Food and Environmental Virology最新文献

Noroviruses, belonging to the family Caliciviridae, are classified into at least ten genogroups (G) based on their major capsid protein (VP1). The common genogroup to be identified in both humans and pigs is GII, although porcine noroviruses (PoNoVs) belong to genotypes of their own (GII.11, GII.18, and GII.19). So far, PoNoVs have not been studied much in Finland, possibly due to their rather symptomless nature in pigs. In the present study, we enrolled a total of 189 fecal samples collected from pigs from Finnish farms. Samples were taken from 12 farms in 2010, 2019 and 2020. We analyzed feces from growing pigs ranging from 2.1 to 6 months of age. RNA was extracted from fecal suspensions using a commercial viral RNA extraction kit, followed by RT (reverse transcription)-qPCR. The genotypes were determined by Sanger sequencing of the PCR fragments amplified by conventional PCR. Of the 12 farms, 6 (50%) had at least one PoNoV-infected pig. Altogether 18 (9.5%) of the 189 pigs tested positive for PoNoVs. Pigs mostly aged over 4 months were infected with PoNoVs. Eventually, 12 positive samples were determined as genotype GII.18. We could demonstrate the presence of PoNoVs in Finnish pigs. In future, more studies in which longer sequences from PoNoV genome can be obtained, are required.

Wastewater-based epidemiology (WBE) is a recognized, dynamic approach to monitoring the transmission of pathogens in communities through urban wastewater. This study aimed to detect and quantify influenza A and B viruses in Italian wastewater during the 2022–2023 season (October 2022 to April 2023). A total of 298 wastewater samples were collected from 67 wastewater treatment plants (WTPs) across the country. These samples were analyzed for influenza A and B viruses (IAV, IBV) using primers originally developed by the Centers for Disease Control and Prevention (CDC) for real-time PCR and adapted for digital PCR. The overall detection rates of IAV and IBV across the entire study period were 19.1% and 16.8%, respectively. The prevalence of IAV in wastewater showed a gradual increase from October to December 2022, peaking at 61% in December. In contrast, IBV peaked at 36% in February 2023. This temporal discrepancy in peak concentrations suggests different seasonal patterns for the two influenza types. These trends mirrored human surveillance data, which showed influenza A cases peaking at 46% in late December and declining to around 2% by April 2023, and influenza B cases starting to increase significantly in January 2023 and peaking at about 14% in March. IAV concentrations ranged from 9.80 × 10² to 1.94 × 10⁵ g.c./L, while IBV concentrations ranged from 1.07 × 10³ to 1.43 × 10⁴ g.c./L. Overall, the environmental data were consistent with the human surveillance trends observed during the study period in the country. These results demonstrate the value of WBE in tracking epidemiological patterns and highlight its potential as a complementary tool to infectious diseases surveillance systems.

Wastewater-based epidemiology (WBE) has emerged as a valuable surveillance tool for SARS-CoV-2 and other pathogens globally, providing insights into community-level infections, including asymptomatic and pre-symptomatic cases. While most WBE programmes focus on quantitative pathogen assessment, next-generation sequencing (NGS) approaches have enabled more detailed analyses, including variant and recombinant genotype identification for viruses like SARS-CoV-2 and poliovirus. Despite recent NGS advancements allowing for the detection of known and novel viruses in wastewater, many of these tools remain underutilised in routine WBE. This short review critically evaluates the applicability of common NGS tools in routine WBE programmes, assessing their capability for identifying emerging threats with epidemic or pandemic potential. Here, we provide evidence-based recommendations for integrating NGS techniques into WBE and the use of results for informed decision-making within a One Health framework, aiming to enhance global infectious disease surveillance and pandemic preparedness.

As SARS-CoV-2 continues to evolve and herd immunity establishes, an increasing number of asymptomatic infections have been reported, increasing the risk of airborne spread of the virus. Most of the studies regarding SARS-CoV-2 RNA presence in air refer to indoor environments, with few studies having reported SARS-CoV-2 RNA in outdoor air. The aim of this study was to assess the presence of SARS-CoV-2 RNA at two different settings, crowded outdoor versus empty outdoor environments in Valladolid, Spain, during winter 2021. Using a Coriolis® air sampler, samples were taken from nine different locations within the city center. RNA extraction and a one-step RT-qPCR were carried out. Six out of the 20 air samples were found to be positive, and they were all obtained from crowded outdoor environments. These results highlight that although in less quantity, SARS-CoV-2 RNA is still present in outdoor air, especially at moments of relaxed mitigation efforts and depending on the number of people present.

