Viruses In Water Research Articles

Combination of Crossflow Ultrafiltration, Monolithic Affinity Filtration, and Quantitative Reverse Transcriptase PCR for Rapid Concentration and Quantification of Model Viruses in Water

We present a rapid and effective adsorption-elution method based on monolithic affinity filtration (MAF) for the concentration and purification of waterborne viruses. The MAF column consists of a hydrolyzed macroporous epoxy-based polymer. High recoveries were achieved by columns for the bacterial virus (bacteriophage) MS2 110 (±19)%, as model organism, as well as for human adenoviruses 42.4 (±3.4)% and murine noroviruses 42.6 (±1.9)%. This new concentration and purification method was combined with crossflow ultrafiltration (CUF). Because of the adsorption of the examined viruses to the macroporous surface of the MAF column at pH 3, concentrated matrix components by CUF can be removed. Bacteriophages MS2 were spiked in tap water and concentrated with the new CUF-MAF concentration method by a volumetric factor of 10(4) within 33 min. Furthermore, the detection limit for quantification of bacteriophage MS2 by quantitative reverse transcriptase PCR (qRT-PCR) could be improved from 79.47 to 0.0056 GU mL(-1) by a factor of 1.4 × 10(4). In a first study, we have shown that this method could also be applied for river water containing naturally MS2 and MS2-like phages.

Freshwater Clams As Bioconcentrators of Avian Influenza Virus in Water

We report experimental evidence for bioconcentration of a low-pathogenicity avian influenza virus (H6N8) in the tissue of freshwater clams. Our results support the concept that freshwater clams may provide an effective tool for use in the early detection of influenza A viruses in aquatic environments.

Pre-analytical and analytical procedures for the detection of enteric viruses and enterovirus in water samples

Practical pre-analytical and analytical procedures were developed and validated for detection of enteric viruses in three water matrices. Both RNA viruses (norovirus, coxsackievirus, echovirus, and rotavirus) and DNA virus (adenovirus 41) were included in the study. The NanoCeram 90mm laminated disc with electropositive filter and procedures of filtration, elution and flocculation were utilized to concentrate known amount of viruses in different water matrices. Real time quantitative PCR was used to evaluate the recovery of virus and cell culture to assess viral infectivity. There was no PCR inhibition using various concentrations and pH of beef extract eluting buffer. A good recovery of the viruses spiked in 10L of deionized water was achieved for serial dilutions of coxsackievirus (41-67%), echovirus (22-90%), norovirus (23-44%) and rotavirus (24-46%). Relatively lower recovery was observed for adenovirus 41 (24-35%). There was no significant difference in viral recovery from deionized, tap and river water samples. The infectivity of recovered adenovirus, coxsackievirus and echovirus was demonstrated using in vitro cell culture. The pre-analytical and analytic procedures attained consistent recovery of RNA and DNA viruses both as infectious viral particles and viral genome, provided effective removal of inhibitory substances, achieved reliable reproducibility, and were relatively inexpensive for monitoring viruses in water.

양전하가 부가된 수처리 필터의 입자제거특성에 관한 연구

The purpose of the present work is to investigate the removal characteristics of positively charged filters for capturing negatively charged particles such as bacteria and virus in water. In order to reduce the pressure drop and increase the filtration efficiency, the filter media, modified by charge modifier having positive functional groups, is developed and evaluated. Improved liquid filters have been developed with the modified surface charge to capture and adsorb particles by electrokinetic interaction between the filter surface and particles contained in an aqueous liquid. The positively charged filter media is composed of glass fiber, cellulose and poly-ethylenimine resin for positively charging with the variation of volume ratio. The zeta potential value of the positively charged filter is +37.92 mV at the glass fiber and cellulose content ratio of 50 : 50 with resin content of 100%, while that of the PSL test particle is -23.5 mV at pH 7. The removal efficiency of the electro-positively charged filter is 98% for PSL particles of 0.11 <TEX>${\mu}m$</TEX>, while that of the negatively charged filter is 7%. The positively charged filter media showed the potential to be an effective method for removing fine particles from the contaminated water for liquid filtration.

Duplex RT-qPCR for the detection of hepatitis E virus in water, using a process control

Human hepatitis E virus (HEV) causes acute hepatitis in humans, predominantly by contamination of food and water. HEV, in particular genotype III, is currently considered to be an emerging pathogen in industrialized countries. Because of the low infectious dose, an efficient and rapid virus concentration method is required to detect low amounts of HEV in food and water samples for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food and water samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of the concentration of viral particles, the extraction of nucleic acid and the presence of the potential inhibitors of the RT-qPCR reaction. The aim of this study was to provide a rapid and sensitive method for detecting HEV in water. The method is based on viral concentration by filtration on membrane filters and direct lysis of adsorbed viruses from filters before RNA extraction and RT-qPCR amplification. We developed a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) was used as a process control. The data show that MNV-1 offers a very reliable and simple way of monitoring false-negative results and is a valuable tool in the routine diagnostic laboratory. The limit of detection (LOD) was in the range of 700 to 3500 HEV genome copies/0.5L bottled water and 3500 HEV genome copies/0.5L tap water.

