Wastewater-based epidemiology applied at the building-level reveals distinct virome profiles based on the age of the contributing individuals.

BACKGROUND: Human viruses released into the environment can be detected and characterized in wastewater. The study of wastewater virome offers a consolidated perspective on the circulation of viruses within a population. Because the occurrence and severity of viral infections can vary across a person's lifetime, studying the virome in wastewater samples contributed by various demographic segments can provide valuable insights into the prevalence of viral infections within these segments. In our study, targeted enrichment sequencing was employed to characterize the human virome in wastewater at a building-level scale. This was accomplished through passive sampling of wastewater in schools, university settings, and nursing homes in two cities in Catalonia. Additionally, sewage from a large urban wastewater treatment plant was analysed to serve as a reference for examining the collective excreted human virome. RESULTS: The virome obtained from influent wastewater treatment plant samples showcased the combined viral presence from individuals of varying ages, with astroviruses and human bocaviruses being the most prevalent, followed by human adenoviruses, polyomaviruses, and papillomaviruses. Significant variations in the viral profiles were observed among the different types of buildings studied. Mamastrovirus 1 was predominant in school samples, salivirus and human polyomaviruses JC and BK in the university settings while nursing homes showed a more balanced distribution of viral families presenting papillomavirus and picornaviruses and, interestingly, some viruses linked to immunosuppression. CONCLUSIONS: This study shows the utility of building-level wastewater-based epidemiology as an effective tool for monitoring the presence of viruses circulating within specific age groups. It provides valuable insights for public health monitoring and epidemiological studies.

Phylodynamics of Merkel-cell polyomavirus and human polyomavirus 6: A long-term history with humans.

New human polyomaviruses have been described in the last years, including the Merkel-cell polyomavirus (MCPyV; Human polyomavirus 5) and the Human polyomavirus 6 (HPyV6). Although their infection is usually asymptomatic, in immunocompromised host can cause life-threatening pathologies, such as the Merkel cell carcinoma, an aggressive skin neoplasia associated to the MCPyV. Despite being prevalent viruses in population, epidemiological data from South America are scarce, as well as the characterization of the viral types circulating and their origin. The aims of this work were to describe MCPyV and HPyV6 from environmental samples with different geographical origin and to analyze their phylogenetic and evolutionary histories, particularly for MCPyV. Partial and complete genome sequences were obtained from sewage samples from Argentina, Uruguay and Spain. A total number of 87 sequences were obtained for MCPyV and 33 for HPyV6. Phylogenetic analysis showed that MCPyV sequences distributed according to their geographic origin in Europe/North America, Africa, Asia, South America and Oceania groups, suggesting that viral diversification might have followed human migrations across the globe. In particular, viruses from Argentina associated with Europe/North America and South America genotypes, whereas those from Uruguay and Spain also grouped with Africa genotype, reflecting the origin of the current population in each country, which could arrive not only during ancient human migration but also during recent migratory events. In addition, the South American group presented a high level of clusterization, showing internal clusters that could be related to specific locations, such as French Guiana and Brazil or the Southern region into South America, such as Argentina and Uruguay, suggesting a long term evolutionary process in the region. Additionally, in this work, we carried out the first analysis about the evolutionary history of MCPyV trough the integration of phylogenetic, epidemiological and historical data. Since a strong association is observed between the phylogenetic relationships and the origin of the sampled population, this analysis was based on the hypothesis of co-divergence between the virus and human populations. This analysis resulted in a substitution rate of 5.1 × 10-8 s/s/y (∼5.1% of divergence per million years) for the complete genome of MCPyV, which is in the range of those estimated for other double-stranded DNA viruses. Regarding HPyV6, a South American group with clusterization was observed (sequences from Uruguay). Meanwhile, sequences from Argentina grouped with European ones (France and Spain) and remained separated from those isolated in China, USA or Australia. The analysis of viruses from the environment allowed us to deep characterize prevalent infections in different geographic regions, reveling that viruses circulating in each population reflected its origin and that there are specific lineages associated with South America.

Evidence of viral dissemination and seasonality in a Mediterranean river catchment: Implications for water pollution management.

