Food and Environmental Virology最新文献

Hepatitis E is an emerging zoonosis caused by the hepatitis E virus (HEV) and is recognised worldwide. Wild boars are considered one of the main reservoirs of the zoonotic HEV-3 genotype. However, HEV-3 has also been detected in many other wildlife species. In this study, we investigated 284 liver and muscle tissue samples from wild boars and 107 liver and muscle tissue samples from four different wild ruminant species (red deer, roe deer, European mouflon and fallow deer) across 35 hunting areas in Slovakia. HEV RNA was detected in 14.2% (95% CI 9.8–18.6%) of the liver and 10.5% (95% CI 0.4–20.6%) of the muscle tissue samples from wild boars but in none of the samples from the wild ruminant species. Phylogenetic analysis based on partial ORF1 and ORF2 of the HEV genome revealed that the Slovak wild boar HEV sequences clustered within the zoonotic genotype HEV-3. Depending on their geographical origin, the obtained sequences clustered into three HEV-3 subtypes: HEV-3a, HEV-3i and HEV-3e. Our findings confirm the circulation of HEV in the wild boar population in the Slovak Republic but not in wild ruminant species.

Wastewater-based epidemiology (WBE) is a well-established tool for monitoring enteric and respiratory viruses. However, its potential application to vector-borne diseases, including dengue virus (DENV), remains largely underexplored. This study investigated the feasibility of using WBE to detect DENV serotype 2 RNA during the largest dengue outbreak recorded to date in Italy (216 confirmed and probable cases), that occurred in the Marche Region (central Italy) from August to October 2024. From 1 to 30 October, during the decline of the epidemic (30 reported cases), 27 wastewater samples were collected from wastewater treatment plants in the affected municipality of Fano and in the nearby urban center of Pesaro. Four viral RNA concentration methods were tested: polyethylene glycol/sodium chloride precipitation, Nanotrap^® Magnetic Virus Particles, electropositive membrane filtration, and solid fraction analysis. Only solid fraction analysis detected DENV-2 RNA in 9 samples, with digital RT-PCR showing greater sensitivity than Real-time RT-PCR. Virus concentrations ranged from 6.1 × 10¹ to 7.9 × 10² genome copies per gram of solid material. This study highlights the feasibility of WBE as a complementary surveillance tool for vector-borne diseases. Future efforts should focus on refining detection methods and in exploring surveillance strategies for timely wastewater sampling to improve early warning and/or outbreak management.

Waterborne pathogens pose a serious threat to public health, emphasizing the need for reliable and efficient water treatment technologies. Wastewater treatment plants employ a range of processes to reduce microbial contamination, with membrane filtration emerging as a promising solution due to its ability to physically remove pathogens without the production of harmful chemical by-products. This study investigates the effectiveness of a gravity-driven membrane (GDM) filtration system for pathogen removal from wastewater and evaluates the associated public health risks with and without treatment. A quantitative microbial risk assessment model was employed to estimate infection probabilities for various waterborne pathogens. The results demonstrated a significant decrease in pathogen concentrations following treatment, with up to a 10⁴-fold reduction in norovirus infection risk. Three critical factors influencing membrane performance were identified: membrane integrity, pore size characteristics, and membrane fouling. Maintaining membrane integrity was found to be essential for ensuring consistent pathogen removal. While nominal pore size is commonly used to predict rejection efficiency, the overall pore size distribution was found to have a greater influence on virus retention. Additionally, although membrane fouling is often considered detrimental, it was shown to enhance virus removal by up to two orders of magnitude. These findings underscore the potential of GDM systems for effective virus removal and highlight the importance of proper membrane design, maintenance, and monitoring in ensuring long-term operational efficiency and maximizing public health protection in wastewater treatment applications.

