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Due to the excretion of SARS-CoV-2 in faeces, the use of wastewater-based epidemiology (WBE) is a useful tool for virus surveillance in large populations. The analysis of this virus includes a concentration step prior to virus detection by RT-qPCR. In addition, the use of massive sequencing allows the detection of specific mutations of clinical importance, as well as the detection of the introduction of new lineages in a specific population. In this chapter, we describe the analysis of SARS-CoV-2 in urban wastewater by the concentration of the samples by precipitation with aluminum chloride, the detection, and quantification of SARS-CoV-2 RNA by RT-qPCR and the genomic sequencing using two different sequencing platforms.

This year-long research analysed emerging risks in influent, effluent wastewaters and biosolids from six wastewater treatment plants in Spain's Valencian Region. Specifically, it focused on human enteric and respiratory viruses, bacterial and viral faecal contamination indicators, extended spectrum beta-lactamases-producing Escherichia coli and antibiotic resistance genes. Additionally, particles and microplastics in biosolid and wastewater samples were assessed. Human enteric viruses were prevalent in influent wastewater, with limited post-treatment reduction. Wastewater treatment effectively eliminated respiratory viruses, except for low levels of SARS-CoV-2 in effluent and biosolid samples, suggesting minimal public health risk. Antibiotic resistance genes and microplastics were persistently found in effluent and biosolids, thus indicating treatment inefficiencies and potential environmental dissemination. This multifaced research sheds light on diverse contaminants present after water reclamation, emphasizing the interconnectedness of human, animal, and environmental health in wastewater management. It underscores the need for a One Health approach to address the United Nations Sustainable Development Goals.

Berries contaminated with human norovirus (HuNoV) have been frequently identified as a cause of foodborne gastroenteritis. To prevent virus transmission while preserving sensory and quality parameters, non-thermal treatments, such as high-pressure processing (HPP), can be applied to the berries and products thereof. Here, strawberry purees contaminated with HuNoV genogroup I (GI.3[P13]) and II (GII.4 Sydney [P16]), along with murine norovirus (MNV) and Tulane virus (TV) serving as surrogates, were exposed to HPP at several pressure-time combinations. Virus inactivation was assessed by cell culture, including the novel human intestinal enteroids (HIE) model for HuNoVs. The infectivity results showed TV more resistant than MNV to HPP, as also confirmed by kinetic mathematical modelling. Results indicated that a holding pressure of 450 MPa and an exposure time of ≥5 min are reliable operational conditions for HPP process to successfully control viral contamination. In addition, the inactivation models deduced from MNV and TV viruses were challenged with experimental HuNoV GII.4 infectivity resulting in bias factors <1 for all treatment conditions. This finding validates the proposed models for the conservative estimation of HuNoV inactivation. Our work offers a blueprint for moving forward with inactivation studies using the HIE system, which provides useful practical information on optimum treatments for the best public health outcomes.

The COVID-19 pandemic has posed a significant global threat, leading to several initiatives for its control and management. One such initiative involves wastewater-based epidemiology, which has gained attention for its potential to provide early warning of virus outbreaks and real-time information on its spread. In this study, wastewater samples from two wastewater treatment plants (WWTPs) located in the southeast of Spain (region of Murcia), namely Murcia, and Cartagena, were analyzed using RT-qPCR and high-throughput sequencing techniques to describe the evolution of SARS-CoV-2 in the South-East of Spain. Additionally, phylogenetic analysis and machine learning approaches were applied to develop a pre-screening tool for the identification of differences among the variant composition of different wastewater samples. The results confirmed that the levels of SARS-CoV-2 in these wastewater samples changed concerning the number of SARS-CoV-2 cases detected in the population, and variant occurrences were in line with clinical reported data. The sequence analyses helped to describe how the different SARS-CoV-2 variants have been replaced over time. Additionally, the phylogenetic analysis showed that samples obtained at close sampling times exhibited a higher similarity than those obtained more distantly in time. A second analysis using a machine learning approach based on the mutations found in the SARS-CoV-2 spike protein was also conducted. Hierarchical clustering (HC) was used as an efficient unsupervised approach for data analysis. Results indicated that samples obtained in October 2022 in Murcia and Cartagena were significantly different, which corresponded well with the different virus variants circulating in the two locations. The proposed methods in this study are adequate for comparing consensus sequence types of the SARS-CoV-2 sequences as a preliminary evaluation of potential changes in the variants that are circulating in a given population at a specific time

