Use of Human Intestinal Enteroids to Evaluate Persistence of Infectious Human Norovirus in Seawater.

Little data on the persistence of human norovirus infectivity are available to predict its transmissibility. Using human intestinal enteroids, we demonstrate that 2 human norovirus strains can remain infectious for several weeks in seawater. Such experiments can improve understanding of factors associated with norovirus survival in coastal waters and shellfish.

Prevalence of human bocavirus infections in Europe. A systematic review and meta-analysis.

Human bocaviruses (HBoVs) are recently described as human emergent viruses, especially in young children. In this study, we undertook a systematic review and meta-analysis to estimate their prevalence in Europe. PubMed, Web of Science and Scopus databases were systematically screened for clinical studies, up to October 2020. Study eligibility criteria were primary full-text articles from clinical studies, conducted using valid screening test methods and published in peer-reviewed journals, in English or Spanish and from European countries. The overall pooled prevalence, prevalence by country as well as the prevalence of HBoV as a single or co-pathogen were estimated using a random-effects model. Sub-group and meta-regression analyses explored potential sources of heterogeneity in the data. A total of 35 studies involving 32,656 subjects from 16 European countries met the inclusion criteria. Heterogeneity (I2  = 97.0%, p < .01) was seen among studies; HBoV prevalence varied from 2.0 to 45.69% with a pooled estimate of 9.57% (95%CI 7.66-11.91%). The HBoV prevalence both as a single infection (3.99%; 95%CI 2.99-5.31%) or as co-infection with other viruses (5.06%; 95%CI 3.88-6.58%) was also analysed. On a geographic level, prevalence by country did not show statistical differences, ranging from 3.24% (Greece) to 21.05% (Denmark). An odds ratio analysis was also included in order to evaluate the relevance of the variable 'age' as a risk factor of HBoV infection in children <5 years old. The OR value of 1.77 (95%CI 1.13-2.77; p < .01) indicated that being <5 years old is a risk factor for HBoV infection. This study showed that HBoV has a moderate prevalence among European countries.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19).

Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries raising great concern. We developed a duplex real-time RT-qPCR assay to detect, discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, and used it to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than clinical epidemiological reporting by the local authorities, first in the southern city of Málaga (Andalucía) in week 20_52 (year_week), and multiple introductions during Christmas holidays were inferred in different parts of the country. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from wastewater treatment plants, which reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that 8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern as soon as they are identified by clinical sequencing and their integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

Detection of SARS-CoV-2 RNA in bivalve mollusks and marine sediments.

The presence of SARS-CoV-2 in wastewater pose the question of whether this new pandemic virus could be released into watercourses and potentially continue to finally reach coastal waters. In this study, we employed two bivalve molluscan species from the genus Ruditapes as sentinel organisms to investigate the presence of SARS-CoV-2 signals in the marine coastal environment. Estuarine sediments from the natural clam banks were also analyzed. Viral RNA was detected by RT-qPCR, targeting IP4, E and N1 genomic regions. Positive samples were also subjected to a PMAxx-triton viability RT-qPCR assay in order to discriminate between intact and altered capsids, obtaining indirect information about the viability of the virus. SARS-CoV-2 RNA traces were detected in 9/12 clam samples by RT-qPCR, from which 4 were positive for two different target regions. Viral quantification ranged from

Making waves: Wastewater-based epidemiology for COVID-19 - approaches and challenges for surveillance and prediction.

The presence of SARS-CoV-2 in the feces of infected patients and wastewater has drawn attention, not only to the possibility of fecal-oral transmission but also to the use of wastewater as an epidemiological tool. The COVID-19 pandemic has highlighted problems in evaluating the epidemiological scope of the disease using classical surveillance approaches, due to a lack of diagnostic capacity, and their application to only a small proportion of the population. As in previous pandemics, statistics, particularly the proportion of the population infected, are believed to be widely underestimated. Furthermore, analysis of only clinical samples cannot predict outbreaks in a timely manner or easily capture asymptomatic carriers. Threfore, community-scale surveillance, including wastewater-based epidemiology, can bridge the broader community and the clinic, becoming a valuable indirect epidemiological prediction tool for SARS-CoV-2 and other pandemic viruses. This article summarizes current knowledge and discusses the critical factors for implementing wastewater-based epidemiology of COVID-19.

