Comparative performance of electronegative membrane filtration and automated concentrating pipette for detection of antibiotic resistance genes and microbial markers in river water samples.

The target viral and bacterial concentrations in river water are essential for environmental monitoring and public health studies. Filtration-based methods are commonly employed, yet challenges arise due to recoverability and filter pore size. This study aimed to compare the performance of electronegative membrane filtration (EMF) and automated Concentrating Pipette (CP) Select (InnovaPrep) methods for quantifying antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial and viral markers in river water samples. Fifty-four river water samples were collected from upstream and downstream locations in a river in Japan. The CP Select method was modified by adding MgCl2 and using different tips. The recovery efficiencies for total coliforms and Escherichia coli were assessed, and class 1 integron-integrase gene (intI1), 16S rRNA, gene encoding sulfonamide resistance (sul1), cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), and Escherichia coli gene (sfmD) were detected. CP Select showed recovery efficiencies of 45 %-63 % for total coliforms and 17 %-35 % for E. coli. The intI1, 16S rRNA, sul1, crAssphage, PMMoV, and sfmD concentrations using the modified CP Select method were 10.1 ±â€¯0.5, 8.7 ±â€¯0.2, 7.7 ±â€¯0.2, 6.7 ±â€¯0.2, 5.4 ±â€¯0.2, and 3.5 ±â€¯0.5 log10 copies/L, respectively. Higher intI1 and sul1 concentrations were observed downstream, with the highest contribution percentage (22 % and 21 %) using CP Select or EMF. The modified CP Select method with 0.05 µm tips yielded more quantifiable results for all target genes and greater PMMoV concentrations (p < 0.05). Positive correlations were found among bacterial, ARG/MGE, and viral markers (Spearman's ρ = 0.71 for 16S rRNA and sfmD, 0.88 for intI1 and sul1, and 0.64 for PMMoV and crAssphage).

Effect of SARS-CoV-2 shedding rate distribution of individuals during their disease days on the estimation of the number of infected people. Application of wastewater-based epidemiology to the city of Thessaloniki, Greece.

During the COVID-19 pandemic, wastewater-based epidemiology has proved to be an important tool for monitoring the spread of a disease in a population. Indeed, wastewater surveillance was successfully used as a complementary approach to support public health monitoring schemes and decision-making policies. An essential feature for the estimation of a disease transmission using wastewater data is the distribution of viral shedding rate of individuals in their personal human wastes as a function of the days of their infection. Several candidate shapes for this function have been proposed in literature for SARS-CoV-2. The purpose of the present work is to explore the proposed function shapes and examine their significance on analyzing wastewater SARS-CoV-2 shedding rate data. For this purpose, a simple model is employed applying to medical surveillance and wastewater data of the city of Thessaloniki during a period of Omicron variant domination in 2022. The distribution shapes are normalized with respect to the total virus shedding and then their basic features are investigated. Detailed analysis reveals that the main parameter determining the results of the model is the difference between the day of maximum shedding rate and the day of infection reporting. Since the latter is not part of the distribution shape, the major feature of the distribution affecting the estimation of the number of infected people is the day of maximum shedding rate with respect to the initial infection day. On the contrary, the duration of shedding (total number of disease days) as well as the exact shape of the distribution are by far less important. The incorporation of such wastewater surveillance models in conventional epidemiological models - based on recorded disease transmission data- may improve predictions for disease spread during outbreaks.

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater.

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

Implementation of a Sensitive Method to Assess High Virus Retention Performance of Low-Pressure Reverse Osmosis Process.