[Background] Breastmilk is considered the gold standard in infant nutrition and provides bioactive compounds to the neonate, among them antibodies and microbiota. In the context of the COVID- 19 pandemics, there were great concerns about a possible mother-to-infant transfer of SARS-CoV-2, since limited knowledge about the safety of breastfeeding after natural infection or vaccination, as well as the transfer of protective antibodies and their neutralization capacity, was available. Additionally, there are concerns about potential short- and long-term adverse effects of SARS-CoV-2 infection and vaccine-induced changes to the breastmilk microbiome composition, which contributes in shaping the early-life microbiome., [Methods] This study included 60 mothers which had a confirmed SARS-CoV-2 infection and also, 86 mothers vaccinated with mRNA-based (Comirnaty, mRNA-1273) and adenoviral-vectored vaccines (ChAdOx1 nCoV-19) were recruited and breastmilk samples were collected longitudinally from baseline up to 30 days after the second dose at seven or eight time points (depending on vaccine type). In COVID-19 lactating mothers, the presence of SARS-CoV-2 was assessed by RT-qPCR targeting the N1 region of the nucleocapsid gene and the envelope (E) gene. In both studies, the levels of SARS-CoV-2 RBD-specific IgA, IgM and IgG were determined by ELISA. The neutralization capacity was tested using pseudotyped vesicular stomatitis virus carrying either the Wuhan-Hu-1, Delta, or BA.1 Omicron spike proteins. To assess the microbiome composition, DNA from breastmilk samples was extracted and the V3-V4 region of the 16S rRNA gene was sequenced using the MiSeq system of Illumina., [Results] After SARS-CoV-2 infection, no virus-specific RNA was detected in breastmilk samples. Determination of antibody levels in mothers with confirmed SARS-CoV-2 infection showed that 82.9% (58 of 70) of milk samples were positive for at least one of the three tested antibody isotypes. Vaccination elicited also a strong induction of SARS-CoV-2-specific antibodies, which was higher in IgG when compared to COVID-19 convalescent women and was strongly increased after the 2nd dose. mRNA-based vaccines induced higher IgG and IgA levels when compared to the adenovirus- vectored vaccine, and women with previous virus exposure increased their IgG antibodies levels after the first dose to a similar level observed in vaccinated women after the second dose. When assessing the neutralization capacity, natural infection resulted in higher neutralizing titers that correlated positively with levels of SARS-CoV-2-specific immunoglobulin A in breastmilk. Breastmilk samples from COVID-19 convalescent mothers infected during the first wave (Wuhan-Hu-1 strain) neutralized less effectively Omicron BA.1 than the Wuhan-Hu-1 variant. In addition, significant differences in the capacity to produce neutralizing antibodies were observed between both mRNA- based vaccines and the adenovirus-vectored ChAdOx1 COVID-19 vaccine. First results of the analysis of the breastmilk microbiome found no significant differences in the mean diversity of species (alpha-diversity) after natural SARS-CoV-2 infection, whereas some specific bacterial groups were increased (e.g. Enterobacteriaceae)., [Conclusions] Overall, our results indicate that breastmilk from naturally infected women or those vaccinated with mRNA-based vaccines contain SARS-CoV-2 neutralizing antibodies that could potentially provide protection to breastfed infants from infection.

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.

Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n = 312) from 24 different wastewater treatment plants were obtained between May 9 (week 19 of 2022) and August 4 (week 31 of 2022). Following concentration of viral particles by a validated aluminum adsorption-precipitation method, a qPCR procedure allowed us to detect MPXV DNA in 56 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 × 103 to 8.7 × 104 genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the reported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.

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.

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.