A Methodology for Network Analysis to Improve the Cyber-Physicals Communications in Next-Generation Networks.

Cyber-physical systems allow creating new applications and services which will bring people, data, processes, and things together. The network is the backbone that interconnects this new paradigm, especially 5G networks that will expand the coverage, reduce the latency, and enhance the data rate. In this sense, network analytics will increase the knowledge about the network and its interconnected devices, being a key feature especially with the increment in the number of physical things (sensors, actuators, smartphones, tablets, and so on). With this increment, the usage of online networking services and applications will grow, and network operators require to detect and analyze all issues related to the network. In this article, a methodology to analyze real network information provided by a network operator and acquire knowledge of the communications is presented. Various real data sets, provided by Telecom Italia, are analyzed to compare two different zones: one located in the urban area of Milan, Italy, and its surroundings, and the second in the province of Trento, Italy. These data sets describe different areas and shapes that cover a metropolitan area in the first case and a mainly rural area in the second case, which implies that these areas will have different comportments. To compare these comportments and group them in a single cluster set, a new technique is presented in this paper to establish a relationship between them and reduce those that could be similar.

Infectivity and RNA Persistence of a Norovirus Surrogate, the Tulane Virus, in Oysters.

Oysters, being filter feeders, can accumulate some human pathogens such as norovirus, a highly infectious calicivirus, most common cause of acute gastroenteritis worldwide. Accumulated virus decays over a period of days to weeks, possibly rendering contaminated oysters safe again. Sensitive molecular methods have been set up for shellfish analysis but without answering the question of infectious virus detection. Using the Tulane virus (TV), a norovirus surrogate that recognizes the same ligand as human norovirus in oyster tissues, the genome and infectious virus decay rates were estimated using inverse linear regression in a Bayesian framework for genome copies. Infectivity decreased faster than genome copies but infectious viruses were detected for several days. Quantifying the decrease in viral infectivity and genome detection in oysters over such a long period may help local authorities to manage production areas implicated in shellfish-borne outbreaks, and thus protect consumers.

Hepatitis A Virus Disinfection in Water by Solar Photo-Fenton Systems.

This study evaluates and compares the effectiveness of solar photo-Fenton systems for the inactivation of hepatitis A virus (HAV) in water. The effect of solar irradiance, dark- Fenton reaction and three different reactant concentrations (2.5/5, 5/10 and 10/20 mg/L of Fe2+/H2O2) on the photo-Fenton process were tested in glass bottle reactors (200 mL) during 6 h under natural sunlight. Disinfection kinetics were determined both by RT-qPCR and infectivity assays. Mean water temperatures ranged from 25 to 27.3 °C, with a maximum local noon UV irradiances of 22.36 W/m2. Photo-Fenton systems yielded increased viral reduction rates in comparison with the isolated effect under the Fenton reaction in darkness (negligible viral reduction) or the solar radiation (0.25 Log of RNA reduction). With the highest concentration employed (10-20 mg/L Fe2+-H2O2), an average RNA reduction rate of ~ 1.8 Log (initial concentration of 105 pfu/mL) and a reduction of 80% in the infectivity capacity were reached. Results showed a strong synergistic effect between Fe2+/H2O2 and sunlight, demonstrating that significant disinfection rates of HAV under photo-Fenton systems may occur with relatively higher efficiency at middle environmental temperatures and without the need for an energy-intensive light source.

Depuration and Relaying: A Review on Potential Removal of Norovirus from Oysters.

Pollution of coastal waters can result in contamination of bivalve shellfish with human enteric viruses, including norovirus (NoV), and oysters are commonly implicated in outbreaks. Depuration is a postharvest treatment involving placement of shellfish in tanks of clean seawater to reduce contaminant levels; this review focuses on the efficacy of depuration in reducing NoV in oysters. There have been many NoV outbreaks from depurated oysters containing around 103 genome copies/g oyster tissue, far exceeding the median infectious dose (ID50). Half of the published NoV reduction experiments showed no decrease in NoV during depuration, and in the remaining studies it took between 9 and 45.5 d for a 1-log reduction-significantly longer than commercial depuration time frames. Surrogate viruses are more rapidly depurated than NoV; the mean number of days to reduce NoV by 1 log is 19, and 7.5 d for surrogates. Thus, surrogates do not appear to be suitable for assessing virological safety of depurated oysters; data on reduction of NoV infectivity during depuration would assist evaluations on surrogate viruses and the impact of methods used. The longer persistence of NoV highlights its special relationship with oysters, which involves the binding of NoV to histo-blood group-like ligands in various tissues. Given the persistence of NoV and on-going outbreaks, depuration as currently performed appears ineffective in guaranteeing virologically safe oysters. Conversely, relaying oysters for 4 wk is more successful, with low NoV concentrations and no illnesses associated with products. The ineffectiveness of depuration emphasizes the need for coastal water quality to be improved to ensure oysters are safe to eat.