Conventional wastewater treatment does not completely remove and/or inactive viruses; consequently, viruses excreted by the population can be detected in the environment. This study was undertaken to investigate the distribution and seasonality of human viruses and faecal indicator bacteria (FIB) in a river catchment located in a typical Mediterranean climate region and to discuss future trends in relation to climate change. Sample matrices included river water, untreated and treated wastewater from a wastewater treatment plant within the catchment area, and seawater from potentially impacted bathing water. Five viruses were analysed in the study. Human adenovirus (HAdV) and JC polyomavirus (JCPyV) were analysed as indicators of human faecal contamination of human pathogens; both were reported in urban wastewater (mean values of 10(6) and 10(5) GC/L, respectively), river water (10(3) and 10(2) GC/L) and seawater (10(2) and 10(1) GC/L). Human Merkel Cell polyomavirus (MCPyV), which is associated with Merkel Cell carcinoma, was detected in 75% of the raw wastewater samples (31/37) and quantified by a newly developed quantitative polymerase chain reaction (qPCR) assay with mean concentrations of 10(4) GC/L. This virus is related to skin cancer in susceptible individuals and was found in 29% and 18% of river water and seawater samples, respectively. Seasonality was only observed for norovirus genogroup II (NoV GGII), which was more abundant in cold months with levels up to 10(4) GC/L in river water. Human hepatitis E virus (HEV) was detected in 13.5% of the wastewater samples when analysed by nested PCR (nPCR). Secondary biological treatment (i.e., activated sludge) and tertiary sewage disinfection including chlorination, flocculation and UV radiation removed between 2.22 and 4.52 log10 of the viral concentrations. Climate projections for the Mediterranean climate areas and the selected river catchment estimate general warming and changes in precipitation distribution. Persistent decreases in precipitation during summer can lead to a higher presence of human viruses because river and sea water present the highest viral concentrations during warmer months. In a global context, wastewater management will be the key to preventing environmental dispersion of human faecal pathogens in future climate change scenarios.

Treating progressive multifocal leukoencephalopathy with interleukin 7 and vaccination with JC virus capsid protein VP1.

Progressive multifocal leukoencephalopathy is a currently untreatable infection of the brain. Here, we demonstrate in 2 patients that treatment with interleukin 7, JC polyomavirus (JCV) capsid protein VP1, and a Toll-like receptor 7 agonist used as adjuvant, was well tolerated, and showed a very favorable safety profile and unexpected efficacy that warrant further investigation.

T cell epitope mapping of JC polyoma virus-encoded proteome reveals reduced T cell responses in HLA-DRB1*04:01+ donors.

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4(+) T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4(+) T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01(+), DRB1*07(+), DRB1*11(+), DRB1*13(+), DRB1*15(+), and DRB1*03(+) individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01(+) individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.

Exploring viral contamination in urban groundwater and runoff.

The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as sewer leakage or urban runoff. However, there is insufficient monitoring of groundwater quality in urban areas. In this study, quantitative polymerase chain reaction (qPCR) was employed to evaluate the presence of human fecal viral indicators and viral pathogens in urban wastewater (n = 13) and groundwater (n = 12) samples from four locations in Barcelona with different degrees of urbanization, as well as in runoff samples (n = 2). Additionally, a target enrichment sequencing (TES) approach was utilized to explore the viral diversity within groundwater and runoff samples, offering insights into viral contamination and potential virus transmission routes in urban areas. Human adenovirus (HAdV) was identified in all wastewater samples, 67 % (8/12) of groundwater samples, and one runoff sample by qPCR indicating human viral fecal contamination. The viral pathogen Norovirus genogroup GI (NoV GI) was detected in wastewater and two winter groundwater samples from highly and medium urbanized areas. NoV genogroup GII (NoV GII), Enterovirus (EV) and SARS-CoV-2 were exclusively detected in wastewater. Human and other vertebrate viruses were detected in groundwater and runoff samples using TES. This study gives insights about the virome present in urban water sources, emphasizing the need for thorough monitoring and deeper understanding to address emerging public health concerns.

Exploring waterborne viruses in groundwater: Quantification and Virome characterization via passive sampling and targeted enrichment sequencing.

