Culture of Human Rotaviruses in Relevant Models Shows Differences in Culture-Adapted and Nonculture-Adapted Strains.

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo-blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models.

Sample and library preparation approaches for the analysis of the virome of irrigation water.

BACKGROUND: The virome (i.e. community of mainly RNA and DNA eukaryotic viruses and bacteriophages) of waters is yet to be extensively explored. In particular, the virome of waters used for irrigation could therefore potentially carry viral pathogens that can contaminate fresh produce. One problem in obtaining viral sequences from irrigation waters is the relatively low amount of virus particles, as well as the presence of human, bacterial and protozoan cells. The present aimed study was to compare different processing, amplification, and sequencing approaches for virome characterization in irrigation waters. RESULTS: Our analyses considered percentages of viral reads, values for diversity indices and number of families found in sequencing results. The results obtained suggest that enrichment protocols using two (bezonase and microccocal nuclease) or four enzymes at once (bezonase, microccocal nuclease, DNAse and RNase), regardless of an Amicon filtration step, are more appropriate than separated enzymatic treatments for virome characterization in irrigation water. The NetoVIR protocol combined with the ScriptSeq v2 RNA-Seq Library (P0-L20 protocol) showed the highest percentages of RNA viruses and identified the higher number of families. CONCLUSION: Although virome characterization applied in irrigation waters is an important tool for protecting public health by informing on circulating human and zoonotic infections, optimized and standardized procedures should be followed to reduce the variability of results related to either the sample itself and the downstream bioinformatics analyses. Our results show that virome characterization can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided that appropriate and rigorous controls are included. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR.

The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. These have finally allowed an estimation of viral infectivity. The present study aims to provide a rapid molecular-based protocol for detection and quantification of viable SARS-CoV-2 from fomites based on the discrimination of non-infectious SARS-CoV-2 particles by platinum chloride (IV) (PtCl4) viability RT-qPCR. An initial assessment compared two different swabbing procedures to recover inactivated SARS-CoV-2 particles from fomites coupled with two RNA extraction methods. Procedures were validated with human (E229) and porcine (PEDV) coronavirus surrogates, and compared with inactivated SARS-CoV-2 suspensions on glass, steel and plastic surfaces. The viability RT-qPCR efficiently removed the PCR amplification signals from heat and gamma-irradiated inactivated SARS-CoV-2 suspensions that had been collected from specified surfaces. This study proposes a rapid viability RT-qPCR that discriminates non-infectious SARS-CoV-2 particles on surfaces thus helping researchers to better understand the risk of contracting COVID-19 through contact with fomites and to develop more efficient epidemiological measures.

Polymers and Biopolymers with Antiviral Activity: Potential Applications for Improving Food Safety.

Gastroenteritis and hepatitis, caused by human noroviruses (HuNoVs) and hepatitis A virus (HAV), respectively, are the most common illnesses resulting from the consumption of food contaminated with human enteric viruses. Food-grade polymers can be tailor designed to improve food safety, either as novel food-packaging materials imparting active antimicrobial properties, applied in food contact surfaces to avoid cross-contamination, or as edible coatings to increase fresh produce's shelf life. The incorporation of antimicrobial agents into food-grade polymers can be used to control the food microbiota and even target specific foodborne pathogens to improve microbiological food safety and to enhance food quality. Enteric viruses are responsible for one fifth of acute gastroenteritis cases worldwide and the development of food-grade polymers and biopolymers with antiviral activity for food applications is a topic of increased interest, both for academia and the food industry, even though developments are still limited. This review compiles existing studies in this widely unexplored area and highlights the potential of these developments to improve viral food safety.

Jaminaea phylloscopi sp. nov. (Microstromatales), a basidiomycetous yeast isolated from migratory birds in the Mediterranean basin.

During a survey of yeasts vectored by migratory birds in the Mediterranean basin, isolations from the cloacae of members of the order Passeriformes collected in Ustica (Italy) were performed. Based on phylogenetic analysis of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer ITS1-5.8S rRNA gene-ITS2 region, five yeast isolates clustered in a new lineage within the Microstromatales clade. The DNA sequences of these isolates differed from those of their closest relatives, Jaminaea angkorensis and Jaminaea lanaiensis, by 20 and 25 nt substitutions in the D1/D2 domain and 119 and 131 nt substitutions in the complete ITS region, respectively. In addition, the five isolates showed phenotypic characteristics not observed in their closest relatives, such as the ability to grow at 44 °C and at pH 2.5, which suggests a possible adaptation to the bird gastrointestinal tract. On the basis of the isolation source, phenotypic features and molecular strain typing carried out with randomly amplified polymorphic DNA (RAPD)-PCR and mini-satellite-primed (MSP)-PCR analysis, the five isolates were characterized as five distinct strains of a novel species formally described as Jaminaea phylloscopi sp. nov., with 551B6T ( = PYCC 6783T = CBS 14087T) as the type strain. The Mycobank accession number is MB811984.