Human enteric viruses are important etiological agents of waterborne diseases. Environmental waters are usually contaminated with low virus concentration requiring large concentration factors for effective detection by (RT)-qPCR. Low-pressure reverse osmosis is often used to remove water contaminants, but very few studies focused on the effective virus removal of reverse osmosis treatment with feed concentrations as close as possible to environmental concentrations and principally relied on theoretical virus removal. The very low viral concentrations usually reported in the permeates (i.e. at least 5 log of removal rate) mean that very large volumes of water need to be analysed to have sufficient sensitivity and assess the process efficiency. This study evaluates two methods for the concentration of adenoviruses, enteroviruses and MS2 bacteriophages at different viral concentrations in large (< 200 L) and very large (> 200 L) volumes. The first method is composed of two ultrafiltration membranes with low-molecular weight cut-offs while the second method primarily relies on adsorption and elution phases using electropositive-charged filters. The recovery rates were assessed for both methods. For the ultrafiltration-based protocol, recovery rates were similar for each virus studied: 80% on average at high virus concentrations (106-107 viruses L-1) and 50% at low virus concentrations (103-104 viruses L-1). For the electropositive-charged filter-based method, the average recoveries obtained were about 36% for ADV 41, 57% for CV-B5 and 1.6% for MS2. The ultrafiltration-based method was then used to evaluate the performance of a low-pressure reverse osmosis lab-scale pilot plant. The retentions by reverse osmosis were similar for all studied viruses and the validated recovery rates applied to the system confirmed the reliability of the concentration method. This method was effective in concentrating all three viruses over a wide range of viral concentrations. Moreover, the second concentration method using electropositive-charged filters was studied, allowing the filtration of larger volumes of permeate from a semi-industrial low-pressure reverse osmosis pilot plant. This reference method was used because of the inability of the UF method to filter volumes on the order of one cubic metre.

Improved virus concentration methods for wash waters from decontamination of permeable and non-permeable surfaces.

Surface decontamination is a method of using wash water to decontaminated surfaces preventing transmission of biological contaminants that can pose potential health risks to responders and the public. However, the risks associated with handling used wash water are largely unknown due to the lack of effective methodology to screen for pathogenic microorganisms present in these samples, especially viral pathogens. This study adapted the dead-end hollow-fiber ultrafiltration (D-HFUF) system to wash waters, including a separate procedure for recovering particle attached viruses. Simulated wash water was created using dechlorinated tap water containing a mild surfactant (0.05 % Tween 80). To determine virus recovery efficiencies, measured amounts of somatic and F+ coliphage were spiked into 2-liter volumes of wash water under the following scenarios: (1) wash water was amended with a measured amount of sterile river sediment with no sediment separation prior to filter concentration; or (2) sediment added to wash water was allowed to settle prior to filter concentrating clarified liquid portions, while precipitated sediment was subjected to viral extraction techniques to recover particle attached virus; and (3) the optimized method was deployed on non-porous and porous surfaces to simulate a decontamination clean-up event. Separation of sediment prior to D-HFUF significantly increased recovery of coliphages, (P = <0.0001) versus filtration of sediment and liquids simultaneously. A tryptic soy broth (TSB) elution solution was significantly more effective (P = ≤0.010) for recovery of both somatic and F+ coliphage, (108 ± 9 % and 92 ± 9 %, respectively), compared to elution buffers containing various surfactants (sodium hexametaphosphate, Tween 80) for recovering particle attached virus. Simulating a biocontaminate clean-up event (using the optimized sediment separation and elution protocol) resulted in coliphage recoveries of 75-96 % (permeable surface) and 71-92 % (non-permeable surface). This procedure can be used to effectively detect viruses in used wash waters aiding in reducing risks to human health during site decontamination.

Development of a simple and low-cost method using Moringa seeds for efficient virus concentration in wastewater.

Effective virus concentration methods are essential for detecting pathogenic viruses in environmental waters and play a crucial role in wastewater-based epidemiology. However, the current methods are often expensive, complicated, and time-consuming, which limits their practical application. In this study, a simple and low-cost method was developed using the extract of Moringa oleifera (MO) seeds (MO method) to recover both enveloped and non-enveloped viruses, including pepper mild mottle virus (PMMoV), murine norovirus (MNV), Aichivirus (AiV), murine hepatitis virus (MHV), and influenza A virus subtype H1N1[H1N1] in wastewater. The optimal conditions for the MO method were determined to be a concentration of MO extract at the UV280 value of 0.308 cm-1 and an elution buffer (0.05 M KH2PO4, 1 M NaCl, 0.1 % Tween80 [v/v]) for recovering the tested viruses in wastewater. Compared to other commonly used virus concentration methods such as InnovaPrep, HA, PEG, and Centricon, the MO method was found to be more efficient and cost-effective in recovering the tested viruses. Moreover, the MO method was successfully applied to detect various types of viruses (PMMoV, AiV, norovirus of genotype II [NoV II], enterovirus [EV], influenza A virus [matrix gene] [IAV], and SARS-CoV-2) in raw wastewater. Thus, the developed MO method could offer a simple, low-cost, and efficient tool to concentrate viruses in wastewater.

