Food and Environmental Virology最新文献

Severe acute respiratory syndrome Coronavirus-2, Norovirus GI and GII have been identified as one of the leading primary agents of lethal diseases such as COVID-19 and acute gastroenteritis, causing critical problems in public health, particularly in children, the elderly, and individuals with weakened immune systems. These pathogens contribute to substantial morbidity, mortality and pose a serious threat to human health, imposing a tremendous economic burden globally. Hence, this study sought to profile the co-occurrence of SARS-CoV-2 and Noroviruses in wastewater collection and treatment facilities within Buffalo City and Amathole District regions, Eastern Cape Province, South Africa. Raw wastewater samples were collected from the wastewater treatment facilities on a weekly basis for a 6-month sampling regime using the grab sampling technique. Total Ribonucleic acids (RNA) were extracted and purified using the commercial Total RNA extraction kits and the extracted RNA sample were further profiled for the presence of SARS-CoV-2 RNA and Noroviruses GI and GII using RT-qPCR. A total of 213 samples were screened using RT-qPCR. SARS-CoV-2 was detected in 146 (69%) samples, Norovirus GI in 58 (27%), Norovirus GII in 25 (12%) and 25 (12%) samples showed co-infection with both GI and GII norovirus targets. The RNA concentration of the extracted samples from wastewater treatment facilities in this study ranged between 102 ng/µl and 12,828 ng/µl. It was observed that the SARS-CoV-2 GC/mL range from 5.22 × 10² to an extreme 2.5650 × 10⁴ GC/mL. The study findings demonstrate a high prevalence of SARS-CoV-2 RNA in wastewater, affirming the utility of wastewater surveillance as an early warning system for potential outbreaks at community levels. Furthermore, the study showed the co-occurrence of SARS-CoV-2 alongside Norovirus GI and GII in wastewater milieu, highlighting an exacerbated potential risk of co-infection and a major threat to public health and environmental safety.

In the Philippines, most of the population relies on decentralized wastewater systems, particularly septic tanks, and the rest on centralized wastewater systems. However, the performance of wastewater treatment systems in the Philippines in reducing viral contaminants remains to be characterized; thus, in this study, the occurrence and reduction of viruses in wastewater before and after treatment were assessed at a wastewater treatment plant (WWTP) that utilizes biological treatment technologies in the Philippines. Influent (n = 18) and effluent (n = 18) samples collected from WWTP from April to August 2024 were centrifuged, followed by viral nucleic acid extraction. Cross-assembly phage (crAssphage) was quantified through quantitative polymerase chain reaction (qPCR), and pepper mild mottle virus (PMMoV), influenza A virus (Inf-A), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and norovirus genogroups I (NoV-GI) and II (NoV-GII) were quantified by reverse transcription-qPCR. All viral targets were quantified in wastewater before and after treatment, except for Inf-A and SARS-CoV-2, which were absent in all effluent samples. The reduction in viral loads in wastewater after treatment was determined, with crAssphage, PMMoV, NoV-GI, and NoV-GII showing high log₁₀ reduction values (LRVs) of 4.35, 4.28, 4.56, and 4.00, respectively, while their lowest LRVs were 2.17, 0.38, 0.72, and − 0.99, respectively. The persistence of crAssphage and PMMoV in wastewater samples and their significant positive associations with NoV-GI and NoV-GII suggest their potential application as indicators of enteric viruses in Philippine wastewater. These findings highlight the importance of viruses in characterizing treatment plant reduction performance and water and wastewater quality in the Philippines.

Pepper mild mottle virus (PMMoV) has emerged as a promising viral indicator for human fecal contamination and evaluating viral removal efficiency in water treatment systems. However, few studies have examined this potential from a genetic perspective over extended timescales. This study investigated PMMoV presence and genetic diversity across multiple water environments in Sweden, including wastewater, lake water, and raw water used for drinking water production, collected between 2016 and 2024. PMMoV was consistently detected in both wastewater and lake water, showing relatively stable concentrations in wastewater between years, and with levels typically 4–7 log₁₀ higher than in lake water. Comparative genotyping of the coat protein (CP) and replication-associated (RP) regions for wastewater samples showed no significant differences. We successfully sequenced 67 isolates from wastewater, 16 from lake water, and 2 from source water at the CP region. Sequence analysis revealed extremely high nucleotide identities (> 96%) with minimal divergence between samples collected six years apart or from different water types. Phylogenetic comparisons with global reference sequences confirmed high genetic conservation worldwide. These findings demonstrate the remarkable genetic stability of PMMoV over time and geography, supporting its reliability as a viral process indicator for water treatment systems. However, the potential for PMMoV to originate from non-human sources suggests it should not be used as a standalone marker for human-specific fecal contamination. Overall, this study highlights the global genetic conservation of PMMoV and its utility for long-term environmental surveillance and water quality assessment.