Raw vegetables irrigated with polluted water that may contain enteric viruses can be associated with foodborne viral disease outbreaks. The objective of this study is to investigate the possible transmission of enteric viruses from irrigation water to lettuce. Therefore, we performed a commercial multiplex real-time PCR assay to monitor the occurrence of enteric viruses in irrigation water samples and in raw vegetables that were cultivated at market gardening sites in Ouagadougou, Burkina Faso. Samples were collected from six market gardening sites located in Ouagadougou. RT-PCR was performed to detect norovirus GI, norovirus GII, rotavirus, enteric adenoviruses F (Serotype 40/41), astrovirus and sapovirus (Genogroups G1, 2, 4, 5). From the 10 irrigation water samples and the 80 lettuce samples, three (30%) and twenty-two (27.5%) were positive for enteric viruses, respectively. Norovirus GII, astrovirus and enteric adenoviruses F (Serotype 40/41) were the most frequently detected viruses in lettuce and irrigation water samples. Our results indicate that raw vegetables may be contaminated with a broad range of enteric viruses, which may originate from virus-contaminated irrigation water, and these vegetables may act as a potential vector of food-borne viral transmission.

Hepatitis E virus (HEV) is primarily transmitted via the fecal–oral route and is considered an anthropozoonosis. Genotypes with zoonotic potential (mainly HEV-3 and HEV-4) can be transmitted through the consumption of raw or undercooked pork, wild boar, deer meat, or processed products. This study aims to explore methodologies for processing meat samples to establish a protocol for HEV detection in meat. The analysis of pre-analytical conditions involved comparing homogenization with PBS versus TRIzol, comparing tissue disruption methods (ultra-turrax versus mortar and pestle), and assessing nucleic acid extraction techniques (spin columns and magnetic beads) across three types of artificially contaminated meat matrices: pork, salmon (fish-meat), and salami. Each test included a process control virus (PP7) and an HEV transcript. Molecular detection was performed via RT-qPCR. Results indicated that TRIzol provided better recovery rates for homogenization, while spin columns were the most effective option for RNA extraction. Both the ultra-turrax homogenizer and the mortar-pestle methods were effective for pork and fish-meat homogenization, while the use of the UT yielded superior results for salami. HEV recovery rates were 36.7%, 26.3%, and 34.1% for salami, salmon, and pork meat, respectively. In conclusion, we reached a simple and reliable protocol for the detection of RNA-HEV from three meat matrices. This method, which includes homogenization with TRIzol, mechanical tissue disruption, and RNA extraction using spin columns followed by real-time PCR, can be applied in future studies to evaluate HEV prevalence in food sources and contribute to the discussion about HEV detection methodologies.

The bourgeoning field of wastewater-based epidemiology (WBE) for the surveillance of several respiratory viruses which includes Influenza A, H1N1pdm09, H3N2, respiratory syncytial viruses (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of interest for public health concerns. However, there are few long-term monitoring studies globally. In this study, respiratory viruses were detected and quantified from 11 sewer sheds by utilizing reverse transcription-quantitative polymerase chain reaction analysis in Pune city, India, from Jan 2022 to Dec 2023. The RNA fragments of respiratory viruses were detected in sewage samples before clinical cases were reported, underscoring the potential of WBE for early detection and monitoring within the population. The Spearman correlation of wastewater viral copies was positively and significantly correlated with the clinically positive case of H1N1pdm09 (ρ = 0.55, p = 1.4 × 10^–9), H3N2 (ρ = 0.25, p = 9.9 × 10^–3), and SARS-CoV-2 (ρ = 0.43, p = 4.1 × 10^–6). The impact of public health interventions on the circulation of infectious respiratory diseases showed a significant difference in the viral load during the period when many preventing measures were carried out against the COVID-19 pandemic (restriction phase), compared to the period when no such preventive measures are followed (no-restriction phase) for Influenza A, H1N1pdm09, H3N2, and RSV with p-value < 0.05, which indicates the influence of health policy implementation in controlling disease spread. The present study provides an effective approach to detecting multiple respiratory viruses from wastewater and provides insights into the epidemiology of respiratory illnesses. The WBE aids in providing information on the spread of pathogens (viruses) in the community, offering a proactive strategy for public health management, allowing for timely interventions and implementing targeted measures to mitigate the spread of these viruses under one health approach.

