \u003cem\u003eInfection\u003c/em\u003e (2022) doi:10.1007/s15010-022-01874-z.\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. 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Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination. \u003cem\u003eNat Rev Genet\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 505\u0026ndash;518 (2022).\u003c/li\u003e\n\u003cli\u003eShchelkunov, S. N. \u003cem\u003eet al.\u003c/em\u003e Analysis of the Monkeypox Virus Genome. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e297\u003c/strong\u003e, 172\u0026ndash;194 (2002).\u003c/li\u003e\n\u003cli\u003eHammarlund, E. \u003cem\u003eet al.\u003c/em\u003e Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox. \u003cem\u003eNat Med\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 1005\u0026ndash;1011 (2005).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2350648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2350648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFear of stigma poses a challenge in tracking the 2022-outbreak of monkeypox virus (MPXV) infection. Patients shed MPXV via skin lesions, gastrointestinal route, and seminal fluids into the wastewater. Monitoring MPXV in wastewater can support tracking transmission. We developed a sensitive NGS panel “P4Mpox22”, to track MPXV in sewage. Since May 2022, we have monitored the sewage of German cities using P4Mpox22 and dPCR to quantify MPXV. Despite only 12 clinical cases reported in the respective sewersheds, we obtained up to 90% MPXV genome coverage. Sewage-derived MPXV genomes cluster with lineage B.1 and exhibit APOBEC-type hypermutations. Using dPCR, we quantified MPXV even in sewersheds with only one clinical case. We show that MPXV sewage monitoring using existing SARS-CoV-2 wastewater surveillance infrastructure could be implemented immediately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne-Sentence Summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman monkeypox virus sequencing in wastewater enables community-wide surveillance while overcoming stigmatization challenges\u003c/p\u003e","manuscriptTitle":"Going beyond SARS-CoV-2: genomic surveillance of monkeypox in German wastewater","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 19:39:35","doi":"10.21203/rs.3.rs-2350648/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b94f58bc-4df3-49f0-8b76-182b87c68685","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":18619422,"name":"Biological sciences/Microbiology/Environmental microbiology/Water microbiology"},{"id":18619423,"name":"Health sciences/Health care/Public health/Epidemiology"}],"tags":[],"updatedAt":"2025-06-18T19:39:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 19:39:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2350648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2350648","identity":"rs-2350648","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}