Aquifers, which provide drinking water for nearly half the world's population, face significant challenges from microbial contamination, particularly from waterborne viruses such as human adenovirus (HAdV), norovirus (NoV) and enterovirus (EV). This study, conducted as part of the UPWATER project, investigates the sources of urban groundwater contamination using viral passive sampling (VPS) and target enrichment sequencing (TES). We assessed the abundance of eight viral pathogens (HAdV, EV, NoV genogroup I and II, rotavirus, influenza A virus, hepatitis E virus and SARS-CoV-2) and investigated the virome diversity of groundwater in the aquifer of the Besòs River Delta in Catalonia. Over a period of 7 months, we collected 114 samples from the aquifer using nylon and nitrocellulose membranes to adsorb viruses over a 10-day period. Human faecal contamination was detected in nearly 50 % of the groundwater samples, with mean HAdV total counts ranging from 1.23E+02 to 3.66E+03 GC, and occasional detections of EV and NoV GI and GII. In addition, deep sequencing revealed a diverse virome in the aquifer, with detection of human pathogens, including adenovirus, astrovirus, calicivirus, enterovirus, herpesvirus, papillomavirus and rotavirus. Time-integrated sampling using VPS increases the likelihood of virus detection and, when combined with TES, can provide a deeper understanding of virus prevalence in this important water compartment. This approach is expected to streamline long-term monitoring efforts and enable small communities or water managers with limited resources to effectively manage their groundwater reservoirs.

CAPTVRED: an automated pipeline for viral tracking and discovery from capture-based metagenomics samples.

Summary: Target Enrichment Sequencing or Capture-based metagenomics has emerged as an approach of interest for viral metagenomics in complex samples. However, these datasets are usually analyzed with standard downstream Bioinformatics analyses. CAPTVRED (Capture-based metagenomics Analysis Pipeline for tracking ViRal species from Environmental Datasets), has been designed to assess the virome present in complex samples, specially focused on those obtained by Target Enrichment Sequencing approach. This work aims to provide a user-friendly tool that complements this sequencing approach for the total or partial virome description, especially from environmental matrices. It includes a setup module which allows preparation and adjustment of the pipeline to any capture panel directed to a set of species of interest. The tool also aims to reduce time and computational cost, as well as to provide comprehensive, reproducible, and accessible results while being easy to costume, set up, and install. Availability and implementation: Source code and test datasets are freely available at github repository: https://github.com/CompGenLabUB/CAPTVRED.git.

Assessing environmental exposure to viruses in wastewater treatment plant and swine farm scenarios with next-generation sequencing and occupational risk approaches.

Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact. HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae. QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus. This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.

Target enrichment metaviromics enables comprehensive surveillance of coronaviruses in environmental and animal samples.

The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples. Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization. In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.

Surveillance of SARS-CoV-2 in sewage from buildings housing residents with different vulnerability levels.

During the last three years, various restrictions have been set up to limit the transmission of the Coronavirus Disease (COVID-19). While these rules apply at a large scale (e.g., country-wide level) human-to-human transmission of the virus that causes COVID-19, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), occurs at a small scale. Different preventive policies and testing protocols were implemented in buildings where COVID-19 poses a threat (e.g., elderly residences) or constitutes a disruptive force (e.g., schools). In this study, we sampled sewage from different buildings (a school, a university campus, a university residence, and an elderly residence) that host residents of different levels of vulnerability. Our main goal was to assess the agreement between the SARS-CoV-2 concentration in wastewater and the policies applied in these buildings. All buildings were sampled using passive samplers while 24 h composite samples were also collected from the elderly residence. Results showed that passive samplers performed comparably well to composite samples while being cost-effective to keep track of COVID-19 prevalence. In the elderly residence, the comparison of sampling protocols (passive vs. active) combined with the strict clinical testing allowed us to compare the sensitivities of the two methods. Active sampling was more sensitive than passive sampling, as the former was able to detect a COVID-19 prevalence of 0.4 %, compared to a prevalence of 2.2 % for passive sampling. The number of COVID-19-positive individuals was tracked clinically in all the monitored buildings. More frequent detection of SARS-CoV-2 in wastewater was observed in residential buildings than in non-residential buildings using passive samplers. In all buildings, sewage surveillance can be used to complement COVID-19 clinical testing regimes, as the detection of SARS-CoV-2 in wastewater remained positive even when no COVID-19-positive individuals were reported. Passive sampling is useful for building managers to adapt their COVID-19 mitigation policies.