Preparation of purified vaccine from local isolate of foot and mouth disease virus and its immune response in bovine calves.

Foot and Mouth Disease (FMD) is globally pandemic which badly affect the economics of livestock based countries like Pakistan. There are different types of Foot and Mouth Disease Virus (FMDV) among these types O is most prevalent in Pakistan. Recently Pakistan is producing approximately fifteen million doses of non-purified FMD vaccine against the demand of 160 million doses annually. More over the Pakistan is still striving for the development and optimization of concentration as well as purification of FMDV. The present project was designed to develop the technology for the purification of FMDV indigenously. The locally isolated and adapted FMDV type O virus was propagated on adherent culture of BHK-21cells to get final volume of virus one liter. This virus suspension was concentrated by peggylation as well as ultra-filtration method. The purification and quantification of concentrated virus was done by size exclusion chromatography. The results showed that peggylation is better method of concentration up to 603.75 µg/ml with 82.80 % recovery rate than ultra-filtration with 43.90 % followed by chromatography for purification. The PD50 was calculated in bovines at 24, 12, 6, 3 and 1.5 µg of FMDV Ag/dose and it revealed that antigen load of 1.98 µg is the dose, where the 50 % of inoculated animals showed the protective antibody level based upon percent inhibition through antibody detecting ELISA. According to the British pharmacopeia, the vaccine should contain 3PD50 which found equivalent to our findings about 6 µg/dose. The group of animal injected with 6/dose (3.23PD50) showed protective titer up to 20th week post priming.

Optimization and performance evaluation of an automated filtration method for the recovery of SARS-CoV-2 and other viruses in wastewater.

A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.

Manipulation, Sampling and Inactivation of the SARS-CoV-2 Virus Using Nonuniform Electric Fields on Micro-Fabricated Platforms: A Review.

Micro-devices that use electric fields to trap, analyze and inactivate micro-organisms vary in concept, design and application. The application of electric fields to manipulate and inactivate bacteria and single-celled organisms has been described extensively in the literature. By contrast, the effect of such fields on viruses is not well understood. This review explores the possibility of using existing methods for manipulating and inactivating larger viruses and bacteria, for smaller viruses, such as SARS-CoV-2. It also provides an overview of the theoretical background. The findings may be used to implement new ideas and frame experimental parameters that optimize the manipulation, sampling and inactivation of SARS-CoV-2 electrically.

Investigation of SARS-CoV-2 RNA contamination in water supply resources of Tabriz metropolitan during a peak of COVID-19 pandemic.

It is crucial to have access to clean water resources during the COVID-19 pandemic for hygiene, since virus infection through wastewater leaks in metropolitan areas can be a threat. Accurate monitoring of urban water resources during the pandemic seems to be the only way to confirm safe and infected resources. Here, in this study, the amount of Severe Acute Respiratory Syndrome Coronavirus 2's Ribonucleic Acid (SARS-CoV-2 RNA) in the Tabriz urban water network located in the northwest of Iran was investigated by an extensive sampling of the city's water sources at a severe peak of the COVID-19 pandemic. The sampling process comprised a range of water sources, including wells, qanats, water treatment facilities, dams, and reservoirs. For each sample, a combination of polyethylene glycol (PEG) and sodium chloride (NaCl) was used for concentration and a laboratory RNA-based method was conducted for quantification. Before applying the extraction and quantification procedure to real samples, the proposed concentration method was verified with synthetic serum samples for the first time. After the concentration, RNA extraction was done by the BehPrep extraction column method, and Reverse Transcription Polymerase Chain Reaction (RT-PCR) detection of the virus was done by Covitech COVID-19 RT-PCR kit. In none of the water supply resources, SARS-COV-2 RNA has been detected except in a sample grabbed from a well adjacent to an urban wastewater discharge point downstream. The results of molecular analysis for the positive sample showed that the CT value and concentration of the virus genome were equal to 32.57 and 5720 copies/L, respectively. Quantitative analysis of real samples shows that the city's water network was safe at the time of the study. However, given that the positive sample was exposed to wastewater leakage, periodic sampling from wells and qanats is suggested during the pandemic until it can be proven that the leakage to these water sources is impossible.

