Food and Environmental Virology最新文献

Viruses can interact with a broad range of inorganic and organic particles in water and wastewater. These associations can protect viruses from inactivation by quenching chemical disinfectants or blocking ultraviolet light transmission, and a much higher dosage of disinfectants is required to inactivate particle-associated viruses than free viruses. There have been only few studies of the association of viruses with particles in wastewater, particularly in secondary treated effluent. As secondary effluent is the source water to the reclaimed water treatment system, this study quantified indigenous enteric viruses, and viral indicators associated with particles in secondary effluents collected from five full-scale water reclamation facilities in the United States. Particle-associated viruses were enumerated using a sequential filtration followed by microfluidic digital PCR. This study showed that enteric viruses and viral indicators (crAssphage and pepper mild mottle virus, PMMoV) were attached to particles of different sizes in secondary effluent. Significantly higher concentrations of RNA viruses including PMMoV, norovirus, and enterovirus were detected in filtrate of the sequential filtration, which contained particles < 0.45 µm. DNA viruses including adenovirus and crAssphage were found to be more associated with larger particles in secondary effluent. Overall, high correlations were observed between viral indicators and enteric viruses, supporting the use of crAssphage and PMMoV to evaluate virus removal efficiency in water and wastewater treatment processes. The association of viruses with particles in wastewater has significant implications on wastewater treatment and disinfection processes as well as virus enumeration in wastewater.

Graphic Abstract

Invasive alien species such as freshwater snails have significantly affected the food, environment, and the health of humans and animals, which have unfortunately received insufficient attention. To facilitate the study of viromes in snail species, we compared the enrichment effect of cesium chloride (CsCl) and sucrose density gradient ultracentrifugations in the recovery of diverse viruses in Pomacea canaliculata and Achatina fulica. First, we showed that CsCl-based ultracentrifugation enriched more virus contigs and reduced the nucleic acid background of the Pomacea canaliculata and was thus beneficial for virus recovery. Further studies comparing CsCl- and sucrose-based density gradient ultracentrifugations revealed that the former enriched more viral contigs and viral families of RNA viruses, while the latter yielded more DNA viruses from both Pomacea canaliculata and Achatina fulica. Certain RNA virus families, such as Rhabdoviridae, Arenaviridae, Hepeviridae, Astroviridae, and Alphatetraviridae, were exclusively enriched by CsCl-based ultracentrifugation. Conversely, several DNA virus families including Bacilladnaviridae, Nudiviridae, Malacoherpesviridae, and Adintoviridae were solely identified using the sucrose-based method. Therefore, the selection of viral enrichment technique (either CsCl or sucrose density gradient ultracentrifugation) should be carefully considered based on the specific virome (DNA or RNA viruses) being studied in mollusk species.

Viral contamination of bivalve molluscs, such as oysters, is a well-recognized food safety risk. The aim of this study was to assess virological hazards in market-ready oysters on the Dutch market. Non-targeted metagenome analysis was first performed on norovirus spiked-in samples showing linear and sensitive detection of norovirus GI.2 and GII.4 down to 14 and 5 genome copies per reaction, respectively. Subsequently, metagenomic measurements were performed to detect vertebrate viral genomes present in 24 undepurated B-area samples and 144 market-ready oyster samples taken in November up to and including February of the years 2015–2021. Genome sequences from fifteen viral species were identified in market-ready oysters which are associated with infections in humans and were detected above the genomic coverage threshold (5%) applied. Among these, the two genera from the Caliciviridae family, norovirus and sapovirus were detected at high prevalence (44 and 30%). Additionally, adeno-associated dependoparvovirus A and B as well as Aichi virus A and B (ribo)nucleic acids were detected (42, 33, 6, and 11%). Nucleic acids from virus species in oysters included potentially hazardous Picobirnavirus, Anellovirus, and multiple Circoviridae and Genomoviridae species. By integrating metagenome analysis into the monitoring process, researchers, food producers and regulatory bodies can gain valuable insights into the viral communities present in the food chain. This allows for the detection of potential pathogenic hazards at an early stage, providing an opportunity for tailored monitoring programs and targeted interventions to maintain the sanitary quality of the production area and safeguard public health.

Hepatitis E virus (HEV) is a zoonotic virus that infects humans when virus-containing pork products are consumed. This study aimed to explore MNV (murine norovirus) and HEV inactivation during cold smoking and ripening/fermentation treatments used for salami-like sausages (mettwurst). MNV inactivation was monitored in culture medium solution and in sausage while being subjected to a salami-like sausage manufacturing process. The inactivation of MNV in the solution was also monitored at room temperature (RT) for four weeks. HEV inactivation was monitored in solution during the cold smoking process and at RT. A TCID₅₀ assay was used to calculate the infectious MNV and HEV titres. MNV survival was modelled using Bayesian inference. MNV load in solution decreased by as much as 4.7 (SD 0.9) log₁₀ TCID₅₀/ml when it was subjected to the cold smoking process. Modelling revealed that 99.999% MNV might be inactivated during the treatment when observed at a level of 95% CI (Bayesian Confidence Interval). On the contrary, MNV load decreased by only 1.8 (0.2) log₁₀ when stored at RT. The low-titre HEV in solution was inactivated (> 1.1 (0.2) log₁₀) when treated, and at RT. However, MNV was resistant in the sausage matrix during the cold smoking process (log₁₀ reduction of 1.9 (0.5) TCID₅₀/ml). Based on modelling, a substantial amount of virus would remain in the product, even when the uncertainty was considered. Hence, viruses, here exemplified by MNV, may not be inactivated from salami-like pork sausages during manufacturing, which poses a risk for consumers in real-life situations.

