Food and Environmental Virology最新文献

Long-term genomic surveillance of SARS-CoV-2 remains important as routine clinical testing and sequencing decline over time after the pandemic. We describe the temporal dynamics, methodological adaptation, and operational continuity of Italy’s national wastewater variant surveillance from October 2021 to December 2025. Across 51 monthly surveys, 6,278 wastewater samples collected from 167 wastewater treatment plants in 18 Regions and 2 Autonomous Provinces were analysed by RT-qPCR and targeted spike sequencing using short- and long-amplicon Sanger and Oxford Nanopore Technology approaches. Overall, 1,186 samples yielded a successful long amplicon and an interpretable Sanger sequence, while 248 regional pools were characterized by ONT during the funded surveillance phase from October 2021 to March 2023. The data documented the replacement of Delta by Omicron, followed by the circulation of BA.1*, BA.2* and BA.4/BA.5* and the subsequent emergence of BQ*, XBB*, EG.5*, and JN.1* lineages, with periods of persistence and co-circulation. The analytical workflow was adapted as lineage-defining mutations changed. Since March 2023, surveillance continued voluntarily through December 2025 using only long-amplicon Sanger sequencing. A qualitative comparison with national periodic flash surveys to estimate the prevalence of SARS-CoV-2 variants on biological samples showed that the two systems broadly captured the same succession of major lineage transitions, although the variant prevalence estimates and wastewater lineage proportions were not directly comparable. These findings support the feasibility of a targeted and adaptable strategy for sustaining nationwide wastewater genomic surveillance across emergency and post-emergency phases and for complementing clinical surveillance when routine testing and sequencing are reduced.

Enteric viruses are major causes of food- and waterborne diseases. Due to the limited cultivability of many human enteric viruses, surrogate viruses are commonly used to model their environmental persistence. This study aimed to evaluate the not-well-understood long-term survival of murine norovirus (MNV), rhesus rotavirus (RRV), and porcine sapovirus (PoSaV), used here as cultivable surrogate viruses for human enteric viruses, in bottled drinking water. Bottled water artificially inoculated with each virus was stored at 4 °C, 25 °C, and 37 °C for up to 365 days, and viral infectivity was quantified using TCID₅₀ and plaque assays, with the latter successfully applied for PoSaV quantification. The initial virus concentrations were 5.2 log₁₀ TCID₅₀/mL for MNV, 6.5 log₁₀ PFU/mL for RRV, and 4.0 log₁₀ PFU/mL for PoSaV. MNV and RRV remained infectious for at least one year at 4 °C, whereas PoSaV gradually declined and became undetectable by day 55. At 25 °C, the viral inactivation rate was accelerated: MNV became undetectable by day 160, RRV by day 320, and PoSaV by day 20. At 37 °C, a rapid inactivation rate was observed for all viruses, with MNV, RRV, and PoSaV becoming undetectable by days 20, 80, and 10, respectively. These results demonstrate the pronounced temperature-dependent survival of enteric viruses in bottled water. It is important to ensure the use of microbiologically clean source for bottled water as well as implement effective food and water safety management to minimise potential viral contamination.

Human norovirus (HuNoV), the leading cause of foodborne illness in the United States and acute gastroenteritis globally, replicates in the zebrafish embryo and larvae model. However, due to challenges in obtaining well-characterized HuNoV positive stool specimens for research, demonstrating replication of Tulane virus (TuV), a surrogate of HuNoV, in the zebrafish embryo and larvae model is valuable to study HuNoV infection mechanisms and subsequent prevention and control strategies. Three nL of TuV (9 log RNA copies/mL or 7 log PFU/mL) were microinjected into either fertilized zebrafish eggs (within 4-hours post-fertilization [hpf]) or the yolk of 3-day post-fertilization larvae. Daily, 10 embryos or larvae were pooled into a single sample, with two samples collected per day up to 5 days post-infection (dpi). Viral RNA was quantified using droplet digital PCR. While TuV replicated in zebrafish embryos, with a 2-log increase by 3 dpi, no replication was observed in zebrafish larvae. Developmental regulation of sialylated glycans in zebrafish was investigated as a potential explanation for the absence of TuV replication in larvae. Zebrafish embryos express α-2,6-linked sialic acid (SA) residues, which decrease from 6 to 48 hpf, while α-2,3 sialylation increases later. Because TuV specifically binds to α-2,6-linked SA but not to α-2,3-linked SA, these developmental changes in sialylation were hypothesized to contribute to the absence of replication in larvae. To test this hypothesis, zebrafish embryos were microinjected with 3 nL of Sambucus nigra lectin (100 mg/mL), which binds α-2,6-linked SA, followed by TuV microinjection. However, lectin treatment did not inhibit replication of TuV, suggesting TuV may utilize other receptors in zebrafish embryos. While TuV replicates exclusively in zebrafish embryos, the previous reports of HuNoV replication in both embryonic and larval stages reveal potential limitations of TuV as a surrogate for evaluating prevention and control strategies against HuNoV transmission through food and environmental routes.

