Human noroviruses and persistent bacterial pathogens remain major food-safety concerns. This screening study compared 40
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.
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.
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
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.
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.
The increased demand for highly pathogenic avian influenza (HPAI) testing in milk highlights the critical need for well-characterized samples that enable meaningful evaluation of current detection methods. In this study, we focus on the development and rigorous characterization of artificially contaminated spiked milk samples, forming the foundation for reliable assessment of assay performance. By using reverse transcriptase digital PCR (RT-dPCR), we obtained precise genome copy (GC) measurements of our virus stock and of spiked samples, producing accurate quantification even at low viral loads. In parallel, the U.S. Department of Agriculture National Veterinary Services Laboratories’ (USDA-NVSL’s) influenza A matrix gene (IAV M) real-time reverse transcriptase (rRT-PCR) assay was employed as the main detection assay, serving as a tool to validate the sample preparation methodology. The goal of this work is to show that robust, reproducible, and analytically traceable samples can be generated to support HPAI method evaluation in proficiency exercises (PEs) distributed nationwide. Low between-sample standard deviations, ranging from 0 to 0.10 rRT-PCR Ct values for replicate samples, fall below the theoretical threshold indicative of heterogeneity, along with RT-dPCR determinations of sample GCs affirm the homogeneity of the developed samples. Parallel analysis of corresponding milk-based and phosphate buffered saline (PBS)-based samples showed no indication of a matrix effect. No significant shifts of Ct value or of measured GCs were observed over time, proving samples to be stable for at least 28 days when stored at -80 °C. The defined assay sensitivity, expressed as the level of detection 50
Wastewater-based epidemiology (WBE) is a promising tool for monitoring respiratory pathogens, yet the rapid in-sewer decay of viruses limits its application in low-incidence settings. This study investigates the decay rates of respiratory syncytial virus (RSV) in wastewater under various conditions to enhance the utility of WBE for forecasting RSV outbreaks. Laboratory-scale recirculating sewer systems were employed to simulate in-sewer decay of RSV at different temperatures, organic concentrations, and total suspended solids (TSS) levels. A first-order decay model revealed a significant temperature dependence, with the decay rate increasing approximately 30-fold from 4 to 35 °C. Conversely, higher TSS concentrations provided substantial protection to RSV particles, extending the T90 by up to sevenfold, whereas SCOD exhibited minimal impact. Based on these findings, a multiple linear regression model was established to identify key predictors. This study underscores the importance of understanding RSV decay kinetics for accurate WBE and highlights the value of kinetic modelling in correcting in-sewer viral loss for optimized wastewater surveillance.
Human Aichi virus (AiV-A1), a member of the Kobuvirus genus associated with viral gastroenteritis, remains largely underdiagnosed in Latin America due to the absence of routine testing and limited epidemiological data. This study provides the first molecular evidence of AiV-A1 circulation in Argentina to date, based on the analysis of wastewater and pediatric stool samples collected in Córdoba between 2012 and 2019. A total of 93 untreated wastewater samples from the main wastewater treatment plant and 155 stool samples from children with acute gastroenteritis (AGE) were screened using a validated nested RT-PCR targeting the 3CD region, followed by sequencing and phylogenetic analysis. AiV-A1 was detected in 58
Hepatitis E virus (HEV) is an important cause of acute viral hepatitis worldwide, with both human-restricted (genotypes 1 and 2) and zoonotic lineages (genotypes 3 and 4). However, information regarding HEV circulation in Central America remains limited. In this study, wastewater-based epidemiological surveillance was conducted in San José, Costa Rica, during 2025 to investigate the presence of HEV in the main wastewater treatment plant serving approximately one million inhabitants. A total of 54 influent and effluent samples were analyzed. HEV RNA was not detected by quantitative RT-PCR; however, RT-nested PCR targeting the ORF1 region identified three positive influent samples. Sequencing yielded two high-quality partial ORF1 sequences suitable for phylogenetic analysis. Both Costa Rican sequences clustered together and showed a close relationship to genotype 3 sequences. To our knowledge, this represents the first report of HEV detected in wastewater in Costa Rica and the first scientific report of environmental detection in Central America. These findings highlight the value of wastewater-based surveillance for identifying circulating viral pathogens in regions where routine clinical testing is limited.