Human respiratory and enteric viruses are responsible for substantial morbidity and mortality worldwide. Wastewater-based epidemiology utilizing next-generation sequencing serves as an effective tool for monitoring viral circulation dynamics at the community level. However, these complex environmental samples are often laden with other microorganisms and host genomic material, which can hinder the sensitivity of viral detection. To address this limitation, targeted enrichment sequencing is emerging as a preferred strategy, facilitating the acquisition of a more comprehensive understanding of specific pathogens. In this study, we evaluated the performance of a targeted enrichment sequencing panel for 42 excreted respiratory viruses (including Picornaviridae, Adenoviridae, Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Orthoherpesviridae, Pneumoviridae, and Parvoviridae families), known as the Respiratory Pathogen ID/AMR enrichment panel (RPIP), coupled with Explify bioinformatics analysis in 3 sewage samples from Uruguay. RPIP panel successfully identified sequences from frequently circulating viruses, along with some that had not been documented previously. We identified and characterized various viruses, including human Enterovirus (Coxsackievirus A1 and A19), Influenza A-H1N1, and full-length sequences of SARS-CoV-2. Additionally, several other viral pathogens were detected, such as human Bocavirus, human Parechovirus, Enterovirus A71, and Enterovirus D68; however, for these viruses further analysis was limited due to the small genomic regions or low-read coverage obtained. While the RPIP panel necessitates substantial sequencing depth and may introduce bias towards the more predominant strains present in the samples, this approach suggests its viability as a genomic epidemiological tool for assessing respiratory and enteric viruses in wastewater.

The transmission and infection of enteric viruses can be influenced by co-existing bacteria within the environment and host. However, the viral binding ligands on bacteria and the underlying interaction mechanisms remain unclear. This study characterized the association of norovirus surrogate Tulane virus (TuV) and murine norovirus (MNV) as well as the human enteric virus Aichi virus (AiV) with six bacteria strains (Pantoea agglomerans, Pantoea ananatis, Bacillus cereus, Enterobacter cloacae, Exiguobacterium sibiricum, Pseudomonas spp.). At room temperature, the viruses bound to all bacteria in strain-dependent rates and remained bound for at least 2 h. The virus association with two gram-positive bacteria B. cereus and E. sibiricum was less efficient than gram-negative bacteria. Next, the bacterial envelope components including lipopolysaccharides (LPS), extracellular polymeric substances (EPS), and peptidoglycan (PG) from selected strains were co-incubated with viruses to evaluate their effect on virus infectivity. All the tested bacteria components significantly increased virus infection to variable degrees as compared to PBS. The LPS of E. coli O111:B4 resulted in the greatest increases of infection by 0.19 log PFU for TuV as determined by plaque assay. Lastly, bacterial whole cell lysate of B. cereus and E. cloacae was examined for their impact on the infectivity of MNV and TuV. The co-incubation with whole cell lysates significantly increased the infectivity of TuV by 0.2 log PFU but not MNV. This study indicated that both the individual bacteria components and whole bacterial cell lysate can enhance virus infectivity, providing key insights for understanding virus–bacteria interaction.

This study aimed to investigate the dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in water samples obtained during the coronavirus disease 2019 pandemic period, employing cross-assembly phage (crAssphage) as a fecal contamination biomarker and next-generation sequencing protocols to characterize SARS-CoV-2 variants. Raw wastewater and surface water (stream and sea) samples were collected for over a month in Rio de Janeiro, Brazil. Ultracentrifugation and negatively charged membrane filtration were employed for viral concentration of the wastewater and surface water samples, respectively. Viruses were detected and quantified by (RT-)qPCR applying TaqMan® system protocols. SARS-CoV-2 RNA signals were detected in 92.5% (37/40) of the wastewater samples and in 31.25% (10/32) of the stream water samples, but not in seawater samples. CrAssphage was detected in 100% of the wastewater samples, 93.75% (30/32) of the stream samples, and in 2/4 of the seawater samples. CrAssphage detection and high concentrations in stream surface waters (median 8.95 log₁₀ gc/L) revealed diffuse contamination by domestic wastewater in a region with high sanitary coverage. The correlations detected between SARS-CoV-2 data and the moving averages of clinical cases per capita over the sampling period were moderate to strong when applying a 13-day offset, regardless of normalization by crAssphage data or not. Sequencing of the receptor-binding domain of the spike protein confirmed the detection of SARS-CoV-2, but did not characterize the circulating variant. On the other hand, the whole genome sequencing protocol identified circulation of the Gamma variant, corroborating the sampling period clinical data.

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.