Human noroviruses and persistent bacterial pathogens remain major food-safety concerns. This screening study compared 40% (v/v) ethanolic extracts (EE) and ultrasound-treated ethanolic extracts (UTE) of the endemic Mount Ida plants Sideritis trojana Ehrend. and Hypericum kazdaghensis Gemici & Leblebici. Feline calicivirus (FCV) and murine norovirus (MNV) served as cultivable human-norovirus surrogates; four reference bacteria were examined by disc diffusion. Ultrasound treatment was associated with higher measured total phenolic content (approximately 2.2- to 3.8-fold) and antioxidant capacity (approximately 1.9- to 2.3-fold) than EE. The largest reduction was 4.71 log₁₀ TCID₅₀/mL for H. kazdaghensis-UTE against FCV after overnight exposure. For MNV, the largest reduction was 2.42 log₁₀ TCID₅₀/mL for S. trojana-EE after overnight exposure. Inhibition-zone diameters ranged from 6.25 to 11.75 mm, including the 6-mm disc. FRhK-4 viability ranged from approximately 56% to 100% across the tested conditions, precluding a general claim of low cytotoxicity. The results provide preliminary, surrogate- and assay-specific evidence of antiviral and antibacterial activity. Because solvent-only and positive controls were not included, causal attribution to plant constituents and comparison with established antimicrobials are not possible. Confirmatory controlled assays are required before practical food-safety use can be proposed.

Viral diseases are among the most serious threats to shrimp aquaculture, causing large-scale mortality and production losses. This review summarizes current knowledge on the major viral diseases affecting cultured penaeid shrimp and freshwater prawns, with particular emphasis on white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV), and Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white tail disease. We present a comparative overview of their genomic features, transmission routes, host range, and pathogenesis. Particular attention is given to the genome organization of WSSV and TSV, including variable regions and structural genes commonly used in molecular detection and strain differentiation. We also discuss mechanisms that may contribute to viral persistence in host populations, such as vertical transmission, chronic infections, and viral integration into host genomes. Finally, the review outlines current disease management strategies, including biosecurity measures, environmental control, and selective breeding. By compiling and contextualizing virological and epidemiological data, this review serves as a reference for disease surveillance and control in shrimp aquaculture.

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Wastewater/sewage represents a highly complex environmental matrix and harbors various viruses including viruses of epidemic and pandemic potential like Influenza A and, SARS-CoV-2. The limitations of available methods for sample concentration are cost, efficiency and time. NGS- metagenomics offer sample to virus genomic characterization for even non-cultivable viruses at highest resolution. In this study, we have collected samples from sewage treatment plants located in Gwalior region of Central India (December 2023 to March 2025). In this study a novel in house nanoweb membrane-based sample enrichment followed by magnetic bead based nucleic acid extraction was optimized in conjunction to shotgun whole genome metagenomics on Nanopore and ion torrent NGS. Both the methods were found comparable with commercially available methods by virus specific TaqMan qPCR. Both the methods were found successful in virus recovery at two log through 10²GC/50 ml feline calicivirus, Influenza A virus, Zika virus, SARS-CoV-2 spiked in sewage matrix alone or in mixture), suggesting the optimized protocol found working for virus characterization at strain level. Here, we have standardized a simple field amenable waste water sample enrichment followed by nucleic acid extraction protocol, that can easily be integrated with latest onsite downstream molecular diagnostic platforms. The developed method is very simple, cost effective and field deployable. This will help to develop a suitable strategic plan for sewage surveillance towards early warning/microbial forensics and future decisions for prevention and therapeutic interventions.

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Human norovirus (HuNoV) and hepatitis A virus (HAV) are highly prevalent and contagious foodborne pathogens that pose a significant threat to global public health. Current molecular detection methods such as RT-qPCR and RT-ddPCR are highly sensitive and specific but time-consuming, require specialized equipment, and are unsuitable for on-site detection. We developed a multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay coupled with CRISPR-Cas12a and lateral flow detection for rapid, simultaneous identification of HuNoV GI, GII, and HAV. Through rigorous in silico design and experimental validation, we optimized primer pools and crRNAs to ensure broad genotype coverage and high specificity. Using 2 µL of input per target per 50 µL reaction, the assay achieved limits of detection of 10¹ copies/µL (2 × 10¹ copies/reaction) for HAV, 10³ copies/µL (2 × 10³ copies/reaction) for GI HuNoV, and 10² copies/µL (2 × 10² copies/reaction) for GII HuNoV, with a total assay time of 50 min from purified RNA to final readout. No cross-reactivity occurred with other common foodborne viruses. Validation using total RNA extracted from shellfish digestive glands artificially spiked with RNA standards provided preliminary evidence supporting the feasibility of the method under laboratory conditions. This portable system shows strong potential as a rapid multiplex molecular detection platform.

