Food and Environmental Virology最新文献

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis globally, with frozen berries frequently implicated in foodborne outbreaks. Current surveillance relies on quantitative reverse transcription PCR (qRT-PCR), which cannot differentiate between infectious and non-infectious viral particles, complicating risk assessment. This study is aimed to establish the minimum viral load on frozen berries detectable by qRT-PCR that corresponds to infectious virus, using murine norovirus (MNV) as a surrogate for HuNoV. Frozen raspberries were artificially inoculated with serial dilutions of MNV (7.1–1.0 log PFU/25 g) and processed using the ISO 15216:2017 method. Infectious virus was quantified by plaque assay, and viral RNA was detected by qRT-PCR. The limit of detection (LOD) for cell culture was 3.1 log PFU/25 g, whereas qRT-PCR extended sensitivity to 1.0 log PFU/25 g (Ct value at 36.7 ± 0.6), representing a 2-log difference. Recovery rates for infectious virus exceeded the ISO 15,216 minimum threshold (1%), and PCR inhibition was negligible. We next examined the extraction efficiency for both infectious MNV and its genetic material from frozen strawberries at inoculation levels higher than the LOD, and observed that the viral recovery from frozen strawberries is very similar to viral recovery from frozen raspberries with no significant differences between them. The disparity between LODs indicates that a substantial proportion of MNV genomes detected by qRT-PCR do not represent infectious particles, aligning with previous findings that one PFU may correspond to multiple genome copies. Given that many surveillance studies report high Ct values (> 35), our data suggest that such detections may not indicate viable virus, underscoring the importance of contextualizing qRT-PCR results with epidemiological evidence. These findings highlight the need for cautious interpretation of surveillance data, particularly for public health decision-making.

Monitoring SARS-CoV-2 RNA in industrial wastewater has emerged as a potential approach to indirectly identify infected individuals within workplace populations. However, wastewater-based surveillance of SARS-CoV-2 in industrial settings has not previously been conducted in Thailand. This study aimed to qualitatively and quantitatively detect SARS-CoV-2 RNA in wastewater discharged from onsite wastewater treatment plants (WWTPs) and to estimate the number of infected individuals. A total of 120 wastewater samples were consecutively collected from 40 onsite WWTPs comprising 38 factories and two non-factory buildings with over a three-month period (April to June 2023), after the Songkran festival, Thailand’s traditional new year celebration. Wastewater samples were concentrated, and the total nucleic acids were extracted. SARS-CoV-2 RNA concentrations were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) targeting the N gene. SARS-CoV-2 RNA was detected in 42.5%, 62.5%, and 40.0% of samples collected in April, May, and June, respectively. The concentrations ranged from 3.5 × 10²–1.1 × 10⁴ copies/L in April, 4 × 10²–1.7 × 10⁵ copies/L in May, and 1.1 × 10³–1.4 × 10⁵ copies/L in June. A higher frequency of SARS-CoV-2 RNA detection was observed in factories having more than 1000 employees. Based on our modified estimation model, the estimated infected individuals were 219, 3145, and 2711 for April, May, and June, respectively. The numbers of estimated infected population closely aligned with the official COVID-19 case trending reported in Pathum Thani province and at the national level during the same period. These findings suggest that wastewater-based epidemiology (WBE) is a reliable tool for measuring, estimating, and assessing COVID-19 dynamics in industrial setting.

Human norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis globally. Histo-blood group antigens (HBGAs) have been recognized as attachment factors for HuNoV. However, the receptors or other potential attachment factors for HuNoV have not been fully elucidated. A bacterial cell surface-displayed system was used to express GII.4 HuNoV protruding domain (GII.4 P) to capture attachment factors from a diarrheal sample of a patient infected with GII.4 HuNoV. A library of candidate HuNoV proteinaceous attachment factors was constructed. Through a database comparison, a literature review, the predicted protein subcellular localization, the protein–protein interactions (PPIs) network analysis, the molecular simulation and the protein docking analysis, four candidate attachment factors were selected for further investigation from the initial 118 candidates. Finally, two proteins, namely myelin-oligodendrocyte glycoprotein (MOG) and leucine-rich repeat-containing protein 15 (LRRC15), were demonstrated strongly binding to GII.4 P by both in vivo and in vitro assays. The binding between MOG/LRRC15 and GII.4 P can be blocked by GII.4 HuNoV antibody. Immunofluorescence showed that MOG/LRRC15 and GII.4 HuNoV co-localized in the cytoplasm in transfected cells. However, GII.4 HuNoV did not proliferate in the newly constructed MOG/LRRC15 overexpressing cells. This study describes attempts to identify HuNoV proteinaceous attachment factors from the diarrheal sample. Findings from this study will aid in understanding of HuNoV infection in humans, enrichment of GII.4 HuNoV from various environments and construction of cell lines for HuNoV cultivation in vitro.

