Food and Environmental Virology最新文献

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.

Since molecular typing method of enterovirus was introduced, many new types have been discovered. However, due to the low epidemic potential, the information on newer enteroviruses remains limited globally. This study aims to investigate the diversity and phylogeny of newer enterovirus types through environmental surveillance utilizing next-generation sequencing (NGS) technology. Sixty-six wastewater samples were collected from two cities in eastern China between 2019 and 2021 and concentrated 100-fold using a negatively charged membrane method. After cell culture, 388 enterovirus isolates representing 14 serotypes were recovered, including Sabin-like poliovirus type 1 (n = 22) and type 3 (n = 57). Concurrently, RNA extraction was performed on all 66 sewage concentrates, and VP1 semi-nested RT-PCR yielded 56 amplicons, which were subsequently subjected to NGS. The NGS analysis identified a total of 33 serotypes, with echovirus 11, coxsackievirus A10, echovirus 18, coxsackievirus B4, and coxsackievirus B5 being the most prevalent, accounting for 29.11%, 9.87%, 8.27%, 8.14%, and 6.10% of the total reads, respectively. Newer identified enterovirus types A76, A89, A90, and C113 were detected in 1 (1.52%), 17 (25.76%), 5 (7.58%), and 6 (9.09%) sewage samples, respectively. Phylogenetic analysis based on the partial VP1 coding region indicated that these local enteroviruses formed distinct lineages separate from previously identified strains. These findings demonstrate that sewage contains enteroviruses with considerable diversity. NGS-based sewage surveillance offers a significant advantage in data output compared to the cell culture method and can be effectively utilized for monitoring newer enterovirus strains.

Graphical Abstract

Norovirus and rotavirus are the major causes of acute gastroenteritis in humans worldwide. Due to their small size, these enteric viruses present in wastewater become aerosolized. The objective of this study was to assess the presence and concentrations of norovirus and rotavirus in aerosol samples collected from wastewater treatment plants (WWTPs) in Thailand. A developed method for concentrating viruses and performing molecular detection was used to determine naturally occurring enteric viruses. Of the 24 collected aerosol samples, 8 (33.3%) and 2 (8.3%) tested positive for norovirus RNA using RT-qPCR and RT-nested PCR, respectively. Based on RT-qPCR, norovirus GII RNA was detected more frequently in aerosol samples (7/24, 29.2%) compared to norovirus GI RNA (2/24, 8.3%). The norovirus GI concentrations were 9.8 × 10² and 3.2 × 10³ genome copies/m³. The norovirus GII concentrations ranged from 1.5 × 10² to 5.5 × 10³ genome copies/m³. RT-nested PCR detected norovirus GII RNA and the rare GII.21 norovirus strains were identified in the two aerosol samples. However, rotavirus RNA was not detected in any of the aerosol samples using either RT-qPCR or RT-nested PCR. This study highlights the quantification and genotyping of norovirus in aerosol samples generated from wastewater, suggesting a potential effect of airborne transmission for WWTPs workers.