With the exception of foreign bodies, formerly Food Contact Materials (FCM) were generally not considered a source of food safety concern. During the past decades, several issues regarding FCM affected the food supply, calling the attention of industries, national bodies, and of course consumers. But, is it already clear what is an FCM? The chapter presents different classifications of FCM depending on the type of contact, type of material, and function. The potential hazards (physical, microbiological, chemical, and allergen) associated FCM and their risk factors are explained according to the type of material (e.g., wood, metal, plastic). The chapter is also reviewing the main standards used in the field (ISO 22000, BRCIoP, and EN 15593) and gives an overview of main regulations applicable. The chapter finishes with five case studies that bring the reader to reality of management of FCM food safety.

La vigilancia microbiológica en aguas residuales puede utilizarse como indicador epidemiológico para la detección de la circulación del virus en la población. Las experiencias previas en la utilización de esta herramienta han demostrado su sensibilidad, incluso cuando la prevalencia de la COVID-19 presenta niveles bajos en la población. Se introducie el proyecto liderado por la Dra. Gloria Sánchez del IATA-Valencia y la Dra. Ana Allende del CEBAS-Murcia, que detectan SARS-CoV-2 en aguas residuales como alerta temprana de la infección

The use of SARS-CoV-2 metagenomics in wastewater can allow the detection of variants circulating at community level. After comparing with clinical databases, we identified three novel variants in the spike gene, and six new variants in the spike detected for the first time in Spain. We finally support the hypothesis that this approach allows the identification of unknown SARS-CoV-2 variants or detected at only low frequencies in clinical genomes.

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.

Medicinal and aromatic plants (MAPs) are potential sources of natural bioactive phytochemical compounds of an incredible worth for the food industry, such as polyphenols. Lamiaceae medicinal and aromatic plants from Granada's high plateau, concretely Origanum bastetanum, Thymus zygis gracilis, Thymus longiflorus, Thymus membranaceus and Ziziphora hispanica, were evaluated under different conventional solid-liquid extraction conditions to obtain extracts enriched in bioactive compounds. Phenolic profile was detected by HPLC-QTOF-MS, identifying a high abundance of bioactive constituents. Furthermore, antioxidant and antiviral activities of the mentioned plants were studied as biological properties of interest for the improvement of food shelf-life. Thus, Origanum bastetanum showed the highest antioxidant potential for all assays. Antiviral activity was also tested against some important foodborne viruses, feline calicivirus (FCV), murine norovirus (MNV) and hepatitis A virus (HAV), with the highest activity obtained for Ziziphora hispanica, Thymus longiflorus and Origanum bastetanum. This research proposes the studied plants as rich sources of bioactive compounds with potential use as preservatives in the food industry.

Wastewater surveillance is a fast and cost-effective tool that enables tracing of both symptomatic and asymptomatic transmission of SARS-CoV-2. In this paper, a pilot program carried out at the University Jaume I for monitoring the trends of the presence of SARS-CoV-2 in wastewater. To the best of our knowledge, this is the first such project conducted on a university campus in Spain. Wastewater samples (n = 838) were collected when students returned to campus, from October 2020 until August 2021, at a confluence sewer point and at the building level including different academic departments and services, the library, administration offices and the university student residence. It has been observed that the probability of SARS-CoV-2 RNA detection in wastewater depended on COVID-19 incidence on campus and visitors/occupants of the buildings i.e., high-, or low-traffic buildings with high or low frequency of potential contacts. Moreover, the third wave in Spain (after Christmas 2020) and an outbreak that occurred at the university student's residence could be carefully followed, allowing confirmation of the end of the outbreak. In addition, viral variants (i.e., mutations and linages) from selected time points were detected by sequencing and gave an indication of the evolution of the virus over time. The results illustrate the potential of wastewater-based epidemiology to provide an early warning for SARS-CoV-2 within the university, especially in buildings with low traffic and more defined populations, like the student residence. The strategy and experience gathered in this study will allow for implementation of improvements for reliable monitoring in the future.

Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n=312) from 24 different wastewater treatment plants were obtained between May 9 (week 22_19) and August 4 (week 22_31), 2022. Following concentration of viral particles by flocculation, a qPCR procedure allowed us to detect MPXV DNA in 63 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 per 103 to 8.7 per 104 genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the reported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.