This study aimed to compare the performance of polyethylene glycol (PEG) precipitation, and Nanotrap^® Microbiome magnetic particle capture workflows for recovering novel fecal marker, pBI143 from 12 wastewater samples collected across six treatment plants in Maryland, USA. Quantitative PCR (qPCR) was used to quantify marker abundance. The Nanotrap workflow yielded significantly higher concentration of pBI143 compared to PEG precipitation workflow (p < 0.05). The Nanotrap workflow used in the study utilized both magnetic nanoparticles A and B, rather than magnetic nanoparticle A alone, highlighting the necessity of optimization based on the intended targets for enhanced recovery. The extracted total nucleic acids by the Nanotrap workflow, were further analyzed to quantify other fecal markers, crAssphage, tomato brown rugose fruit virus (ToBRFV), and pepper mild mottle virus (PMMoV). No significant differences in the concentrations of pBI143, crAssphage, and ToBRFV (p > 0.05) were observed, whereas the concentration of PMMoV was significantly lower than that of the three fecal markers (p < 0.05). Based on the concentration alone, pBI143, ToBRFV, and crAssphage were found to be a better alternative to PMMoV as an endogenous fecal marker.

The alarming findings presented in the latest WHO report on the global antimicrobial resistance crisis have redirected scientific attention toward phage-based approaches as a renewed line of defense against multidrug-resistant (MDR) bacteria. In this study, four bacteriophages infecting a MDR-Escherichia coli strain were isolated from water sources and subjected to detailed phenotypic and genomic characterization. All phages efficiently inhibited MDR-E. coli at MOIs of 0.1 and 0.01, showing high stability across a broad temperature (4–65 °C) and pH (4–10) range. TEM analysis revealed that all phages exhibited a podovirus-morphotype. At 4 °C, titers remained stable for 6 months, with only a 1–2 log reduction -over a year. Notably, phage Sem4 exhibited markedly stronger lytic activity than the phage cocktail, fully suppressing bacterial growth. In tap water, phage Sem4 treatment reduced bacterial counts from 10⁷ to 7 × 10⁴ CFU/mL within 8 h, with no detectable colonies at 24–48 h. Genomic analysis showed that these phages possess linear dsDNA genomes of 44,244–45,205 bp, with ~ 45% GC content. Phylogenetic and comparative analyses classified them as novel Vectrevirus members within the Molineuxvirinae subfamily of the Autographiviridae family, sharing less than 95% intergenomic similarity with known Vectrevirus species. No genes associated with antibiotic resistance, toxins, or lysogeny were detected. These findings underscore the potential of - phages as a promising candidate for the development of next-generation biocontrol strategies, especially marking the efficiency of Sem4 in water sanitation systems, paving the way for sustainable and targeted interventions against MDR bacterial contamination.