Lactococcal 949 group phages recognize a carbohydrate receptor on the host cell surface.

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages.

Transfer, composition and technological characterization of the lactic acid bacterial populations of the wooden vats used to produce traditional stretched cheeses.

The biofilms of 12 wooden vats used for the production of the traditional stretched cheeses Caciocavallo Palermitano and PDO Vastedda della valle del Belìce were investigated. Salmonella spp. and Listeria monocytogenes were never detected. Total coliforms were at low numbers with Escherichia coli found only in three vats. Coagulase-positive staphylococci (CPS) were below the enumeration limit, whereas lactic acid bacteria (LAB) dominated the surfaces of all vats. In general, the dominance was showed by coccus LAB. Enterococci were estimated at high numbers, but usually between 1 and 2 Log cycles lower than other LAB. LAB populations were investigated at species and strain level and for their technological properties relevant in cheese production. Eighty-five strains were analysed by a polyphasic genetic approach and allotted into 16 species within the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. Enterococcus faecium was found in all wooden vats and the species most frequently isolated were Enterococcus faecalis, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus acidilactici and Streptococcus thermophilus. The study of the quantitative data on acidification rate, autolysis kinetics, diacetyl production, antibacterial compound generation and proteolysis by cluster and principal component analysis led to the identification of some strains with promising dairy characteristics. Interestingly, a consistent percentage of LAB was bacteriocin-like inhibitory substances (BLIS) producer. Thus, the microbial biofilms of the wooden vats analysed in this study might contribute actively to the stability of the final cheeses.

Antiviral Activity of Natural Compounds for Food Safety.

Gastroenteritis and hepatitis are the most common illnesses resulting from the consumption of food contaminated with human enteric viruses. Several natural compounds have demonstrated antiviral activity against human enteric viruses, such as human norovirus and hepatitis A virus, while little information is available for hepatitis E virus. Many in-vitro studies have evaluated the efficacy of different natural compounds against human enteric viruses or their surrogates. However, only few studies have investigated their antiviral activity in food applications. Among them, green tea extract, grape seed extract and carrageenans have been extensively investigated as antiviral natural compounds to improve food safety. Indeed, these extracts have been studied as sanitizers on food-contact surfaces, in produce washing solutions, as active fractions in antiviral food-packaging materials, and in edible coatings. The most innovative applications of these antiviral natural extracts include the development of coatings to extend the shelf life of berries or their combination with established food technologies for improved processes. This review summarizes existing knowledge in the underexplored field of natural compounds for enhancing the safety of viral-contaminated foods and underscores the research needs to be covered in the near future.

Challenges for estimating human norovirus infectivity by viability RT-qPCR as compared to replication in human intestinal enteroids.

Viability RT-qPCR, a molecular detection method combining viability marker pre-treatment with RT-qPCR, has been proposed to infer infectivity of viruses which is particularly relevant for non-culturable viruses or sophisticated cell culture systems. Being human noroviruses (HuNoV) most frequently associated with foodborne outbreaks, this study compared different viability techniques and infectivity in human intestinal enteroids (HIE) to ultimately determine whether the molecular approaches could serve as rapid assays to predict HuNoV inactivation in high-risk food. To this end, the performance of three viability RT-qPCR assays with different intercalating markers ((Viability PCR Crosslinker Kit (CL), propidium monoazide (PMAxx™), and platinum chloride (PtCl4)) in estimating survival of HuNoV exposed to thermal and high pressure (HPP) treatments was compared to replication tested in the HIE cell culture model. A nearly full-length genomic molecular assay coupled with PMAxx™ to infer HuNoV thermal inactivation was also assessed. The experimental design included HuNoV genogroup I.3 [P13], GII.4 Sydney [P16], GII.6 [P7], along with Tulane virus (TV) serving as surrogate. Finally, viability RT-qPCR was tested in HPP-treated strawberry puree, selected as a food matrix with high viral contamination risk. PMAxx™ and CL performed evenly, while PtCl4 affected HuNoV infectivity. Taking all experimental data together, viability RT-qPCR was demonstrated to be an improved method over direct RT-qPCR to estimate viral inactivation at extreme thermal (95 °C) and HPP (450 MPa) exposures, but not under milder conditions as amplification signals were detected. Despite its complexity and limitations, the HIE demonstrated a more robust model than viability RT-qPCR to assess HuNoV infectivity.

Leveraging Plasma-Activated Seawater for the Control of Human Norovirus and Bacterial Pathogens in Shellfish Depuration.