Berries have been implicated as the probable vehicle of infection in multiple outbreaks of norovirus and hepatitis A virus (HAV). These foods often receive minimal or no processing and may be exposed to virus contamination at each stage of production. In an increasingly globalized world, berries have a wide distribution and can give rise to the spread of diseases in distant parts of the world. With the aim of describing the virological quality of the berries cultivated in Argentina, a total of 184 soft fruits of different varieties (strawberries, blueberries, raspberries, blackberries, currants, pomegranate arils, cassis, and elder) were collected during the periods 2016-2018 and 2020. Viral particles were eluted and concentrated by polyethylene glycol precipitation according to ISO 15216-2:2019 guidelines. Genome detection of norovirus (NoV) genogroups I (GI) and II (GII), HAV, rotavirus, and enterovirus was performed by real-time RT-PCR with TaqMan probes. Positive samples were amplified by conventional RT-PCR and the amplicons were purified and sequenced in both directions. Phylogenetic analysis was performed using the Neighbor-Joining method based on the evolutionary model Kimura-2-parameters. NoV GII.6 was detected in 1/184 (0.5%) of the soft fruits, corresponding to a raspberry sample obtained during the fall of 2017. No presence of other human enteric viruses was found in the other berries analyzed. The collected data are the first in Argentina in relation to the prevalence of enteric viruses in berries and is useful as reference data for a risk assessment of soft fruits as vehicles of foodborne pathogenic viruses.

Objectives To compare the RNA loads of severe acute respiratory syndrome coronavirus 2 in nasopharyngeal specimens collected from patients with breakthrough coronavirus disease 2019 (COVID-19) caused by the Delta variant with those in specimens collected from patients with breakthrough COVID-19 caused by the Omicron variant. Methods A retrospective, observational study was conducted, including 240 consecutive adult out-patients, of whom 121 (74 females; median age, 40 years) had COVID-19 due to the Omicron variant and 119 (65 females; median age, 48 years) had COVID-19 caused by the Delta variant. The viral RNA load was quantitated using the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MS, USA). The viability platinum chloride reverse transcription-PCR assay was used to discriminate between potentially infectious viral particles and free (encapsidated) viral RNA. Results Overall, the viral RNA loads were significantly higher (p 0.003) for the Omicron variant (median, 8.1 log10 copies/mL; range, 4.0–10.9 log10 copies/mL) than for the Delta variant (median, 7.5 log10 copies/mL; range, 3.0–11.6 log10 copies/mL). A trend towards higher viral loads was noticed for Omicron compared with that for Delta across the following time frames since vaccination: 16–90 days (median, 6.83 vs. 5.88 log10 copies/mL, respectively; range, 3.91–10.68 vs. 3.67–9.66 log10 copies/mL, respectively; p 0.10), 91–180 days (median, 8.09 vs. 7.46 log10 copies/mL, respectively; range, 4.30–10.92 vs. 3.03–11.56 log10 copies/mL, respectively; p 0.003) and 181–330 days (median, 8.56 vs. 8.10 log10 copies/mL, respectively; range, 6.51–10.29 vs. 3.03–10.61 log10 copies/mL, respectively; p 0.11). The platinum chloride treated or untreated reverse transcription-PCR cycle threshold ratio for the nucleocapsid gene as the target was slightly higher for Omicron than for Delta (median, 0.62 vs. 0.57, respectively; range, 0.57–0.98 vs. 0.61–0.87, respectively), although statistical significanc

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.

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.

