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Assessing the presence of viruses in large-volume samples involves cumbersome methods that require specialized training and laboratory equipment. In this study, a large volume concentration (LVC) method, based on dead-end ultrafiltration (DEUF) and Wet Foam Elution™ technology, was evaluated in different type of waters and different microorganisms. Its recovery efficiency was evaluated through different techniques (infectivity assays and molecular detection) by spiking different viral surrogates (bacteriophages PhiX174 and MS2 and Coxsackie virus B5 (CVB5) and Escherichia coli (E. coli). Furthermore, the application of a secondary concentration step was evaluated and compared with skimmed milk flocculation. Viruses present in river water, seawater and groundwater samples were concentrated by applying LVC method and a centrifugal ultrafiltration device (CeUF), as a secondary concentration step and quantified with specific qPCR Human adenoviruses (HAdV) and noroviruses (NoVs). MS2 was used as process control, obtaining a mean viral recovery of 22.0 ± 12.47%. The presence of other viruses was also characterized by applying two different next-generation sequencing approaches. LVC coupled to a secondary concentration step based on CeUF allowed to detect naturally occurring viruses such as HAdV and NoVs in different water matrices. Using HAdV as a human fecal indicator, the highest viral pollution was found in river water samples (100% of positive samples), followed by seawater (83.33%) and groundwater samples (66.67%). The LVC method has also proven to be useful as a virus concentration method in the filed since HAdV and NoVs were detected in the river water and groundwater samples concentrated in the field. All in all, LVC method presents high concentration factor and a low limit of detection and provides viral concentrates useful for subsequent molecular analysis such as PCR and massive sequencing. ; This study was partly supported by grant PCI2019-103643, RTI2018-097346-B-I00 and AGL2017-86797-C2-1-R AGL funded by MCIN/AEI/10.13039/501100011033/FEDER "A way of making Europe", by the European Union. Sílvia Bofill-Mas is a Serra-Hunter Fellow at the University of Barcelona. Eva Forés is an APIF fellow at the University of Barcelona. Marta Itarte is a fellow of the Catalan Government "AGAUR" (FI) at the University of Barcelona. We thank Rosina Girones for her scientific assessment and Mauricio Córdova for his collaboration in the application of the method in situ. ; Peer reviewed

SARS-CoV-2 variants are emerging worldwide, and monitoring them is key in providing early warnings. Here, we summarize the different analytical approaches currently used to study the dissemination of SARS-CoV-2 variants in wastewater and discuss their advantages and disadvantages. We also provide preliminary results of two sensitive and cost-effective approaches: variant-specific reverse transcription-nested PCR assays and a nonvariant-specific amplicon deep sequencing strategy that targets three key regions of the viral spike protein. Next-generation sequencing approaches enable the simultaneous detection of signature mutations of different variants of concern in a single assay and may be the best option to explore the real picture at a particular time. Targeted PCR approaches focused on specific signature mutations will need continuous updating but are sensitive and cost-effective. ; This study was partially supported by the Catalan Government through 2020PANDE00044, and CDTI project PROCEED, EXP - 00132149/COI-20201289. Sílvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. Eva Forés is an APIF fellow at the University of Barcelona. Marta Itarte is a fellow of the Catalan Government 'AGAUR' (FI) at the University of Barcelona. We also gratefully acknowledge the authors, originating and submitting laboratories of the SARS-CoV-2 sequences from GISAID's EpiCov Database (https://www.epicov.org/epi3/) used in the phylogenetic analyses; access to the individual isolates is facilitated through the GISAID website (https://www.gisaid.org/). Graphical Abstract was created with Biorender.com. ; Peer reviewed

Quantitative measurements of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw wastewater have been implemented worldwide since the beginning of the pandemic. Recent efforts are being made to evaluate different viral concentration methodologies to overcome supplier shortages during lockdowns. A set of 22-wastewater samples seeded with murine hepatitis virus (MHV), a member of the Coronaviridae family, and the bacteriophage MS2, were used to characterize and compare two ultrafiltration-based methods: a centrifugal ultrafiltration device (Centricon® Plus-70) and the automated concentrating pipette CP-Select™. Based on the recovery efficiencies, significant differences were observed for MHV, with Centricon® Plus-70 (24%) being the most efficient method. Nevertheless, concentrations of naturally occurring SARS-CoV-2, Human adenoviruses and JC polyomaviruses in these samples did not result in significant differences between methods suggesting that testing naturally occurring viruses may complement the evaluation of viral concentration methodologies. Based on the virus adsorption to solids and the necessity of a pre-centrifugation step to remove larger particles and avoid clogging when using ultrafiltration methods, we assessed the percentage of viruses not quantified after ultrafiltration. Around 23% of the detected SARS-CoV-2 would be discarded during the debris removal step. The CP-Select™ provided the highest concentration factor (up to 333×) and the lowest LoD (6.19 × 103 GC/l) for MHV and proved to be fast, automatic, highly reproducible and suitable to work under BSL-2 measures. ; This work was partially funded by the projects PCI2019-103643, RTI2018-097346-B-I00, and AGL2017-86797-C2-1-R projects, all funded by the Spanish Ministry of Science, Innovation and Universities. S. Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. This study is performed with partial support from the Water Research Institute of the University of Barcelona. Graphical abstract was created with Biorender.com. ICRA authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124). ICRA is part of the CERCA program. We would like to thank Dr. Zammit for his critical revision of the manuscript. ; Peer reviewed