Human enteric viruses frequently encounter diverse microbial communities in wastewater, where interactions with bacteria and other microorganisms can profoundly influence their environmental persistence and transmission. Increasing evidence demonstrates that bacteria can bind and stabilize enteric viruses, thereby enhancing thermal inactivation and prolonging infectivity. These interactions not only shape viral fate during wastewater treatment but also may affect the sensitivity and interpretation of wastewater-based epidemiology, an essential tool for public health surveillance. Yet significant knowledge gaps remain regarding the molecular mechanisms that govern bacterial-viral interactions and their roles in broader wastewater microbiomes. Together, advancing our understanding of transkingdom interactions in wastewater is essential for improving treatment processes, strengthening environmental surveillance, and reducing the risk of virus transmission through contaminated water systems.
In April 2024, two cases of hepatitis A (HA) were linked to the consumption of oysters produced in the same region, which triggered a multidisciplinary study. A network was quickly set up in a one-health approach including epidemiological and clinical data, as well as analysis of locally collected shellfish and wastewater samples. By the end of the outbreak, up to 17 HA cases met the case definition: all shared a same genotype IA strain and have eaten oysters. The prompt response enabled the identification of oyster samples positive for the HAV genome at the start of the monitoring period; these oyster samples subsequently tested negative during the following two months of the sampling campaign. Monitoring three nearby wastewater treatment plants revealed that the contamination was limited to a single area, allowing for more effective monitoring of shellfish. None of the identified cases lived in the area served by the treatment plant, and no clinical cases were detected among the local population, either before or after the outbreak. The decline of HAV concentration in sewage signed the end of the outbreak. While predicting contamination of shellfish growing areas is complicated, sewage surveillance can help establishing an alert system to prevent shellfish contamination, which is an important issue for silent diseases.
Increasing water scarcity and climate-related extremes are amplifying the need for safe water reuse, however, wastewater treatment plants (WWTPs) may not fully remove viral pathogens, posing potential risks to freshwater environments. This study assessed human enteric and respiratory viruses using (RT)-qPCR, along with faecal indicators using culture-based methods, at WWTPs in two Mediterranean protected areas (Albufera Natural Park and the Ebro River region) across three campaigns. A total of influent (n = 33), reclaimed wastewater (n = 33), biosolids (n = 32), surface water (n = 33), and sediment (n = 29) samples were analysed. Influent wastewater contained a wide range of viral and faecal indicators, with biosolids showing similar levels. Although reclaimed water demonstrated reductions in Escherichia coli and somatic coliphages, these reductions remained below the ≥ 5 and ≥ 6 Log10 threshold required under EU water reuse regulations, respectively. In surface waters, human enteric viruses were detected at low concentrations, with human norovirus genogroup II (HuNoV GII) showing the highest incidence (36.36
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.
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
This study explores the antiviral potential of kumis, a traditional fermented mare’s milk beverage, against SARS-CoV-2 using K18-hACE2 transgenic mice. Kumis samples fermented in leather, wood, and plastic containers were tested to assess their immunomodulatory and antiviral effects. Mice were intranasally infected with live SARS-CoV-2 and orally administered kumis daily for five consecutive days, starting one day prior to viral challenge, and monitored for a total of 15 days. Kumis-treated mice exhibited significantly reduced weight loss at specific time points (days 12 and 15 post-infection; p = 0.0310). Survival was numerically higher in kumis-treated groups (Plastic 5/6, Wooden 5/6, Leather 4/6) compared to controls (3/6), but this difference did not reach statistical significance (p = 0.6649). Histopathological and virological analyses supported the protective effects, with the plastic-fermented sample showing the most pronounced benefit. The study also highlights the importance of fermentation environments on the microbial composition and therapeutic efficacy of kumis. Although kumis is not a direct antiviral drug, it can serve as a supportive food that enhances immunity and provides protection during viral illnesses.
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.
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