Hepatitis A (HepA) is a viral hepatitis caused by the hepatitis A virus (HAV), and currently Mexico is considered to have an intermediate-HAV endemicity. Wastewater-based epidemiology (WBE) is a tool for monitoring infectious diseases but has been poorly evaluated in intermediate-HAV-endemicity settings. From June 2024 to June 2025, 24-hour composite wastewater samples were collected twice a week from three wastewater treatment plants in the metropolitan area of San Luis Potosí, Mexico. Wastewater samples were ultracentrifuged, RNA was extracted, and HAV and crAssphage (for normalization) were detected by qPCR and weighted by the wastewater treatment plant’s inflow. We compared these results with HepA hospital admissions from eight public hospitals in the city during the same period. We compared HAV genotype sequences obtained from wastewater and patients. We processed 244 wastewater samples over 46 weeks, of which 35.1% tested positive for HAV. Seventy-three HepA hospital admissions were reported during this period. The Spearman correlation between HAV viral load and hospital admissions, along with its displacement over 10 weeks, showed the strongest correlation between Lag 4 to Lag 6. We genotyped six HAV strains from wastewater samples and two from HepA patients. Phylogenetic analysis revealed that the patient strains were distinct but identical or closely related to the circulating wastewater strains. In conclusion, WBE HAV surveillance may predict an increase in HepA admissions four to six weeks in advance. Diverse, but closely related HAV strains constantly circulate in intermediate HAV-endemicity settings.

Rotavirus SA11 (RV SA11), a simian strain and member of group A, is widely used for RV research due to its attenuation and similarity to human strains. This study investigated the effects of food acidifying agents and common food processing and preservation conditions on the survivability of RV SA11. Acetic, citric, and lactic buffers (adjusted to pH 4.0) as simulated buffer systems and real food models, including traditional pickle, orange juice, and a fermented yogurt drink, were inoculated with RV SA11 at an initial titer of 7.22 log₁₀ TCID₅₀/ml. Viral titers were determined after exposing samples to refrigeration (4 °C for 2 and 4 days), room temperature (20 °C for 2 and 6 h), heating (90 °C for 10 and 30 min), and microwave treatment (0.5 and 2 min). At 4 °C, a 4.99 log₁₀ TCID₅₀/ml reduction was observed in the lactic acid system after 4 days, while complete inactivation occurred within 2 days in the citric and 4 days in the acetic systems. At 20 °C, viral titers decreased by 4.45, 3.99, and 3.06 log₁₀ TCID₅₀/ml in lactic, citric, and acetic systems, respectively. Heating at 90 °C completely inactivated the virus within 10 min in lactic and 30 min in acetic systems, while microwave exposure for 2 min eliminated infectivity in both acetic and citric buffers. Viral reductions in real foods after 6 h at 20 °C were lower than in simulated buffers (a range of 2.45 to 3.39 log₁₀ TCID₅₀/ml). Overall, these findings show that food composition and processing conditions significantly influence rotavirus persistence and inactivation in food matrices.

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Monitoring human fecal contamination in wastewater is crucial for public health and environmental safety. Traditional fecal indicator bacteria (FIB), such as Escherichia coli and Enterococci, lack source specificity between human and non-human sources. Tomato brown rugose fruit virus (ToBRFV) has emerged as a promising viral indicator, characterized by high environmental stability, specificity for human feces, and widespread occurrence in wastewater systems. This study evaluates ToBRFV as a novel RNA-based viral indicator for wastewater surveillance in Korea. Wastewater samples were collected from six wastewater treatment plants (WWTPs) in Incheon between July 2024 and April 2025. The results showed that ToBRFV was detected in all influent samples (240/240), with concentrations ranging from 7.42 to 9.91 log₁₀ gene copies/L and low coefficients of variation (3.36%-6.39%). The observed concentrations were significantly higher than those reported from other regions in previous studies, confirming high abundance and stable detection of ToBRFV. This study represents the first quantitative assessment of ToBRFV in Korean wastewater using digital PCR. These findings suggest that ToBRFV has potential as a reliable human fecal contamination indicator and normalization marker for wastewater-based epidemiology (WBE) applications in Korean wastewater systems.