[ES] El SARS-CoV-2 (del inglés severe acute respiratory síndrome coronavirus 2) es un virus respiratorio causante de la COVID-19. Como ya ocurre con otros virus respiratorios, el SARS-CoV-2 se excreta también en heces lo que nos permite poder detectarlo y cuantificarlo en las aguas residuales con técnicas de biología molecular como la RT-qPCR. Gracias a la detección del ARN de SARS-CoV-2 en aguas residuales podemos utilizar los resultados como una herramienta epidemiológica complementaria a las ya utilizadas a nivel clínico, pudiendo así adelantarnos a la situación que se va a dar en clínica y ejercer una mejor y rápida respuesta sanitaria, previa a la aparición de casos clínicos severos. Por otro lado, el bacteriófago crAssphage se encuentra de forma natural en el tracto gastrointestinal de los seres humanos y, por tanto, se excreta en las heces. Esta característica nos permite utilizarlo como indicador de contaminación fecal vírica en el agua. Se han realizado estudios (Wilder et al. 2021; Haramoto et al. 2018; Polo et al. 2020) los cuales demuestran que esta técnica de co-cuantificación con crAssphage en aguas residuales ayuda a estandarizar los resultados debido al factor de dilución que presentan las muestras de agua residual al mezclarse con agua pluviales. El objetivo del trabajo se centró tanto en la detección y cuantificación de SARS-CoV-2 para observar su dinámica poblacional y su incidencia, como en normalizar los resultados obtenidos con el indicador de contaminación fecal vírica crAssphage en aguas residuales procedentes de colectores de la ciudad de Valencia. Para ello se utilizaron técnicas de biología molecular, como la RT-qPCR para SARS-CoV-2 y la qPCR para crAssphage, para la detección y cuantificación de ambos virus en muestras de colectores de la provincia de Valencia y un control de proceso (PEDV) para evaluar la eficiencia del proceso de concentración y extracción. Los resultados obtenidos se utilizaron para seguir la evolución de SARS-CoV-2 en, [EN] Detection and quantification of SARS-CoV-2 and normalization of the results with the indicator of fecal viral contamination crAssphage. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a respiratory virus that causes COVID-19. As with other respiratory viruses, SARS-CoV-2 is also excreted in faeces, which allows us to detect and quantify it in wastewater with molecular biology techniques such as RT-qPCR. Due to the detection of SARS-CoV-2 RNA in wastewater, we can use the results as an epidemiological tool complementary to those already used at the clinical level, thus being able to anticipate the situation that will occur in the clinic and exercise a better and rapid health response, prior to the appearance of severe clinical cases. On the other hand, the bacteriophage crAssphage is found naturally in the gastrointestinal tract of humans and is therefore excreted in faeces. This characteristic allows us to use it as an indicator of viral fecal contamination in water. Studies have been carried out (Wilder et al. 2021; Haramoto et al. 2018; Polo et al. 2020) which show that this co-quantification technique with crAssphage in wastewater helps to standardize the results due to the dilution factor presented by the wastewater samples when mixed with rainwater. The objective of the work focused both on the detection and quantification of SARS-CoV-2 to observe its population dynamics and its incidence, and on normalizing the results obtained with indicator of viral fecal contamination crAssphage in wastewater. For this, molecular biology techniques were used, such as RT-qPCR for SARS-CoV2 and qPCR for crAssphage, for the detection and quantification of both viruses in samples from collectors in the city of Valencia and a process control (PEDV) to monitor the efficiency of the concentration and extraction process. The results obtained were used to follow the evolution of SARS-CoV-2 in the city of Valencia. In this study, a total of 53 samples were analyz

La irrupción del coronavirus SARS-CoV-2 y de la pandemia de covid-19 ha planteado un enorme desafío a la ciencia. Buscar soluciones a un virus que ha causado, hasta el momento, 130 millones de infectados y ha provocado casi 3 millones de muertes. Para desentrañar las claves de esta nueva amenaza global, el Consejo Superior de Investigaciones Científicas, el CSIC, puso en marcha en marzo de 2020 la Plataforma Temática Interdisciplinar Salud Global. Su objetivo es coordinar a cientos de equipos de diversas disciplinas en busca de soluciones a la pandemia, creando nuevas redes de colaboración con organismos públicos de investigación, universidades, la clínica y el sector empresarial. La plataforma ha reunido a expertos de la investigación en biomedicina (virólogos, inmunólogos, genetistas y biotecnólogos), junto a químicos y físicos, además de sociólogos y demógrafos. Su trabajo es determinar las características del virus y de la covid-19, y diseñar estrategias que permitan hacer frente a esta y futuras pandemias. Tras un año de investigación, la Plataforma Salud Global ha obtenido conocimientos fundamentales en todos los aspectos de la pandemia: prevención, contención, diagnóstico, terapia y vacunas, e impacto social.