Digital PCR for Quantifying Norovirus in Oysters Implicated in Outbreaks, France.

Using samples from oysters clearly implicated in human disease, we quantified norovirus levels by using digital PCR. Concentrations varied from 43 to 1,170 RNA copies/oyster. The analysis of frozen samples from the production area showed the presence of norovirus 2 weeks before consumption.

Prevalence and Genetic Diversity of Human Sapoviruses in Shellfish from Commercial Production Areas in Galicia, Spain.

The prevalence of human forms of Sapovirus, an emerging pathogen of human gastroenteritis, was investigated in an 18-month survey from class B mollusc-harvesting areas in two Galician rias (northwest Spain). The detection and quantification of Sapovirus was performed by reverse transcription-real-time PCR, according to the recently developed standard method ISO/TS 15216-1:2013, and genotyping by reverse transcription-nested PCR. The bivalve species studied were wild and cultured mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and Venerupis decussata), and cockles (Cerastoderma edule). Sapovirus was detected in 30 out of 168 samples (17.9%), with cockles being the species with the highest prevalence of positives (28.1%), followed by clams (22.6%), wild mussels (14.3%), and cultured mussels (12.9%). The estuary in the south of the region demonstrated a higher percentage of positive samples (21.8%) than the one in the north (14.4%). Viral contamination levels for the positive samples ranged between 1.9 × 10(3) and 1.4 × 10(5) RNA copies/g of digestive tissue. Thirteen Sapovirus sequences could be obtained based on partial capsid gene sequence and were classified into four genotypes: GI.1 (2 samples), GI.2 (8 samples), GIV.1 (2 samples), and GV.1 (1 sample).

Solar water disinfection (SODIS): Impact on hepatitis A virus and on a human Norovirus surrogate under natural solar conditions.

This study evaluates the effectiveness of solar water disinfection (SODIS) in the reduction and inactivation of hepatitis A virus (HAV) and of the human Norovirus surrogate, murine Norovirus (MNV-1), under natural solar conditions. Experiments were performed in 330 ml polyethylene terephthalate (PET) bottles containing HAV or MNV-1 contaminated waters (10(3) PFU/ml) that were exposed to natural sunlight for 2 to 8 h. Parallel experiments under controlled temperature and/or in darkness conditions were also included. Samples were concentrated by electropositive charged filters and analysed by RT-real time PCR (RT-qPCR) and infectivity assays. Temperature reached in bottles throughout the exposure period ranged from 22 to 40ºC. After 8 h of solar exposure (cumulative UV dose of ~828 kJ/m2 and UV irradiance of ~20 kJ/l), the results showed significant (P<0.05) reductions from 4.0 (+/-0.56)x10(4) to 3.15 (+/-0.69)x10(3) RNA copies/100ml (92.1%, 1.1 log) for HAV and from 5.91 (+/-0.59)x10(4) to 9.24 (+/-3.91)x10(3) RNA copies/100 ml (84.4%, 0.81 log) for MNV-1. SODIS conditions induced a loss of infectivity between 33.4% and 83.4% after 4 to 8 h in HAV trials, and between 33.4% and 66.7% after 6 h to 8 h in MNV-1 trials. The results obtained indicated a greater importance of sunlight radiation over the temperature as the main factor for viral reduction.

Mathematical model for viral depuration kinetics in shellfish: an useful tool to estimate the risk for the consumers.