This study focuses on the characterization of human viral diversity within a major Wastewater Treatment Plant (WWTP) in the central area of Tianjin, China, with a particular emphasis on adenoviruses due to their robust survivability and potential health implications. By employing metagenomic sequencing, targeted sequencing, quantitative polymerase chain reaction (qPCR) analysis, and cell culture methodologies, we identified a broad spectrum of human viruses, encompassing 25 viral families and 45 viral genera, including enteric, respiratory, and contact viruses. Our findings demonstrate that adenoviruses were stably present in sewage from January to May and retained their infectious activity. The temporal analysis of viral loads across different months revealed no significant changes, suggesting a consistent risk of exposure. Additionally, our phylogenetic analysis of adenoviruses isolated from A549 cell cultures provides insights into their genotypic diversity and potential transmission dynamics. This comprehensive assessment underscores the critical need for improved viral surveillance in urban wastewater systems to mitigate public health risks and highlights the importance of advanced treatment technologies to address the challenges posed by adenoviruses and other pathogenic viruses in municipal wastewater.

Surveillance in wild birds is essential for the timely detection of high pathogenicity avian influenza (HPAI) strains. As flocks congregate in large numbers in wetlands and may potentially contaminate the environment with pathogens, the monitoring of such water bodies represents an attractive opportunity to complement animal testing and to improve surveillance for avian influenza. To increase sensitivity, water concentration is often required but available methods based on (ultra)filtration and precipitation are mostly limited by the use of pumping equipment and by the need to identify the representative sample volumes. In contrast, passive samplers (PS) offer a cost-effective and scalable solution that requires basic devices for the deployment of adsorbent materials and minimal training for their installation in the field. This study evaluated nine materials for their virus adsorption efficiency in brackish and freshwater. Cotton gauze, nitrocellulose, and nylon showed the best performance across different deployment times, with the highest recovery after 24 h. Shorter (3 h) and longer (7 days) deployments also proved effective, accommodating different sampling regimens according to the logistical needs. Importantly, PS revealed their efficacy in adsorbing also deteriorated virions or in dynamic ecosystems subjected to changes in water volumes. Field trials in wetlands corroborated laboratory findings and demonstrated that PS allowed detecting avian influenza virus (AIV, including HPAI strains) genome in water bodies, yielding consistent results with active surveillance in wild birds. By offering a simple, cost-effective, and versatile solution, PS represent a promising tool for environmental AI monitoring and can successfully complement existing avian influenza surveillance activities.

Environmental monitoring of avian influenza virus (AIV) in live poultry markets (LPMs) is vital for health risk forewarning, and survey of AIV environmental prevalence and biomolecular analysis of keystone subtypes remain essential. In this study, 970 biosamples were collected from air, poultry, water, and other environmental specimens of LPMs in Changsha from 2021 to 2023, and environmental prevalence survey of AIV and biomolecular analysis of H9N2 subtype were then conducted. Real-time fluorescent quantitative PCR was employed to detect the AIV and its subtypes (H5/H7/H9), revealing the positive rates of 68.45% for AIV and 53.51% for H9 subtype across all samples. Notably, aerosol samples had the positive rates of 89.00% for AIV and 72.50% for H9 subtype. Correlation analysis further revealed a negative correlation between the operation of exhaust facilities and the positive rates of AIV and H9 subtype. Subsequently, 30 AIV strains were isolated via chicken embryo inoculation, 12 of which were sequenced and identified to be H9N2 isolates. Genetic evolution analysis further indicated that these isolates were assigned into the G57 genotype. Furthermore, homology modeling revealed that mutations of V²²³A and N²²⁴H were identified at the receptor binding site of HA protein in several novel AIV isolates, which may lead to the variation in spatial conformation. Overall, the findings investigate the prevalence of AIV in LPMs and biomolecular attributes of H9N2 subtype, and provide some theoretical reference for future health risk assessment and preventive strategies of AIV.

Wastewater-based epidemiology (WBE) has traditionally served as a tool for monitor pathogens, biomarkers, and consumption of pharmaceuticals or illicit drugs. In particular, enteric viruses have been extensively studied in wastewater due to their high titer of excretion. In this study, we investigated the presence of six clinically significant enteric viruses in twelve different areas of a Spanish middle-size city (Burgos), over a 3-year period from November 2021 to November 2024 (n = 600). Viral concentration was performed using an aluminum-based adsorption-precipitation method, followed by nucleic acid extraction and quantification via RT-qPCR. Process controls were included in each experiment to ensure assay accuracy and to calculate viral recovery rates, providing reliable estimates of enteric virus concentrations. The findings revealed that norovirus genogroup II was the most prevalent virus detected in 97.50% of the samples, followed by human astroviruses (90.00%), norovirus genogroup I (85.33%), rotavirus (83.83%), hepatitis E Virus (12.17%), and hepatitis A Virus (0.33%). Spatial heterogeneity in viral distribution was observed among sampling sites, along with temporal and seasonal variations between the COVID-19 pandemic and the post-pandemic periods. A positive correlation was found between enteric viruses and SARS-CoV-2, with both groups of viruses generally displaying stable co-existence. In our hands, this study represents the first long-term WBE analysis of enteric viruses conducted in a middle-sized city, providing valuable insights into the distribution, dynamics, and behavior of major enteric viruses across an extended temporal frame and different areas of the city, spanning both pandemic and post-pandemic contexts.

Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR–Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25 g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.

Environmental matrices (EMs) are important reservoirs for the human bocavirus (HBoV). This study aimed to determine HBoV prevalence in EMs and model its association with SDG6.3.1 (wastewater production (WWp), collection (WWc), treatment (WWt), and reuse (WWr)). HBoV data-mined from EMs were fitted to a random-intercept-logistic regression/1000-bootstrapped-based meta-regressions. HBoV global prevalence in EMs was 42.19% (95%CI: 28.07–57.72), and varied non-statistically across continents (North America (66.59%), Africa (42.32%), Europe (41.95%), Asia (39.96%), South America (20.55%)), economies (high-income (45.04%) > upper-middle-income (42.56%) > lower-middle-income (41.35%)), WHO regions (Western Europe (41.95%) > Middle East & North Africa (41.35%) > East Asia & Pacific (39.96%) > North America (66.59%) > Latin America & Caribbean (20.55%)) but significantly across dwelling settings (urban: 52.02% versus rural: 20.29%) and sample types (raw sewage (RS, 73.16%) > treated sewage (TS, 43.47%) > sewage sludge (SS, 19.87%) > sediment (13.24%) > surface waters (18.55%)). HBoV subtypes circulating in EMs varied among sample types (HBoV1 in TS (40.74%) > RS (22.45%) > surface water (9.09%); HBoV2 in RS (75.42%) > TS (54.82%) > surface water (18.24%); HBoV3 in RS (64.74%) > TS (58.95%) > surface water (6.48%) > SS (3.49%)). HBoV prevalence in EMs had direct relationship (p ≤ 0.05) with SDG6.3.1 variables (WWp: F_1;35 = 4.5822), and WWr: F_1;35 = 4.3735; WWt: F_1;35 = 3.9517; and WWc: F_1;35 = 3.3510) accounted for 17.13%, 15.79%,15.65%, and 12.92% of the estimate variance, respectively. In conclusion, HBoV prevalence is high in EMs globally, across regions, sample types and showed considerable affinity with SDG6.3.1 variables.

Hepatitis E virus (HEV) infection is an emerging zoonosis that can be transmitted to humans through the consumption of raw or undercooked pork products. Several methods have been described to detect the virus in food, but there are few data on qualitative and quantitative performance characteristics. In this study, we have developed an optimised method for quantitative detection of HEV in pork sausage based on a combination of previously published protocols. The method utilises sample disruption and phase separation with tri-reagent and 1-bromo-3-chloropropane followed by RNA concentration with isopropanol precipitation. We validated the method for use on reverse transcription quantitative real-time PCR (RT-qPCR) and reverse transcription droplet digital (RT-ddPCR). The 95% limit of detection and limit of quantification was 200 copies/g for both RT-qPCR and RT-ddPCR. RT-ddPCR technology has previously shown promise as a more precise alternative to RT-qPCR. However, we found no evidence for improved performance using RT-ddPCR instead of RT-qPCR for this method. Additionally, we further verified the performance of the HEV RT-PCR assay using the WHO International Standard and Reference Panel for HEV RNA. Finally, we assessed different combinations of RNA concentration protocols and RT-PCR detection strategies. This showed that isopropanol precipitation of viral RNA was at least twice as efficient as magnetic silica bead-based extraction when an inhibitor-tolerant RT-qPCR detection strategy was used. In summary, we present an efficient and well-characterised method for quantitative detection of HEV in pork sausage. Such methods are valuable to provide high-quality data for risk assessments and food monitoring.