Background During the COVID-19 pandemic in 2020, breastfeeding in women positive for SARS-CoV-2 was compromised due to contradictory data regarding potential viral transmission. However, growing evidence confirms the relevant role of breast milk in providing passive immunity by generating and transmitting specific antibodies against the virus. Thus, our study aimed to develop and validate a specific protocol to detect SARS-CoV-2 in breast milk matrix as well as to determine the impact of maternal SARS-CoV-2 infection on presence, concentration, and persistence of specific SARS-CoV-2 antibodies. Study design/Methods A prospective multicenter longitudinal study in Spain was carried out from April to December 2020. A total of 60 mothers with SARS-CoV-2 infection and/or recovered from COVID-19 were included (n=52 PCR-diagnosed and n=8 seropositive). Data from maternal-infant clinical records and symptomatology were collected. A specific protocol was validated to detect 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 COVID-19 patients and from 13 women before the pandemic were also evaluated. Results All breast milk samples showed negative results for SARS-CoV-2 RNA presence. We observed high intra- and inter-individual variability in the antibody response to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Protease domain (MPro) antibodies were also detected in milk. In general, 82.9 % of the milk samples were positive for at least one of the three antibody isotypes, being 52.86 % of those positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2 – 87.5 %), whereas it raised continuously for IgG (47.8 % the first ten days to 87.5 % from day 41 up to day 206 post-PCR confirmation). Conclusions Considering t

One solution to current water scarcity is the reuse of treated wastewater. Water reuse systems have to be examined as a whole, including the efficacy of water-reclamation treatments and the operation steps from the wastewater inlet into the WWTP to the irrigation endpoint, including the irrigated crop. In this study, the monitoring of human enteric viruses and coliphages were assessed in two water reused systems. The presence of hepatitis A virus (HAV) and human noroviruses genogroups I and II (GI and GII) were analyzed by real-time RT-PCR (RT-qPCR) in water (n = 475) and leafy green samples (n = 95). Total coliphages were analyzed by the double-layer agar plaque technique. The prevalence of HAV in water samples was very low (c.a. 2%), mostly linked to raw sewage, while for leafy green samples, none was positive for HAV. In leafy greens, prevalence of norovirus was low (less than 5–6%). The highest reductions for norovirus were observed in samples taken from the water reservoirs used by the growers near the growing field. The virus die-off during water storage due to solar radiation could be considered as an additional improvement. Reclamation treatments significantly reduced the prevalence and the counts of noroviruses GI and GII and coliphages in reclaimed water. However, the coliphage reductions (c.a. 5 log) do not comply with the specifications included in the new European regulation on reclaimed water (≥6.0 log). Correlations between noroviruses GI and GII and coliphages were found only in positive samples with high concentrations (>4.5 log PFU/100 mL). A high percentage of samples (20–25%) negative for total coliphages showed moderate norovirus counts (1–3 logs), indicating that coliphages are not the most suitable indicator for the possible presence of human enteric viruses.

Background Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. Methods We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. Findings We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. Interpretation The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.

Nursing homes (NH) have been severely affected by the coronavirus disease 2019 (COVID-19) pandemic, largely due to their congregate nature and the vulnerability of residents [1]. Advanced age, frailty and concurrence of underlying chronic health conditions place NH residents at high risk for developing severe forms of COVID-19 and for death. Long-lasting virus shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in urine and faeces has been documented in both symptomatic and asymptomatic infected adults [2]. As a result, near-source tracking in the sewers serving particular buildings (i.e. campus dormitories, workplaces, correctional facilities, schools) has emerged as an appealing non-invasive tool that, when combined with subsequent targeted population screening when SARS-CoV-2 is detected, may enable rapid identification and control of facility outbreaks [3]. In this pilot study, we provide evidence demonstrating the feasibility and utility of this wastewater-based epidemiological approach for early identification of isolated cases or outbreaks of SARS-CoV-2 infection in NH.