The global emergence of SARS-CoV-2 has highlighted the urgent need for effective disinfection strategies to mitigate virus transmission. Electrolyzed water (EW), an eco-friendly and cost-effective biocidal agent, has garnered attention for its broad-spectrum antimicrobial activity. This study evaluates the virucidal, bactericidal, and fungicidal capacities of EW with diverse pH, with a focus on its effectiveness against SARS-CoV-2 and other pathogens. EW was generated under controlled conditions with adjustable pH (4.5 and 6.1) and free available chlorine (FAC) concentrations (300–1000 ppm). The biocidal activity was tested on surfaces and in solution following standardized protocols. Results demonstrated that oxidized EW at optimized concentrations achieved a ≥ 4 log reduction in bacterial populations and effectively inactivated enveloped and non-enveloped viruses, but this effect depends on FAC, pH, and contact time. Notably, EW reduced SARS-CoV-2 by > 4 log in solution and surfaces, in only 2 and 5 min, respectively. These findings highlight the potential of EW as a sustainable, multi-application disinfectant to combat emerging pathogens like SARS-CoV-2, ensuring public health safety.

Noroviruses are the leading cause of gastroenteritis outbreaks in humans worldwide. Their unique properties ensure stability over extended periods under adverse conditions, which enhances their risk as food and water contaminants. In recent years, intensive research has focused on the natural antimicrobial potential of plant metabolites as disinfectants against environmental pathogens. The oregano essential oil (OEO) has gained attention due to its valuable properties, including antimicrobial, antioxidant, antiviral, and antifungal activities. However, the susceptibility of OEO to degradation and oxidation under environmental or storage conditions, coupled with its low water solubility, has limited its practical applications. Nanoencapsulation has emerged as a promising strategy to overcome these limitations by prolonging shelf life, improving stability, enabling controlled release, and expanding its potential uses. In this study, we evaluated the virucidal potential of chitosan-based polymeric nanoparticles incorporating Origanum vulgare essential oil against murine norovirus 1 (MNV-1) for food and environmental applications. To assess the virucidal effect of the OEO nanoparticles, the reduction in viral infectivity was determined by comparing the TCID₅₀/mL values of untreated viral suspensions with those treated with the tested compounds at varying concentrations. The results demonstrated effective viral inactivation at all tested concentrations, with the undiluted formulation (40 mg/mL incorporated OEO) achieving the highest inactivation rate (99.72%). The blank formulation showed no significant virucidal activity, while the pure OEO exhibited cytotoxicity at most tested concentrations. These findings support the development of a biotechnological disinfectant with potential applications in both environmental and controlled conditions.

Currently, the predominant commercially available disinfectants and sanitizers are formulated with active ingredients including alcohol, halogenated compounds (e.g., sodium hypochlorite), surfactants, oxidizing agents (eg., hydrogen peroxide), and quaternary ammonium compounds. Natural products represent an alternative since they are often effective microbicides and are generally recognized as safe (GRAS) due to their long history of use. The purpose of this study was to evaluate the efficacy of several organic acids and plant essential oils against three bacteriophages (MS2, ΦX174, PR772) with different genome characteristics (e.g., RNA versus DNA, single-stranded versus double-stranded) and against an enveloped and non-enveloped mammalian virus (human coronavirus 229E and feline calicivirus, respectively). The active components of essential oils (carvacrol, cinnamaldehyde, eugenol, thymol) and organic acids (acetic, salicylic, tartaric) demonstrated virucidal activity against the three bacteriophages (typically 1.00 log₁₀ to > 5.00 log₁₀ reductions within 10 min); however, ΦX174 exhibited lesser susceptibility (< 1.0 log₁₀ reduction with all but salicylic acid) and PR772 was the most resistant to cinnamaldehyde. It is unclear why ΦX174, a ssDNA virus, was more resistant to these antimicrobials than the ssRNA (MS2) and dsDNA (PR772) viruses. Carvacrol, eugenol, and thymol were also highly effective against both mammalian viruses (≥ 1.49 and > 3.00 log₁₀ reductions within one and 10 min, respectively). Salicylic acid was the most effective antimicrobial tested with > 3.84 log₁₀ reductions observed with all the viruses in one minute. Based on these results, several of the organic acids and essential oil components included appear to be viable eco-friendly alternatives to currently used disinfectants/sanitizers that are effective against viruses possessing diverse genomic and morphological characteristics.