Comparison of Models for Quantification of Tomato Brown Rugose Fruit Virus Based on a Bioassay Using a Local Lesion Host.

Considering the availability of serological and molecular biological methods, the bioassay has been paled into insignificance, although it is the only experimental method that can be used to demonstrate the infectivity of a virus. We compared goodness-of-fit and predictability power of five models for the quantification of tomato brown rugose fruit virus (ToBRFV) based on local lesion assays: the Kleczkowski model, Furumoto and Mickey models I and II, the Gokhale and Bald model (growth curve model), and the modified Poisson model. For this purpose, mechanical inoculations onto Nicotiana tabacum L. cv. Xanthi nc and N. glutionosa L. with defined virus concentrations were first performed with half-leaf randomization in a Latin square design. Subsequently, models were implemented using Python software and fitted to the number of local lesions. All models could fit to the data for quantifying ToBRFV based on local lesions, among which the modified Poisson model had the best prediction of virus concentration in spike samples based on local lesions, although data of individual indicator plants showed variations. More accurate modeling was obtained from the test plant N. glutinosa than from N. tabacum cv. Xanthi nc. The position of the half-leaves on the test plants had no significant effect on the number of local lesions.

Evaluation of a simple ultrafiltration method for concentration of infective canine parvovirus and feline coronavirus from cell culture supernatants.

Enrichment of viral infectious titers following its propagation by cell culture is desirable for various experimental studies. The performance of an ultrafiltration (UF) process to concentrate infectious titers of non-enveloped Canine parvovirus 2 (CPV-2) and enveloped Feline coronavirus (FCoV) obtained from cell culture supernatants was evaluated in this study, and compared with ultracentrifugation (UC) process. A mean gain of > 1.0 log10 TCID50/mL was obtained for CPV-2 with UF, which was comparable with the gain obtained by UC. On the other hand, the gain was lower (0.7-1.0 log10 TCID50/mL) for FCoV with UF in contrast to UC (> 2.0 log10 TCID50/mL). However, the lower retentate volume following UC (∼120 fold) compared to that following UF (∼10 fold) for either of the viruses suggests a trend of increased infectious titer retention in UF concentrates relative to UC concentrates. The simplistic UF process evaluated here thus has the potential for use in applications requiring increased infectious titers of CPV-2 and FCoV.

Development of a magnetic nanoparticle-based method for concentrating SARS-CoV-2 in wastewater.

Several virus concentration methods have been developed to increase the detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater, as part of applying wastewater-based epidemiology. Polyethylene glycol (PEG) precipitation method, a method widely used for concentrating viruses in wastewater, has some limitations, such as long processing time. In this study, Pegcision, a PEG-based method using magnetic nanoparticles (MNPs), was applied to detect SARS-CoV-2 in wastewater, with several modifications to increase its sensitivity and throughput. An enveloped virus surrogate, Pseudomonas phage φ6, and a non-enveloped virus surrogate, coliphage MS2, were seeded into wastewater samples and quantified using reverse transcription-quantitative polymerase chain reaction to assess the recovery performance of the Pegcision. Neither increasing MNP concentration nor reducing the reaction time to 10 min affected the recovery, while adding polyacrylic acid as a polyanion improved the detection sensitivity. The performance of the Pegcision was further compared to that of the PEG precipitation method based on the detection of SARS-CoV-2 and surrogate viruses, including indigenous pepper mild mottle virus (PMMoV), in wastewater samples (n = 27). The Pegcision showed recovery of 14.1 ± 6.3 % and 1.4 ± 1.0 % for φ6 and MS2, respectively, while the PEG precipitation method showed recovery of 20.4 ± 20.2 % and 18.4 ± 21.9 % (n = 27 each). Additionally, comparable PMMoV concentrations were observed between the Pegcision (7.9 ± 0.3 log copies/L) and PEG precipitation methods (8.0 ± 0.2 log copies/L) (P > 0.05) (n = 27). SARS-CoV-2 RNA was successfully detected in 11 (41 %) each of 27 wastewater samples using the Pegcision and PEG precipitation methods. The Pegcision showed comparable performance with the PEG precipitation method for SARS-CoV-2 RNA concentration, suggesting its applicability as a virus concentration method.