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Both highly pathogenic avian influenza virus (HPAIV) and its viral RNA have been detected in raw milk during the ongoing outbreak in dairy cows. While pasteurization effectively inactivates the virus in milk, comparatively little has been published on AIV inactivation in fermented dairy products made from raw milk. We evaluated the viability of two isolates of low pathogenic avian influenza virus (LPAIV) during fermentation of kefir to assess the potential for viral persistence. Raw (unpasteurized) milk (ca. 3.0% fat) was inoculated with ca. 7.5 log₁₀ CFU per mL of active commercial kefir starter culture. Next, raw milk (125 mL) was inoculated with 1.0 mL of either strain A/rgGyrfalconHAxPR8/2014 H5N1 or A/turkey/Wisconsin/1968 H5N9 to achieve an average initial level of ca. 5.0 log₁₀ 50% egg infectious doses (EID₅₀) per mL. The inoculated raw milk (ca. pH 6.55 ± 0.05) was fermented at 25 °C for ca. 36 h to an average endpoint pH of pH 4.44 ± 0.05. Results confirmed that fermentation of raw milk to produce kefir delivered an appreciable reduction of both LPAIV strains, that being a ca. 4.1 or 3.1 log₁₀ EID₅₀ per mL decrease of strains H5N1 or H5N9, respectively, from their initial levels in raw milk. In the absence of a starter culture, due to fermentation by the indigenous flora the pH of the raw milk after incubation at 25 °C for ca. 36 h also significantly decreased on average to ca. pH 5.3:, and levels of strains H5N1 and H5N9 were reduced by 4.1 and 3.5 log₁₀ EID₅₀ per mL, respectively. Our findings established that fermentation of raw milk (with or without added kefir starter culture) to ≤ pH 5.3 over ca. 36 h at 25 °C appreciably decreases titers (≥ 3.1 log₁₀ EID₅₀ per mL) of AIV strains H5N1 and H5N9 and, therefore, lowers risk associated with its consumption.

To investigate the contamination status of foodborne viruses in fresh fruits, vegetables, and their ready-to-eat (RTE) products sold in Shijiazhuang, China, a total of 365 samples were collected from farmers’ markets, fresh produce stores, and large supermarkets between 2024 and 2025. Samples were tested for Norovirus (NoV) GI, NoV GII, Hepatitis A virus (HAV), Paslahepevirus balayani (HEV), Classic Human astrovirus (HAstV), and Sapovirus (SaV) using quantitative multiplex real-time reverse transcriptase-polymerase chain reaction (MRT-qPCR). Overall, foodborne viruses were detected in 93 samples (25.48%, 93/365). Detection rates of NoV GI, NoV GII, HAV, HEV, HAstV, and SaV were 11.78% (43/365), 9.04% (33/365), 0.27% (1/365), 4.93% (18/365), 0.27% (1/365), and 2.19% (8/365), respectively, with Norovirus being the dominant contaminant. The mixed contamination rate was 2.47% (9/365), mainly involving dual-virus contamination. Contamination was highest in coriander (32.50%, 13/40), followed by cherry tomatoes (31.82%, 21/66) and blueberries (30.77%, 20/65). The detection rate of foodborne viruses in fresh produce (26.89%, 89/331) was higher than that in RTE products (11.76%, 4/34) (χ² = 7.170, P < 0.05), and significantly higher in summer (42.31%, 33/78) than in other seasons (20.91%, 60/287) (χ² = 14.80, P < 0.01). Additionally, there was no significant difference in contamination rates among samples collected from different sources (χ² = 2.95, P > 0.05). This study provides the first characterization of foodborne virus contamination in commercially available fresh fruits and vegetables and their RTE products in Shijiazhuang, highlighting potential public health risks and supporting strengthened food safety surveillance.

Several studies investigated the presence of SARS-CoV-2 in water samples and raw urban wastewater collected from urban sewage treatment plants (STPs) during the COVID-19 pandemic and correlated viral load and epidemiological data. However, limited information is available regarding the potential of urban ditches as sampling sites for epidemiological surveillance. This study aimed to detect SARS-CoV-2 RNA in urban ditches and evaluate its correlation with epidemiological data. A total of 168 samples were collected from January 2021 to February 2022 in three urban ditches and two STPs in the metropolitan region of Vitória, Espírito Santo (ES). Physicochemical and microbiological water quality parameters were analyzed, and SARS-CoV-2 RNA was detected using a TaqMan RT-qPCR system. SARS-CoV-2 was detected in 145/168 of the samples, with 87.2% (75/86) found in the urban ditches and 85.3% (70/82) in the STPs. The viral load in the three urban ditches ranged from 1.59 × 10⁴ to 2.56 × 10⁴ genome copies/litre (GC/L) while in the two STPs, it ranged from 7.79 × 10³ to 1.26 × 10⁴ GC/L. A moderate to strong positive correlation was observed between viral load and confirmed COVID-19 cases, particularly for samples collected from urban ditches (ditch 1, r = 0.58; ditch 2, r = 0.78; ditch 3, r = 0.86). The results indicate greater correlation of SARS-CoV-2 RNA concentration in ditches of low sanitation areas as compared with STPs, showing that ditches could be an important and equivalent sampling point for viral epidemiological studies.