Comparative inactivation of murine norovirus, human adenovirus, and human JC polyomavirus by chlorine in seawater.

Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log(10) GC reductions and a 2.3- and 2.4-log(10) PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log(10) GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log(10) GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration.

A novel tool for specific detection and quantification of chicken/turkey parvoviruses to trace poultry fecal contamination in the environment.

Poultry farming may introduce pathogens into the environment and food chains. High concentrations of chicken/turkey parvoviruses were detected in chicken stools and slaughterhouse and downstream urban wastewaters by applying new PCR-based specific detection and quantification techniques. Our results confirm that chicken/turkey parvoviruses may be useful viral indicators of poultry fecal contamination.

Central role of JC virus-specific CD4+ lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome.

Progressive multi-focal leucoencephalopathy and progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are caused by infection of the central nervous system with the JC polyoma virus. Both are complications of monoclonal antibody therapy in multiple sclerosis and other autoimmune diseases. Progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome can obscure the diagnosis of progressive multi-focal leucoencephalopathy and lead to severe clinical disability and possibly death. Different from progressive multi-focal leucoencephalopathy, in which demyelination results from oligodendrocyte lysis by JC virus in the absence of an immune response, tissue destruction in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome is caused by a vigorous immune response within the brain. The cells and mediators that are involved in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are as yet poorly understood. We examined two patients with multiple sclerosis, who developed progressive multi-focal leucoencephalopathy and later progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome under natalizumab therapy. Due to initially negative JC viral deoxyribonucleic acid testing in the cerebrospinal fluid, a diagnostic brain biopsy was performed in one patient. Histopathology revealed brain inflammation characterized by a prominent T cell infiltrate (CD4(+)> CD8(+) T cells), but also B/plasma cells and monocytes. Despite very low JC viral load, both patients showed high intrathecal anti-JC virus antibodies. Brain-infiltrating CD4(+) T cells were studied regarding antigen specificity and function. CD4(+) T cells were highly specific for peptides from several JC virus proteins, particularly the major capsid protein VP1. T cell phenotyping revealed CD4(+) Th1 and bifunctional Th1-2 cells. The latter secrete large amounts of interferon-γ and interleukin-4 explaining the strong brain inflammation, presence of plasma cells and secretion of intrathecal anti-VP1 antibodies. The functional phenotype of brain-infiltrating JC virus-specific CD4(+) T cells was confirmed and extended by examining brain-derived JC virus-specific CD4(+) T cell clones. Our data provide novel insight into the pathogenesis of progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome and indicate that JC virus-specific CD4(+) T cells play an important role in both eliminating JC virus from the brain, but also in causing the massive inflammation with often fatal outcome.

Viral metagenomics reveals persistent as well as dietary acquired viruses in Antarctic fur seals.

Viruses linked to animals inhabiting Antarctic latitudes remain poorly studied. Remote environments hosting large pinniped populations may be prone to exposure of immunologically naïve animals to new infectious agents due to increasing human presence or introduction of new animal species. Antarctic fur seals (Arctocephalus gazella) inhabiting the Western Antarctic Peninsula and the South Shetland Islands are challenged because of climate change and increased anthropogenic activity. In the present study, the fecal and serum virome of A. gazella was characterized by applying target enrichment next generation sequencing. The resulting viromes were dominated by CRESS-DNA sequences. Viruses known to infect vertebrate and invertebrate hosts were also observed in fecal samples. Fur seal picornavirus was present in all the fecal pools studied suggesting it is a prevalent virus in these species. Six different viruses presenting similarities with previously described A. gazella viruses or other otariids and mammal viruses were identified as potential new A. gazella viruses. Also, diet-derived viruses such as crustacean viruses were present in fecal content. Penguin viruses, but not fish viruses, were also detected. Obtained results contribute to a better understanding of the viral community present in these species, which is relevant for its conservation.

Evaluation of a virus concentration method based on ultrafiltration and wet foam elution for studying viruses from large-volume water samples.

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.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance.

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses.

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

Looking for a needle in a haystack. SARS-CoV-2 variant characterization in sewage.

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.