Two large wastewater treatment plants (WWTP), covering around 2.7 M inhabitants, which represents around 85% of the metropolitan area of Barcelona, were sampled before, during and after the implementation of a complete lockdown. Five one-step RT-qPCR assays, targeting the polymerase (IP2 and IP4), the envelope E and the nucleoprotein (N1 and N2) genome regions, were employed for SARS-CoV-2 RNA detection in 24-h composite wastewater samples concentrated by polyethylene glycol (PEG) precipitation. SARS-CoV-2 was detected in a sewage sample collected 41 days ahead of the declaration of the first COVID-19 case. The evolution of SARS-CoV-2 genome copies in wastewater evidenced the validity of water-based epidemiology to anticipate COVID-19 outbreaks, to evaluate the impact of control measures and even to estimate the burden of shedders, including presymptomatic, asymptomatic, symptomatic and undiagnosed cases. For this latter objective, a model was applied for the estimation of the total number of shedders, evidencing a high proportion of asymptomatic infected individuals. In this way, an infection prevalence of 2.0-6.5% was figured. On the other hand, a proportion of around 0.12% and 0.09% of the total population was determined to be required for positive detection in the two WWTPs. At the end of the lockdown, SARS-CoV-2 RNA apparently disappeared in the WWTPs but could still be detected in grab samples from four urban sewers. Sewer monitoring allowed for location of specific hot spots of COVID-19, enabling the rapid adoption of appropriate mitigation measures.

In the last decades, berries have been identified as important vehicles for the transmission of foodborne viruses and different strategies are being explored to eliminate or reduce viral contamination in these fruits. The aim of this work was to develop novel edible coatings with antiviral properties for inactivating and reducing murine norovirus (MNV). Firstly, the effect of gelatin (G) addition on Persian gum (PG) films was studied in terms of microstructural, mechanical, optical, and water barrier properties. The following PG:G ratios were considered: 100:0, 75:25, 50:50, 25:75, and 0:100. Microstructure analysis revealed the compatibility of both hydrocolloids since no phase separation was observed. The addition of G to PG films provided stiffer and more deformable films than pure PG, with lower water vapor permeability values. Specifically, films prepared with 50:50 PG:G ratio presented better mechanical and barrier performance. Interestingly, pure PG showed antiviral activity on murine norovirus, probably due to the presence of some impurities (mainly tannins). Adding allyl isothiocyanate (AITC) enhanced the PG antiviral activity at refrigerated temperatures in blueberries, not being affected by the AITC concentration. This effect was not observed at ambient temperature, probably due to the volatilization of AITC.

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.

The present study reports a multivariable optimization of ultrasound-assisted extraction of phenolic compounds from arrayan leaves. The parameters ethanol percentage in extraction solvent, temperature and extraction time, and mass to solvent ratio were optimized applying full factorial and central composite designs. According to the models, the optimal extraction conditions were: 42% of ethanol, extraction time of 27 min, extraction temperature of 50ºC and a mass to solvent ratio of 1:33.4 g mL-1. Under these conditions, a total phenolic content (TPC) of 128.16±1.18 and 593.64±6.49 mg of gallic acid equivalent per g of dry weight (DW) were determined in raw extract (RE) and polyphenols-rich extract (PRE), which was obtained by semi-preparative chromatography on XAD-7 column. Antioxidant capacity determined via FRAP analysis showed values of 1349.53±28.99 and 6175.47±127.64 µmol Trolox equivalent per g of DW for RE and PRE, and DPPH IC50 values of 831.40±0.80 and 132.21±2.51 µg mL-1, respectively. Polyphenols profile analyzed by liquid chromatography-mass spectrometry showed the presence of quercetin 3-ß-D-glucoside, myricetin, quercetin, kaempferol 3-glucoside and gallic acid; the latter compounds are reported for the first time in arrayan leaves. Due to the presence of polyphenols, antiviral activity was assayed over norovirus and hepatitis A showing a significant dose-dependent effect.

Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.