Viral Safety Issues in the Production and Manufacturing of Human Immunoglobulin Preparations from Equine Plasma/Serum.

The current Russian and foreign pharmacopoeias either do not provide any information about existing types of viral diseases in horses or do not present it in full. Data of modern domestic and foreign literature was used to prepare the most complete list of viruses that cause equine diseases including 36 infectious agents, 25 of which are pathogenic for humans, 13 of the 25 of which are widespread throughout Russia. Information is provided on the magnitudes of the disease incubation periods (which are most often within one month), the external clinical signs of these diseases (which can also be asymptomatic), and the maximum possible concentrations of viruses in the blood of horses with these diseases (which can reach 8 log conventional units/mL of blood). This information is offered for use in critical production stages of heterologous immunoglobulin drugs for medical use to assure viral safety.

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis.

BACKGROUND: In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. METHODS: To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. RESULTS: We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. CONCLUSIONS: ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial.

Application of iron flocculation to concentrate white spot syndrome virus in seawater.

The iron flocculation method, which comprises the Fe-virus flocculate formation-filtration-resuspension steps, is extensively used to concentrate and precipitate viruses distributed in water. To apply this method to concentrate white spot syndrome virus (WSSV) in seawater, viral genomic and infective recovery yields were compared between polyethylene sulfone (PES) and polycarbonate (PC) membrane filters and two types of resuspension buffers (oxalate and ascorbate). Viral genome quantitation was determined above a 95 % limit of detection (11.48 viral DNA copies/µL) using quantitative real-time PCR. From WSSV-spiked seawater (100-106 viral DNA copies/mL), the viral genomic recovery yields of the PES-Oxalate, PC-Oxalate, PES-Ascorbate, and PC-Ascorbate conditions were 78.67 % ± 12.90 %, 84.53 % ± 24.30 %, 85.59 % ± 16.98 %, and 93.74 % ± 7.44 %, respectively. The detectable Fe-virus flocculates collected by the PC membrane were approximately 101 WSSV DNA copies/mL of seawater, a value more than 10-fold higher than that compared to the PES membrane filter (102 WSSV DNA copies/mL), regardless of the resuspension buffer types. WSSV resuspended with oxalate buffer caused mass mortality among whiteleg shrimp (Litopenaeus vannamei), inducing the expression of the virus envelope protein, VP28, similar to that of a native virus, suggesting stable viral activity during the resuspension process. Based on the PES-Ascorbate, WSSV particles could be successfully concentrated in seawater from shrimp farms with white spot disease outbreaks (approximately 102 WSSV DNA copies/mL). Collectively, these findings indicate that the simple and efficient method of iron flocculation is sufficient to concentrate WSSV in seawater and could be used as a non-invasive approach and one of the reasonable diagnostic processes for white spot disease surveillance.

Detection of SARS-CoV-2 RNA throughout wastewater treatment plants and a modeling approach to understand COVID-19 infection dynamics in Winnipeg, Canada.

Although numerous studies have detected SARS-CoV-2 RNA in wastewater and attempted to find correlations between the concentration of SARS-CoV-2 RNA and the number of cases, no consensus has been reached on sample collection and processing, and data analysis. Moreover, the fate of SARS-CoV-2 in wastewater treatment plants is another issue, specifically regarding the discharge of the virus into environmental settings and the water cycle. The current study monitored SARS-CoV-2 RNA in influent and effluent wastewater samples with three different concentration methods and sludge samples over six months (July to December 2020) to compare different virus concentration methods, assess the fate of SARS-CoV-2 RNA in wastewater treatment plants, and describe the potential relationship between SARS-CoV-2 RNA concentrations in influent and infection dynamics. Skimmed milk flocculation (SMF) resulted in 15.27 ± 3.32% recovery of an internal positive control, Armored RNA, and a high positivity rate of SARS-CoV-2 RNA in stored wastewater samples compared to ultrafiltration methods employing a prefiltration step to eliminate solids in fresh wastewater samples. Our results suggested that SARS-CoV-2 RNA may predominate in solids, and therefore, concentration methods focusing on both supernatant and solid fractions may result in better recovery. SARS-CoV-2 RNA was detected in influent and primary sludge samples but not in secondary and final effluent samples, indicating a significant reduction during primary and secondary treatments. SARS-CoV-2 RNA was first detected in influent on September 30th, 2020. A decay-rate formula was applied to estimate initial concentrations of late-processed samples with SMF. A model based on shedding rate and new cases was applied to estimate SARS-CoV-2 RNA concentrations and the number of active shedders. Inferred sensitivity of observed and modeled concentrations to the fluctuations in new cases and test-positivity rates indicated a potential contribution of newly infected individuals to SARS-CoV-2 RNA loads in wastewater.

