Food and Environmental Virology最新文献

Capsid Integrity qPCR (CI-qPCR) assays offer a promising alternative to cell culture-based infectivity assays for assessing pathogenic human virus viability in wastewater. This study compared three CI-qPCR methods: two novel (Crosslinker, TruTiter) and one established (PMAxx dye). These methods were evaluated on heat-inactivated and non-heat-inactivated ‘live’ viruses spiked into phosphate-buffered saline (PBS) and wastewater, as well as on viruses naturally present in wastewater samples. The viral panel included Human adenovirus 5 (HAdV), enterovirus A71 (EV), hepatitis-A virus (HAV), influenza-A H3N2 (IAV), respiratory syncytial virus A2 (RSV), norovirus GI, norovirus GII, and SARS-CoV-2. All three methods successfully differentiated between degraded, heat-inactivated, and live viruses in PBS. While all three methods were comparable for HAdV and norovirus GI, PMAxx detected significantly lower gene copies for EV and IAV. In spiked wastewater, PMAxx yielded significantly lower gene copies for all heat-inactivated viruses (HAdV, EV, HAV, IAV, and RSV) compared to the Crosslinker and TruTiter methods. For viruses naturally present in wastewater (un-spiked), no significant difference was observed between PMAxx and TruTiter methods. Intact, potentially infectious viruses were detected using both PMAxx and TruTiter on untreated and treated wastewater samples. A comparative analysis of qPCR data and TEM images revealed that viral flocculation of IAV may interfere with capsid integrity assays using intercalating dyes. In summary, our findings not only advance the development of more effective methods for assessing viral viability in wastewater, but also highlight the potential of CI-qPCR techniques to enhance early warning systems for emerging pathogens, thereby strengthening public health preparedness and response strategies.

The attempt to investigate hepatitis E virus (HEV) contamination in naturally growing mangrove bivalve mollusks captured for local sale in a touristic area of Maranhão state in Brazil revealed the detection of rat hepatitis E virus (ratHEV). Using international standard protocols for processing and nucleic acid extraction, we analyzed 89 bivalve samples (Mytella falcata and Crassostrea rhizophorae) with two broadly reactive assays: heminested pan-Hepeviridae (ORF-1) and probe-based HEV-1 to HEV-4 (ORF-2/ORF-3). Heminested reactions presented 2 (2.2%) amplifications of the expected size. Nucleotide identities ranged from 86.6 to 89.0% with ratHEV isolates from wild rats, pigs, one human case and sewage reported in different countries. Regarding the phylogenetic tree, the sequences grouped with genotype HEV-C1. This first report of ratHEV detection in bivalve mollusks may be a starting point for further research on virus variability, distribution, host range and especially the possible role of contaminated shellfish as a vehicle for ratHEV transmission.

Wastewater-based surveillance has emerged as a powerful approach to monitoring infectious diseases within a community. Typically, wastewater samples are concentrated before viral analyses to improve sensitivity. Current concentration methods vary in time requirements, costs, and efficiency. Here, we evaluated the concentration efficiency and bias of a novel viral concentration approach, Nanotrap Microbiome Particles (NMP), in wastewater. NMP concentration efficiency was target-specific, with significantly lower concentrations of the bacterial indicator HF183 and viral indicator Carjivirus (formerly crAssphage) relative to direct extraction (1.2 × 10⁵ vs. 3.4 × 10⁵ GC/mL and 2.0 × 10⁵ vs. 1.2 × 10⁵ GC/mL, respectively), but significantly higher concentrations of the viral fecal indicator Pepper Mild Mottle Virus (PMMoV) relative to direct extraction (1.4 × 10⁵ vs. 8.4 × 10³ GC/mL). Targeted metagenomic sequencing showed that NMP resulted in significantly more unique species reads per sample than direct extractions (p < 0.001) by detecting species that went undetected by direct extractions. Key viral families identified with high abundances were Adenoviridae, Caliciviridae, Herpesviridae, Papillomaviridae, and Polyomaviridae. NMP showed differential ability for concentrating clinically relevant viral families, suggesting that the technology should be evaluated and optimized for specific viral targets before implementation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus are primarily transmitted through droplets or aerosols from patients. The inactivation effects of existing virus control techniques may vary depending on the environmental factors. Therefore, it is important to establish a suitable evaluation system for assessing virus control techniques against airborne viruses for further real-world implementation. This study aimed to assess the inactivating effects of chemical substances on SARS-CoV-2 and influenza A virus in the air using an established evaluation system. A mixture containing SARS-CoV-2 and influenza A virus in diluted saliva was nebulized into the designed 1 m³ chamber, and the virucidal effects of hypochlorous acid, chlorine dioxide, and ozone in the air samples at 23 ± 1 °C with 50 ± 5% relative humidity were determined using the plaque assay. Both viral infectivity titers decreased depending on chemical substance concentration and exposure time. The concentrations of hypochlorous acid, chlorine dioxide, and ozone in the air reached an approximately 2-log reduction of SARS-CoV-2 infectivity titer within 10 min at 0.02, 1.0, and 1.0 ppm, respectively. SARS-CoV-2 persisted in the air even under conditions where the influenza A virus was inactivated below the detection limits. These findings demonstrate that hypochlorous acid, chlorine dioxide, and ozone are effective in inactivating SARS-CoV-2 and influenza A virus in the air.

Wastewater surveillance for pathogens is important to monitor disease trends within communities and maintain public health; thus, a quick and reliable protocol is needed to quantify pathogens present in wastewater. In this study, a method using a commercially available magnetic carbon bead-based kit, i.e., the Carbon Prep (C.prep) method (Life Magnetics), was employed to detect and quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bacteriophage Phi6 and pepper mild mottle virus (PMMoV) in wastewater samples. The performance of this method was evaluated by modifying several steps and comparing it with the polyethylene glycol (PEG) precipitation method to demonstrate its applicability to virus detection in wastewater. The protocol of the C.prep method, based on the manufacturer’s instructions, could not detect SARS-CoV-2 RNA, while the optimized protocol could detect it in the tested samples at concentrations that were not significantly different from those obtained using the PEG precipitation method. However, the optimized C.prep method performed more poorly in recovering Phi6 and detecting PMMoV than the PEG precipitation method. The results of this study indicated that the full workflow of the C.prep method was not sufficient to detect the target viruses in wastewater and that an additional RNA extraction step was needed to increase its detection sensitivity.

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.