Hepatitis E virus (HEV) is a cause of enterically transmitted hepatitis around the world. Because of the high frequency of asymptomatic infections, the magnitude of HEV infection is underestimated. Wastewater monitoring could be useful to improve our knowledge on HEV epidemiology. In this study, we analyzed the capacity of wastewater surveillance to give an insight into the circulation and the diversity of HEV in two French cities. HEV RNA was detected and quantified by digital PCR in 115 untreated composite wastewater samples collected weekly at the inlet of wastewater treatment plants (WWTPs), 58 at Toulouse WWTP and 57 at Dunkerque WWTP. Plasma HEV RNA in blood donors was detected by a commercial assay (Roche Cobas) over the same period in the same area. HEV diversity was analyzed using long-read single-molecule real-time sequencing (Pacific Biosciences). HEV RNA was detected in 88% and 95% wastewater samples collected at Toulouse (Occitanie region, Southern France) and Dunkerque (Hauts-de-France region, Northern France) WWTPs, respectively. HEV RNA concentration ranged between 4.1 and 5.7 log copies/L and was almost similar between the two sites. A long orf2 fragment of HEV genome (1030 nucleotides) was obtained and sequenced in 45% and 70% of positive HEV RNA wastewater samples collected at Toulouse site and Dunkerque site, respectively. Out of 31 strains identified in Toulouse wastewater, 24 were HEV-3c (77%), 6 were HEV-3f (19%), and 1 was HEV-3h (3%). Out of 55 strains identified in Dunkerque, 30 were HEV-3c (55%) and 25 were HEV-3f (45%). All HEV RNA-positive samples from blood donors that could be genotyped during the study period contained HEV-3. Subtype distribution in 51 blood donors living in Toulouse did not differ from that in Toulouse wastewater. The HEV-3 subtype distribution in 51 Hauts-de-France region blood donors and in Dunkerque wastewater were different, but the predominant subtype was the same (HEV-3c). Lastly, we explored the link between the measurement of viral loads in wastewater and the extent of infection in the served population. Although a good correlation between the peaks of positive HEV RNA estimated in wastewater samples and that observed in blood donors was observed with a lag of + 3 weeks for Toulouse, the correlation was weaker for Dunkerque. Wastewater surveillance system applied locally could be very useful for assessing the HEV infection status of a population.

The effects of allyl isothiocyanate (AITC) on enteric viruses, specifically hepatitis A virus (HAV) and murine norovirus (MNV) as a norovirus surrogate, were evaluated at different concentrations, temperatures, and exposure time. AITC at 0.1 and 0.5% was mixed with each virus and incubated at 10, 25, and 37 °C for 2 h or overnight. AITC demonstrated a concentration-, temperature-, and time-dependent antiviral effect, with the lowest concentration resulting in a modest decrease in viral titer. However, at the highest concentration and 37 °C during overnight incubation, reductions of 3.75 log TCID50/mL for MNV and below the limit of detection for HAV were reported. Additionally, efficacy of AITC was evaluated on human norovirus (HuNoV) GI suspensions using an in situ capture quantitative reverse transcription polymerase chain reaction (RT-qPCR) method. The results indicated that HuNoVs are susceptible to AITC at 37 °C, which partially inhibits the interaction between the viral capsid and its receptor. Furthermore, AITC was tested as a natural disinfectant for produce with treatment times of 15 and 30 min, with no statistically significant changes in viral titers. Although further optimization of AITC application is required, these findings suggest that AITC has potential as a tool to reduce enteric virus contamination on food and food-contact surfaces.

This study presents a comprehensive assessment of viral contamination and antibiotic resistance genes (ARGs) presence in mussels (Mytilus galloprovincialis) (n = 60) collected from retail stores in the Campania region (Italy). High prevalence of human noroviruses (HuNoV) genogroup I (GI) (77%) and genogroup II (GII) (40%), rotaviruses (RV) (60%), and astroviruses (HAstV) (25%) was found, with average levels of 4.34, 5.09, 5.05, and 4.00 Log genome copies (GC)/g, respectively. All samples tested negative for hepatitis A and E viruses. Viral faecal contamination indicators, including somatic coliphages (88%, 3.62 mean Log plaque forming units (PFU)/100 g) and crAssphage (50%, 3.72 mean Log GC/g), showed strong correlations (ρ > 0.65, p-value < 0.05) with HuNoV GII, HAstV, and RV concentrations in mussels. The study also investigated the presence of respiratory viruses, with all samples testing negative for SARS-CoV-2, respiratory syncytial virus, and influenza A virus.

Furthermore, a capsid-integrity RT-qPCR assay was applied to selected positive samples, confirming the presence of potentially infectious viruses and underscoring the associated risks to consumers.

Additionally, ARGs were detected by qPCR, targeting beta-lactams, quinolones, and chloramphenicol resistance genes in both the total and the bacteriophage fractions of selected samples.

Overall, this study emphasizes the importance of continued surveillance and strategic interventions to mitigate public health risks associated with the consumption of contaminated bivalve molluscan shellfish (BMS), which may imply the dissemination of infectious enteric viruses and ARGs within communities.