Enteric virus depuration from shellfish is a complex biological process that may be influenced by biological properties of the mollusc and/or virus species. On the basis of previous experimental data, a mathematical model was developed to characterize the kinetics of viral elimination during the depuration process. The experimental data consisted on twenty depuration trials, each with 60 kg of Manila clams (Venerupis philippinarum) and mediterranean mussels (Mytilus galloprovincialis) previously subjected to bioaccumulation with HAV or MNV-1 (as a surrogate for human norovirus), that were performed in an experimental depuration system during 7 days. It was observed that although viral loads decay along depuration, a residual viral load remains at the end of the process suggesting a decomposition of viral load in a diluted load (susceptible of depuration) and a non-diluted load (unavailable to depurate). The model yielded a general equation, which can predict the viral load at any depuration time knowing the specific filtration rate, dependent on the bivalve species, and specific viral properties. The mathematical model can be combined with quantitative risk assessment calculations to determine the safety of the depurated shellfish, which can be very helpful not only for shellfish producers but also to public health authorities.

Solar water disinfection (SODIS): impact on hepatitis A virus and on a human Norovirus surrogate under natural solar conditions

This study evaluates the effectiveness of solar water disinfection (SODIS) in the reduction and inactivation of hepatitis A virus (HAV) and of the human Norovirus surrogate, murine Norovirus (MNV-1), under natural solar conditions. Experiments were performed in 330 ml polyethylene terephthalate (PET) bottles containing HAV or MNV-1 contaminated waters (103 PFU/ml) that were exposed to natural sunlight for 2 to 8 h. Parallel experiments under controlled temperature and/or in darkness conditions were also included. Samples were concentrated by electropositive charged filters and analysed by RT-real time PCR (RT-qPCR) and infectivity assays. Temperature reached in bottles throughout the exposure period ranged from 22 to 40ºC. After 8 h of solar exposure (cumulative UV dose of ~828 kJ/m2 and UV irradiance of ~20 kJ/l), the results showed significant (P < 0.05) reductions from 4.0 (±0.56) ×104 to 3.15 (±0.69) × 103 RNA copies/100 ml (92.1%, 1.1 log) for HAV and from 5.91 (±0.59) × 104 to 9.24 (±3.91) × 103 RNA copies/100 ml (84.4%, 0.81 log) for MNV-1. SODIS conditions induced a loss of infectivity between 33.4% and 83.4% after 4 to 8 h in HAV trials, and between 33.4% and 66.7% after 6 h to 8 h in MNV-1 trials. The results obtained indicated a greater importance of sunlight radiation over the temperature as the main factor for viral reduction (AU)

No disponible

Detection and quantification of hepatitis A virus and norovirus in Spanish authorized shellfish harvesting areas.

An 18-month survey was conducted in ten class "B" harvesting areas from two Galician Rias (NW of Spain), the most important bivalve production area in Europe, to determine the prevalence of hepatitis A virus (HAV) and human norovirus (NoV), including genogroups I (GI) and II (GII). Quantification was performed by reverse transcription real-time PCR (RT-qPCR), according to the recently developed standard method ISO/TS 15216-1:2013. Four bivalve species were studied, including wild and cultured mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and Venerupis decussata) and cockles (Cerastoderma edule). Overall, 55.4% of samples were contaminated by at least one of the studied viruses, being detected the simultaneous presence of two or three viruses in 11.3% of the cases. NoV GI was the most prevalent virus (32.1%), followed by NoV GII (25.6%) and HAV (10.1%). Cultured mussels showed the highest percentage of positive samples (61.4%), followed by cockles (59.4%), wild mussels (54.3%) and clams (38.7%). Viral contamination levels for most of the positive samples ranged from 10(2) to 10(3) RNA copies/g of digestive tissue (RNAc/g DT). The presence of viral contamination was statistically higher (P<0.0001) in warm months (April to September) than in cold months (October to March). The data presented here may contribute to the development of more representative sampling strategies, in monitoring and management of shellfish growing areas as well as being useful in a future scenario in which viral critical values are adopted in legislation.

Effectiveness of depuration for hepatitis A virus removal from mussels (Mytilus galloprovincialis).