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Conventional UV-C (254 nm) inactivation technologies have limitations and potential operator-safety risk. To overcome these disadvantages, novel UV-C light-emitting diodes (LED) are developed and investigated for their performance. This study aimed to determine the inactivation of human norovirus (HuNoV) surrogates, Tulane virus (TV), and feline calicivirus (FCV-F9), by UV-C (254 nm) in comparison to UV-C LED (279 nm) in phosphate-buffered saline (PBS) and coconut water (CW). Five-hundred microliters of FCV-F9 (~ 5 log plaque forming units (PFU)/mL) or TV (~ 6 log PFU/mL) were added to 4.5 mL PBS or CW in continuously stirred glass beakers and exposed to 254 nm UV-C for 0 up to 15 min (maximum dosage of 33.89 mJ/cm²) or 279 nm UV-C LED for 0 up to 2.5 min (maximum dosage of 7.03 mJ/cm²). Recovered viruses were assayed in duplicate from each treatment replicated thrice. Mixed model analysis of variance was used for data analysis. Significantly lower D₁₀ values were obtained in PBS and CW (p ≤ 0.05) for both tested viruses using UV-C LED (279 nm) where FCV-F9 showed D₁₀ values of 7.08 ± 1.75 mJ/cm² and 3.75 ± 0.11 mJ/cm², while using UV-C (254 nm) showed D₁₀ values of 13.81 ± 0.40 mJ/cm² and 6.43 ± 0.44 mJ/cm² in PBS and CW, respectively. Similarly, lower D₁₀ values were obtained for TV of 3.91 ± 1.03 mJ/cm² and 4.26 ± 1.02 mJ/cm² with 279 nm UV-C LED and were 18.76 ± 3.16 mJ/cm² and 10.21 ± 1.48 mJ/cm² with 254 nm UV-C in PBS and CW, respectively. Viral resistance to these treatments was fluid-matrix dependent. These findings indicate that use of 279 nm UV-C LED is more effective in inactivating HuNoV surrogates than conventional 254 nm UV-C in the tested fluids.

Human norovirus (HuNoV) is the leading cause of foodborne illness in the developed world and a major contributor to gastroenteritis globally. Its low infectious dose and environmental persistence necessitate effective disinfection protocols. Sodium hypochlorite (NaOCl) bleach is a widely used disinfectant for controlling HuNoV transmission via contaminated fomites. This study aimed to evaluate the susceptibility of HuNoV genotypes (n = 11) from genogroups I, II, and IV to NaOCl in suspension. HuNoV was incubated for 1 and 5 min in diethyl pyrocarbonate (DEPC) treated water containing 50 ppm, 100 ppm, or 150 ppm NaOCl, buffered to maintain a pH between 7.0 and 7.5. Neutralization was achieved by a tenfold dilution into 100% fetal bovine serum. RNase pre-treatment followed by RT-qPCR was used to distinguish between infectious and non-infectious HuNoV. Statistical methods, including imputation, machine learning, and generalized linear models, were applied to process and analyze the data. Results showed that NaOCl reduced viral loads across all genotypes, though efficacy varied. Genotypes GI.1, GII.4 New Orleans, and GII.4 Sydney were the least susceptible, while GII.6 and GII.13 were the most susceptible. All NaOCl concentrations above 0 ppm were statistically indistinguishable, and exposure duration did not significantly affect HuNoV reduction, suggesting rapid inactivation at effective concentrations. For instance, some genotypes were completely inactivated within 1 min, rendering extended exposure unnecessary, while other genotypes maintained the initial concentration at both 1 and 5 min, indicating a need for longer contact times. These findings underscore the critical role of HuNoV genotype selection in testing disinfection protocols and optimizing NaOCl concentrations. Understanding HuNoV susceptibility to NaOCl bleach informs better disinfection strategies, aiding public health and food safety authorities in reducing HuNoV transmission and outbreaks.

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.