Evaluation of reduction efficiencies of pepper mild mottle virus and human enteric viruses in full-scale drinking water treatment plants employing coagulation-sedimentation-rapid sand filtration or coagulation-microfiltration.

Here, we evaluated the reduction efficiencies of indigenous pepper mild mottle virus (PMMoV, a potential surrogate for human enteric viruses to assess virus removal by coagulation-sedimentation-rapid sand filtration [CS-RSF] and coagulation-microfiltration [C-MF]) and representative human enteric viruses in four full-scale drinking water treatment plants that use CS-RSF (Plants A and B) or C-MF (Plants C and D). First, we developed a virus concentration method by using an electropositive filter and a tangential-flow ultrafiltration membrane to effectively concentrate and recover PMMoV from large volumes of water: the recovery rates of PMMoV were 100% when 100-L samples of PMMoV-spiked dechlorinated tap water were concentrated to 20 mL; even when spiked water volume was 2000 L, recovery rates of >30% were maintained. The concentrations of indigenous PMMoV in raw and treated water samples determined by using this method were always above the quantification limit of the real-time polymerase chain reaction assay. We therefore were able to determine its reduction ratios: 0.9-2.7-log10 in full-scale CS-RSF and 0.7-2.9-log10 in full-scale C-MF. The PMMoV reduction ratios in C-MF at Plant C (1.0 ± 0.3-log10) were lower than those in CS-RSF at Plants A (1.7 ± 0.5-log10) and B (1.4 ± 0.7-log10), despite the higher ability of MF for particle separation in comparison with RSF owing to the small pore size in MF. Lab-scale virus-spiking C-MF experiments that mimicked full-scale C-MF revealed that a low dosage of coagulant (polyaluminum chloride [PACl]) applied in C-MF, which is determined mainly from the viewpoint of preventing membrane fouling, probably led to the low reduction ratios of PMMoV in C-MF. This implies that high virus reduction ratios (>4-log10) achieved in previous lab-scale virus-spiking C-MF studies are not necessarily achieved in full-scale C-MF. The PMMoV reduction ratios in C-MF at Plant D (2.2 ± 0.6-log10) were higher than those at Plant C, despite similar coagulant dosages. In lab-scale C-MF, the PMMoV reduction ratios increased from 1-log10 (with PACl [basicity 1.5], as at Plant C) to 2-4-log10 (with high-basicity PACl [basicity 2.1], as at Plant D), suggesting that the use of high-basicity PACl probably resulted in higher reduction ratios of PMMoV at Plant D than at Plant C. Finally, we compared the reduction ratios of indigenous PMMoV and representative human enteric viruses in full-scale CS-RSF and C-MF. At Plant D, the concentrations of human norovirus genogroup II (HuNoV GII) in raw water were sometimes above the quantification limit; however, whether its reduction ratios in C-MF were higher than those of PMMoV could not be judged since reduction ratios were >1.4-log10 for HuNoV GII and 2.3-2.9-log10 for PMMoV. At Plant B, the concentrations of enteroviruses (EVs) and HuNoV GII in raw water were above the quantification limit on one occasion, and the reduction ratios of EVs (>1.2-log10) and HuNoV GII (>1.5-log10) in CS-RSF were higher than that of PMMoV (0.9-log10). This finding supports the usefulness of PMMoV as a potential surrogate for human enteric viruses to assess virus removal by CS-RSF.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers.

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.