The COVID-19 pandemic has underscored the importance of alternative epidemiological tools capable of providing real-time, population-level insights into infectious disease dynamics. Wastewater-based epidemiology (WBE) has emerged as a powerful, non-invasive method for tracking viral transmission within communities. While initially focused on SARS-CoV-2, WBE now offers the potential for multiplex surveillance of a broader range of respiratory pathogens. This study applied WBE to monitor the circulation of six major respiratory viruses such as Human adenovirus (HAdV), SARS-CoV-2, Influenza A and B, Respiratory Syncytial Virus (RSV) A/B, and Rhinoviruses, over a six-month period (ISO weeks 2022-40 to 2023-13) in the city of Patras, Greece. Weekly composite samples from a central wastewater treatment plant were analyzed via quantitative PCR, and viral genome concentrations were normalized per 100,000 inhabitants. The results revealed distinct circulation patterns: HAdV and SARS-CoV-2 were detected consistently throughout the period, while Influenza A peaked during the winter months, followed by Influenza B. RSV, however, appeared earlier in the season (first detectable from ISO week 2022-46) and overlapped with Influenza. Rhinoviruses displayed intermittent peaks, indicating multiple waves or persistent low-level transmission. Correlation analyses showed strong positive associations between influenza viruses, RSV, and SARS-CoV-2, suggesting synchronized seasonal trends. Hierarchical cluster analysis classified the viruses into three distinct groups: (1) an epidemic cluster including Influenza A/B, RSV, and Rhinoviruses; (2) a persistently present cluster represented by HAdV; and (3) a separate episodic cluster characterized by SARS-CoV-2. These groupings reflect differences in viral epidemiology and shedding behaviour. This study confirms the effectiveness of WBE in tracking the temporal dynamics of multiple respiratory viruses and provides evidence of its utility in supplementing traditional clinical surveillance systems. The consistent detection of underreported pathogens such as HAdV underscores the added value of environmental monitoring for public health preparedness and early warning applications. These findings advocate for the integration of WBE into routine respiratory virus surveillance frameworks.

Wastewater-based epidemiology (WBE) has emerged as a valuable tool for monitoring community-level infections, capturing both symptomatic and asymptomatic cases, as demonstrated during the SARS-CoV-2 pandemic. However, WBE implementation is challenged by the complex nature of wastewater, which contains PCR inhibitors and varies significantly between wastewater treatment plants (WWTPs) due to differences in influent sources, industrial discharge, and environmental conditions. Furthermore, the lack of standardized protocols for virus concentration limits the practical and reliable use of WBE. Timely collection, concentration, and analysis of wastewater are crucial for detecting pathogens and informing public health decisions, yet most currently used protocols have been empirically established without a detailed comparison of methods under realistic surveillance conditions. This study compared two virus concentration approaches, filtration and pelleting, to determine which method yields the highest relative detection of SARS-CoV-2 RNA. Wastewater samples from six WWTPs were processed using different concentration methods, with and without an inhibitor removal step. WWTPs were selected based on historical SARS-CoV-2 RNA detection data, with three characterized by consistently high viral levels during peak infection periods and three with low or undetectable levels. In this context, method comparisons are extended to more accurately reflect real-world monitoring programs. In sites with high RNA concentrations, pellets and 100 kDa and 30 kDa ultrafiltration methods yielded significantly higher SARS-CoV-2 RNA concentrations than the volume-intensive method routinely used in national surveillance. PCR inhibitor removal further improved detection, particularly in pellets, which offer a practical alternative for low-resource settings. In contrast, low-RNA concentration sites showed no significant differences between methods. However, PCR inhibitor removal enhanced detection across all methods. These findings demonstrate that viral RNA detection varies between concentration approaches and that removing PCR inhibitors substantially improves detection. Overall, standardized and optimized protocols for wastewater concentration and PCR inhibitor removal are essential for generating reliable WBE data.

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