Cold plasma is a promising alternative for water treatment owing to pathogen control and a plethora of issues in the agriculture and food sectors. Shellfish pose a serious risk to public health and are linked to large viral and bacterial outbreaks. Hence, current European regulations mandate a depuration step for shellfish on the basis of their geographical growth area. This study investigated the inactivation of relevant viral and bacterial pathogens of three plasma-activated seawaters (PASWs), and their reactive oxygen and nitrogen species (RONS) composition, as being primarily responsible for microbial inactivation. Specifically, F-specific (MS2) and somatic (φ174) bacteriophage, cultivable surrogate (murine norovirus, MNV, and Tulane virus, TV), and human norovirus (HuNoV GII.4) inactivation was determined using plaque counts and infectivity assays, including the novel human intestinal enteroid (HIE) model for HuNoV. Moreover, the kinetic decay of Escherichia coli, Salmonella spp., and Vibrio parahaemolyticus was characterized. The results showed the complete inactivation of phages (6-8 log), surrogates (5-6 log), HuNoV (6 log), and bacterial (6-7 log) pathogens within 24 h while preventing cytotoxicity effects and preserving mussel viability. Nitrites (NO2-) were found to be mostly correlated with microbial decay. This research shows that PASWs are a suitable option to depurate bivalve mollusks and control the biohazard risk linked to their microbiological contamination, either viral or bacterial.

Human intestinal enteroids and predictive models validate the operational limits of sanitizers used for viral disinfection of vegetable process wash water.

Vegetables are globally associated with a considerable number of foodborne outbreaks caused by viral infections, specifically human norovirus. In fresh produce industry, washing represents a critical step for food safety as process wash water (PWW) needs to be maintained at appropriate microbial quality to prevent water-mediated cross-contamination. This study aimed to explore the disinfection efficacy of chlorine (free chlorine, FC), chlorine dioxide (ClO2) and peracetic acid (PAA) in PWW against infectious human norovirus and Tulane virus (TV). First, we tested the extent of TV inactivation in baby leaf, bell pepper, and vegetables mix PWW and monitored the viral decay by cell culture. Then, inactivation kinetics were defined for infectious human norovirus exposed to FC, ClO2 and PAA in baby leaves PWW using the human intestinal enteroids (HIE) system. Finally, kinetic inactivation models were fitted to TV reduction and decay of sanitizers to aid the implementation of disinfection strategies. Results showed that >8 log10 human norovirus and 3.9 log10 TV were inactivated by 20 ppm FC within 1 min; and by 3 ppm ClO2 in 1 min (TV) or 5 min (norovirus). PAA treatment at 80 ppm reduced ca. 2 log10 TV but not completely inactivated the virus even after 20 min exposure, while 5 min treatment prevented norovirus replication in HIE. TV inactivation in PWWs was described using an exponential decay model. Taking these data together, we demonstrated the value of applying the HIE model to validate current operational limits for the most commonly used sanitizers. The inactivation kinetics for human norovirus and TV, along with the predictive model described in this study expand the current knowledge to implement post-harvest produce safety procedures in industry settings.

Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater.

Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples.

The International Virus Bioinformatics Meeting 2023.

The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.

New Challenges for Detection and Control of Foodborne Pathogens: From Tools to People.

Contamination of foods by human pathogenic microorganisms is a major concern to both food safety and public health [...].

Monitoring Human Viral Pathogens Reveals Potential Hazard for Treated Wastewater Discharge or Reuse.

Wastewater discharge to the environment or its reuse after sanitization poses a concern for public health given the risk of transmission of human viral diseases. However, estimating the viral infectivity along the wastewater cycle presents technical challenges and still remains underexplored. Recently, human-associated crAssphage has been investigated to serve as viral pathogen indicator to monitor fecal impacted water bodies, even though its assessment as biomarker for infectious enteric viruses has not been explored yet. To this end, the occurrence of potentially infectious norovirus genogroup I (GI), norovirus GII, hepatitis A virus (HAV), rotavirus A (RV), and human astrovirus (HAstV) along with crAssphage was investigated in influent and effluent water sampled in four wastewater treatment plants (WWTPs) over 1 year by a PMAxx-based capsid integrity RT-qPCR assay. Moreover, influent and effluent samples of a selected WWTP were additionally assayed by an in situ capture RT-qPCR assay (ISC-RT-qPCR) as estimate for viral infectivity in alternative to PMAxx-RT-qPCR. Overall, our results showed lower viral occurrence and concentration assessed by ISC-RT-qPCR than PMAxx-RT-qPCR. Occurrence of potentially infectious enteric virus was estimated by PMAxx-RT-qPCR as 88-94% in influent and 46-67% in effluent wastewaters with mean titers ranging from 4.77 to 5.89, and from 3.86 to 4.97 log10 GC/L, with the exception of HAV that was sporadically detected. All samples tested positive for crAssphage at concentration ranging from 7.41 to 9.99 log10 GC/L in influent and from 4.56 to 6.96 log10 GC/L in effluent wastewater, showing higher mean concentration than targeted enteric viruses. Data obtained by PMAxx-RT-qPCR showed that crAssphage strongly correlated with norovirus GII (ρ = 0.67, p < 0.05) and weakly with HAstV and RV (ρ = 0.25-0.30, p < 0.05) in influent samples. In effluent wastewater, weak (ρ = 0.27-0.38, p < 0.05) to moderate (ρ = 0.47-0.48, p < 0.05) correlations between crAssphage and targeted viruses were observed. Overall, these results corroborate crAssphage as an indicator for fecal contamination in wastewater but a poor marker for either viral occurrence and viral integrity/infectivity. Despite the viral load reductions detected in effluent compared to influent wastewaters, the estimates of viral infectivity based on viability molecular methods might pose a concern for (re)-using of treated water.