The efficacy and kinetic of depuration of hepatitis A virus (HAV) were evaluated under experimental conditions with Mediterranean mussels (Mytilus galloprovincialis) subjected previously to bioaccumulation processes. Seven independent trials (70kg of mussels each) were performed in a closed experimental system using two different water temperatures (13 and 17°C) during 7days. The real time RT-PCR technique with TaqMan probes was used for viral quantification. Qualitative infectivity assays were conducted to test the presence of infectious viral particles at the end of the depuration period. The depuration trials showed an average reduction of HAV levels of aproximately 1.1 Log units (>90%). However, the average final viral loads in shellfish samples remain at relatively high levels (6.5×10(3) RNA copies/g digestive tissue) and still infectious. A positive correlation between the initial and the final numbers of the viral RNA copies was observed. The reduction of HAV showed a two-phase removal kinetic, an initial logarithmic trendline, with a rapid reduction of viruses during the first 24-48h of depuration, and a subsequent stabilization with a slower depuration rate until the end of the process.

Depuration kinetics of hepatitis A virus in clams.

The efficacy and dynamic of depuration for the removal of hepatitis A virus (HAV) contamination were evaluated under experimental conditions using Manila clams previously subjected to bioaccumulation with this virus. Five independent trials were assayed in a closed experimental system with a total volume of approximately 1750 l, using clam batches of 60 Kg. The reverse transcriptase-real time PCR (RT-qPCR) technique was utilized for viral quantification. Infectivity assays were conducted at the end of depuration. Although the final viral loads in shellfish after 7 days remained relatively high and still infectious, an average reduction in HAV levels of 1.44 log units (approx. 93.1%) was observed. This reduction showed a two-phase removal kinetic, with an initial rapid reduction of viruses during the first 72 h of depuration, with a 0.6 log units (69%) of average decrease in HAV RNA copies/g digestive tissue, and a subsequent stabilization with a slower depuration rate in the remaining days.

Depuration kinetics of murine norovirus in shellfish.

This study evaluates and compares the uptake rates and depuration kinetics of murine norovirus (MNV-1), as a human norovirus surrogate, in Manila clams (Venerupis philippinarum) and Mediterranean mussels (Mytilus galloprovincialis). Ten trials of 70kg/trial (five with each mollusk) were performed. Mollusks were subjected to a controlled bioaccumulation step of 24h with 102pfu MNV-1/mL seawater. Then, mollusks were relocated in an experimental depuration system for 7days. Viral contamination was quantified after bioaccumulation and then daily during depuration by reverse transcription-real time PCR (RT-qPCR) with TaqMan probes. Infectivity assays were conducted to test the presence of infectious viral particles at the end of the depuration period. Results showed significant differences in the uptake and removal viral rates between molluscan species. The average viral uptake for clams and mussels were 5.4×106 and 4.0×105RNA copies/g digestive tissue respectively, representing an uptake rate >90% higher in clams. The average reductions with regard to the initial levels were 60.5% for clams and 91.6% for mussels. On the other hand, a similar logarithmic trend line in MNV-1 depuration kinetics was observed in both bivalves, with two differentiated phases: an initial rapid reduction of viruses during the first 24-72h of depuration, and a subsequent stabilization with a slower depuration rate. All trials with clams and mussels showed significant viral reductions but remaining virus were still infectious at the end of the process.

Assessment of human enteric viruses in cultured and wild bivalve molluscs.

Standard and real-time reverse transcription-PCR (rRT-PCR) procedures were used to monitor cultured and wild bivalve molluscs from the Ría de Vigo (NW Spain) for the main human enteric RNA viruses, specifically, norovirus (NoV), hepatitis A virus (HAV), astrovirus (AsV), rotavirus (RT), enterovirus (EV), and Aichi virus (AiV). The results showed the presence of at least one enteric virus in 63.4% of the 41 samples analyzed. NoV GII was the most prevalent virus, detected in 53.7% of the samples, while NoV GI, AsV, EV, and RV were found at lower percentages (7.3, 12.2, 12.2, and 4.9%, respectively). In general, samples obtained in the wild were more frequently contaminated than those from cultured (70.6 vs. 58.3%) molluscs and were more readily contaminated with more than one virus. However, NoV GI was detected in similar amounts in cultured and wild samples (6.4 x 10(2) to 3.3 x 10(3) RNA copies per gram of digestive tissue) while the concentrations of NoV GII were higher in cultured (from 5.6 x 10(1) to 1.5 x 10(4) RNA copies per gram of digestive tissue) than in wild (from 1.3 x 10(2) to 3.4 x 10(4) RNA copies per gram of digestive tissue) samples.