The limited information about the routes of the transmission of SARS-CoV-2 within the ongoing pandemic scenario mobilized the administration, industry and academy to develop sanitation and disinfection systems for public and private spaces. Ozone has been proposed as an effective disinfection method against enveloped and non-enveloped viruses, including viruses with similar morphology to SARS-CoV-2. Due to this efficacy, numerous gaseous and aqueous phase ozone applications have emerged potentially to inhibit virus persistence in aerosols, surfaces, and water. In this work, a numerical model, a RANS CFD model for ozone dispersion inside tram and underground coach has been developed including the chemical self-decomposition and surface reactions of the ozone. The CFD model has been developed for a real tram coach of 28.6 × 2.4 × 2.2 m (L × W × H) using 1.76 million nodes and the Menter’s shear stress transport turbulence model. The model predicts the O3 concentration needed to meet disinfection criteria and the fluid dynamics inside the public transport coach. The effectiveness of the system has been validated with laboratory and field tests in real full-scale coach using porcine epidemic diarrhea virus (PEDV) and murine norovirus (MNV-1) as SARS-CoV-2 and human norovirus surrogates, respectively. Lab-scale experiments on plastic surfaces demonstrated O3 disinfection (100 ppm, 95% RH, 25 min) inactivate > 99.8% MNV-1 and PEDV. Additionally, field tests in real full-scale coach demostrate the efficacy of the system as > 98.6% of infectious MNV-1 and > 96.3% PEDV were inactivated.

In this work, a bioactive persimmon extract was produced from discarded fruits. A central composite design was used to evaluate the effect of different extraction parameters and ripeness stages of persimmon fruits on the total phenolic content and antioxidant activity of the resulting extracts. Significantly greater phenolic contents were obtained from immature persimmon (IP) fruits. The optimum IP extract with the conditions set by the experimental design was industrially up-scaled and its composition and functional properties were evaluated and compared with those obtained under lab-scale conditions. Both extracts contained significant protein (>20%) and phenolic contents (∼11–27 mg GA/g dry extract) and displayed significant antiviral activity against murine norovirus and hepatitis A virus. Moreover, the extract showed no toxicity and significantly reduced the fat content and the cellular ageing of Caenorhabditis elegans (C. elegans) without affecting the worm development. These effects were mediated by down-regulation of fat-7, suggesting an anti-lipogenic activity of this extract.

The virological quality of process water (PW) used by the produce industry has received limited attention. As a first step to overcoming technical limitations in monitoring viruses in PW, the analytical performance of ultrafiltration was assessed to concentrate viral particles from 20 L of spiked PW. The selected method used for sample concentration of PW was carefully validated, thus enabling the accurate quantification and estimation of viral titers of human enteric viruses and phages. PW from the produce industry was collected periodically from the washing tanks of commercial facilities. The analysis of coliphages was performed by plaque assay, while the occurrence of enteric viruses and crAssphage was determined by molecular techniques. Significant differences in the physicochemical composition of PW, mostly due to the different nature of fresh produce types and differences in the sanitizer used in commercial operation, were observed. Accumulation of crAssphage and coliphages was observed in PW, but correlation with human enteric viruses was not possible due to the low prevalence of these pathogens in the PW analyzed. The obtained results showed that depending on the type of product washed, the product/water ratio and the residual concentrations of the sanitizers, the prevalence and concentration of bacteriophages changed significantly.

Betulinic acid, which is found in transgenic roots of Senna obtusifolia (L.) H.S.Irwin & Barneby, is a pentacyclic triterpene with distinctive pharmacological activities. In this study, we report the differences in the content of betulinic acid and selected anthraquinones in transgenic S. obtusifolia hairy roots with overexpression of the PgSS1 gene (SOPSS2 line) and in transformed hairy roots without this genetic construct (SOA41 line). Both hairy root lines grew in 10 L sprinkle bioreactor. Additionally, the extracts obtained from this plant material were used for biological tests. Our results demonstrated that the SOPSS2 hairy root cultures from the bioreactor showed an increase in the content of betulinic acid (38.125 mg/g DW), compared to the SOA41 hairy root line (4.213 mg/g DW). Biological studies have shown a cytotoxic and antiproliferative effect on U-87MG glioblastoma cells, and altering the level of apoptotic proteins (Bax, p53, Puma and Noxa). Antimicrobial properties were demonstrated for both tested extracts, with a stronger effect of SOPSS2 extract. Moreover, both extracts showed moderate antiviral properties on norovirus surrogates.