Hepatitis E virus (HEV), human astrovirus (HAstV), and sapovirus (SaV) are pathogens associated with foodborne disease outbreaks. We developed a rapid and sensitive quadruple real-time fluorescence quantitative PCR (RT-qPCR) method for the simultaneous detection of HEV, HAstV, and SaV, using MS2 phage as a process control virus. We optimized the experimental parameters, detection limits for HEV, HAstV, SaV, and MS2 RNA were 10³ copies/μL, 10³ copies/μL, 10² copies/μL, and 10³ copies/μL, respectively, with intra-method and inter-method coefficients of variation below 3.0%, indicating good reproducibility and a total detection time of less than 90 min. We collected 354 bivalve shellfish samples from various regions in Hebei Province. After optimizing the proteinase K-PEG 8000 precipitation-chloroform extraction method for viral nucleic acid extraction, we applied the quadruple real-time RT-qPCR for simultaneous detection. The positive rates were 9.60% (34/354) for HEV, 3.67% (13/354) for HAstV, and 6.78% (24/354) for SaV, with mixed contaminations observed for HEV and HAstV (0.28%), HEV and SaV (2.54%), and HAstV and SaV (0.56%). In addition, a single real-time RT-qPCR was performed on 200 randomly selected samples and showed an overall agreement with the quadruple method of 98.67%, 100% positive agreement, 98.54% negative agreement and a Kappa value of 0.922. In conclusion, this quadruple real-time RT-qPCR method offers rapid screening for HEV, HAstV, and SaV in bivalve shellfish.

Monitoring viral loads in sewage can reflect the prevalence of infections within communities to a certain extent. Methods for concentrating and enriching viruses in sewage are also rapidly evolving. The magnetic bead method has been widely adopted for nucleic acid extraction due to its simplicity and high efficiency. In this study, we designed three pre-treatment procedures (T1: sedimentation for 30 min; T2: low-speed centrifugation at 2500×g for 5 min; T3: high-speed centrifugation at 8000×g for 5 min) to identify the optimal pre-treatment for enhancing viral nucleic acid concentration efficiency using the magnetic bead method. Spiked recovery tests were employed to compare the concentration efficiency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage between the magnetic bead method and the traditional polyethylene glycol (PEG) precipitation method. Real sewage samples were further used to evaluate both methods for enriching enveloped viruses (SARS-CoV-2, influenza virus) and non-enveloped viruses (Norovirus, Rotavirus, Adenovirus). Results demonstrated that low-speed centrifugation (T2) served as the optimal pre-treatment for the magnetic bead-based concentration. The high-throughput magnetic bead method achieved significantly higher recovery rates for SARS-CoV-2, Norovirus, and Adenovirus compared to PEG precipitation. Thus, the high-throughput magnetic bead method can be widely applied for the concentration and concentration of diverse viruses in sewage.

Cosavirus (CoSV) and Salivirus (SalV) are two novel picornaviruses that are associated with human diseases. However, epidemiological data on these viruses in China remain limited. In this study, we conducted the first simultaneous investigation of CoSV and SalV in a city of eastern China by wastewater-based epidemiology (WBE) method. From 2021 to 2023, a total of 21 influent wastewater samples were collected. After virus concentration and molecular detection, 71.43% (15/21) and 80.95% (17/21) of samples tested positive for CoSV and SalV, respectively. The sequences of CoSV were determined to be CoSV-A and CoSV-D, and SalV were all genotyped to be SalV-A1. Phylogenetic analysis showed that UTR region of CoSV-D and 3D region of CoSV-A can be divided into several clusters. UTR region of CoSV-A, UTR and 3D region of SalV-A1 were not clustered. Furthermore, the sequences in this study were closely related to some strains from human samples in China and wastewater samples in other countries. These findings reveal that CoSV and SalV circulated at a high prevalence in the local population and demonstrate that WBE is an effective method for novel picornaviruses research.

Viruses are key components of soils, influencing microbial dynamics and biogeochemical cycles. Here, we used shotgun metagenomics to analyze the virome of hydrocarbon-contaminated and uncontaminated soils from King George Island, Antarctica. Viral sequences were obtained from metagenomes of all soils; however, the relative abundance of viruses was higher in hydrocarbon-contaminated soils compared to uncontaminated soils. Our results indicate that viral distribution correlates with polycyclic aromatic hydrocarbons (PAHs) in King George Island soil (p = 0.05). Taxonomic analysis of viral contigs revealed viruses from realms Duplodnaviria, Varidnaviria, Monodnaviria, and Riboviria. While Caudoviricetes (realm Duplodnaviria) represented more than 90% of the relative abundance of viruses found in hydrocarbon-contaminated soils, this class represented less than 62% of the viruses of uncontaminated soils. Most viral hosts detected in King George Island soils belonged to hydrocarbon-degrading bacterial genera from the phyla Pseudomonadota and Actinomycetota. Hydrocarbon contamination resulted in the enrichment of well-characterized viruses at the expense of previously uncharacterized viral taxa, which were predominantly detected in non-contaminated soils. Among them, Gordonia-related Stormageddonvirus was the most abundant viral entity identified in King George Island soil. Viral auxiliary metabolic genes (AMGs) associated with nitrogen and phosphate cycles were found in different Caudoviricetes-related contigs. Our results further indicate that Caudoviricetes abundance is correlated with the carbon: phosphate (C:P) ratio in King George Island soils. We propose the Caudoviricetes may actively contribute to microbial competition for phosphorus in hydrocarbon-contaminated soils. These findings shed light on the intricate interplay between viruses, microbial hosts, and environmental contamination in Antarctic soils.