Spatial and temporal distribution of SARS-CoV-2 diversity circulating in wastewater.

Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.7 lineage and the ability of the technique to anticipate the detection of certain mutations before they are detected in clinical samples. The study proves the usefulness of sewage sequencing to track Variants of Concern that can complement clinical testing to help in decision-making and in the analysis of the evolution of the pandemic.

SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain.

OBJECTIVES: To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies. DESIGN AND PATIENTS: This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included. SETTING: Seven health centres from different provinces in Spain. MAIN OUTCOME MEASURES: Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)-IgA, IgG and IgM-in breast milk samples from patients with COVID-19. RESULTS: All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation). CONCLUSIONS: Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244.

The International Virus Bioinformatics Meeting 2022.

The International Virus Bioinformatics Meeting 2022 took place online, on 23-25 March 2022, and has attracted about 380 participants from all over the world. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The participants created a highly interactive scientific environment even without physical face-to-face interactions. This meeting is a focal point to gain an insight into the state-of-the-art of the virus bioinformatics research landscape and to interact with researchers in the forefront as well as aspiring young scientists. The meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions. The main topics were: SARS-CoV-2, viral emergence and surveillance, virus-host interactions, viral sequence analysis, virus identification and annotation, phages, and viral diversity. This report summarizes the main research findings and highlights presented at the meeting.

Bias of library preparation for virome characterization in untreated and treated wastewaters.

The use of metagenomics for virome characterization and its implementation for wastewater analyses, including wastewater-based epidemiology, has increased in the last years. However, the lack of standardized methods can led to highly different results. The aim of this work was to analyze virome profiles in upstream and downstream wastewater samples collected from four wastewater treatment plants (WWTPs) using two different library preparation kits. Viral particles were enriched from wastewater concentrates using a filtration and nuclease digestion procedure prior to total nucleic acid (NA) extraction. Sequencing was performed using the ScriptSeq v2 RNA-Seq (LS) and the NEBNext Ultra II RNA (NB) library preparation kits. Cleaned reads and contigs were annotated using a curated in-house database composed by reads assigned to viruses at NCBI. Significant differences in viral families and in the ratio of detection were shown between the two library kits used. The use of LS library showed Virgaviridae, Microviridae and Siphoviridae as the most abundant families; while Ackermannviridae and Helleviridae were highly represented within the NB library. Additionally, the two sequencing libraries produced outcomes that differed in the detection of viral indicators. These results highlighted the importance of library selection for studying viruses in untreated and treated wastewater. Our results underline the need for further studies to elucidate the influence of sequencing procedures in virome profiles in wastewater matrices in order to improve the knowledge of the virome in the water environment.

Comparing analytical methods to detect SARS-CoV-2 in wastewater.

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene glycol (PEG) precipitation protocol. Moreover, two different RNA extraction methods were compared in this study: a commercial manual spin column centrifugation kit and an automated protocol based on magnetic silica beads. Overall, the evaluated concentration methods did not impact the recovery of gamma-irradiated SARS-CoV-2 nor MgV, while extraction methods showed significant differences for PEDV. Mean recovery rates of 42.9 ± 9.5%, 27.5 ± 14.3% and 9.0 ± 2.2% were obtained for gamma-irradiated SARS-CoV-2, PEDV and MgV, respectively. Limits of detection (LoD95%) for five genomic SARS-CoV-2 targets (N1, N2, gene E, IP2 and IP4) ranged from 1.56 log genome equivalents (ge)/mL (N1) to 2.22 log ge/mL (IP4) when automated system was used; while values ranging between 2.08 (N1) and 2.34 (E) log ge/mL were observed when using column-based extraction method. Different targets were also evaluated in naturally contaminated wastewater samples with 91.2%, 85.3%, 70.6%, 79.4% and 73.5% positivity, for N1, N2, E, IP2 and IP4, respectively. Our benchmarked comparison study suggests that the aluminum precipitation method coupled with the automated nucleic extraction represents a method of acceptable sensitivity to provide readily results of interest for SARS-CoV-2 WBE surveillance.