Fluorescence instrument for in situ monitoring of viral abundance in water, wastewater and recycled water

In a world of advanced molecular methods quantifying viruses in water remains one of the most inefficient and costly. Using a general molecular DNA/RNA probe - SYBR Gold combined with differential filtration a fast, cost effective and sensitive method is presented to determine the concentration of viruses in water in situ or on-line. The approach differentiates the nucleotide size fractions that are stained with SYBR Gold to show only those associated with Viral DNA and RNA. There was a linear relationship between the fluorescence maxima for SYBR Gold added to wastewater and viral numbers determined with direct counting using epifluorescent microscopy (r(2)=0.97) and for a range of diverse natural water samples (r(2)=0.86). The method was applied to water from the tropics and Antarctica, marine and freshwater environments where natural viral abundances ranged from 10(6) to 10(8) virusesmL(-1). The method takes into account the background fluorescence that represented 70% of total fluorescence and any auto-fluorescence due to other dissolved organic carbon. While DNAse II lowered the background fluorescence associated with free DNA and RNA it could not be eliminated. The technique presented is suitable for monitoring in situ viral numbers in natural water bodies and engineered water treatment processes. This on-line viral monitoring design has the potential to replace human viral pathogen indicators.

Role of Water Fleas (Daphniamagna) in the Accumulation of Avian Influenza Viruses from the Surrounding Water

Objective: To determine the role of water fleas in accumulating avian influenza viruses (AIV) from the surrounding water and to estimate their role as a vector of AIV. Methods: Water fleas were exposed to H4N6 and H5N1 AIV-contaminated water in a closed system. The potential of water fleas to take up and retain the viruses was estimated by quantitative real-time RT-PCR (qRRT-PCR) and titration on cell culture. Results: Contamination trials showed that significantly higher amounts of viral RNA were detectable per gram of water fleas as compared to per milliliter of the surrounding water at 1, 4, and 6 days of incubation. Viral infectivity was only detectable in the water samples collected immediately after mixing the virus in water containing the water fleas, while no virus was detectable in any of the water fleas or water samples collected afterwards. Conclusions: Water fleas are able to accumulate AIV from surrounding water based upon the qRRT-PCR detection of viral RNA. Additional studies are necessary to investigate the inactivation potential of water fleas on viral infectivity.

Persistence of the 2009 Pandemic Influenza A (H1N1) Virus in Water and on Non-Porous Surface

Knowledge of influenza A virus survival in different environmental conditions is a key element for the implementation of hygiene and personal protection measures by health authorities. As it is dependent on virus isolates even within the same subtype, we studied the survival of the 2009 H1N1 pandemic (H1N1pdm) virus in water and on non-porous surface. The H1N1pdm virus was subjected to various environmental parameters over time and tested for infectivity. In water, at low and medium salinity levels and 4°C, virus survived at least 200 days. Increasing temperature and salinity had a strong negative effect on the survival of the virus which remained infectious no more than 1 day at 35°C and 270 parts per thousand (ppt) of salt. Based on modeled data, the H1N1pdm virus retained its infectivity on smooth non-porous surface for at least 7 days at 35°C and up to 66 days at 4°C. The H1N1pdm virus has thus the ability to persist in water and on glass surface for extended periods of time, even at 35°C. Additional experiments suggest that external viral structures in direct contact with the environment are mostly involved in loss of virus infectivity.

Prevalence of protozoa, viruses, coliphages and indicator bacteria in groundwater and river water in the Kathmandu Valley, Nepal

Limited information is available on the prevalence of waterborne pathogens in aquatic environments in developing countries. In this study, water samples were collected from nine shallow wells and a river in the Kathmandu Valley, Nepal, during the rainy season in 2009 and were subjected to detection of waterborne protozoa, viruses and coliphages using a recently developed method for simultaneous concentration of protozoa and viruses in water. Escherichia coli and total coliforms were also tested as indicator bacteria. At least one type of the five pathogens tested ( Cryptosporidium, Giardia, human adenoviruses, and noroviruses of genogroups I and II) was detected in five groundwater samples (56%) (1000 ml each) from shallow wells. Compared with groundwater samples, the pathogens were more abundant in the river water sample (100 ml); the concentrations of Cryptosporidium and Giardia were 140 oocysts/l and 8500 cysts/l, respectively, and the mean threshold cycle (Ct) values in real-time RT-PCR were 34.3, 36.8 and 34.0 for human adenoviruses and noroviruses of genogroups I and II, respectively. Genotyping of F-RNA coliphages by real-time RT-PCR was successfully used to differentiate human and animal faecal contamination in the samples. Moreover, for the groundwater samples, protozoa and viruses were detected only in E. coli-positive samples, suggesting that E. coli may be an appropriate indicator of pathogen contamination of valley groundwater.

Inactivation of Bacterial Viruses in Water Using Deep Ultraviolet Semiconductor Light-Emitting Diode

Disinfection of bacterial viruses MS2, Qβ, and φX174 in water was successfully carried out using deep ultraviolet light-emitting diodes (DUV-LEDs) operated at 280 nm and 255 nm. It was shown that t...

Dynamics of diatom viruses on the western coast of Japan

Diatoms are prominent primary producers that play an important role in global carbon cycles. Diatom dynamics are, therefore, important for biogeochemistry, fisheries and earth science. Viral infection is now assumed to be one of the most significant factors affecting diatom dynamics. However, few studies, based on practical field data, have focused on the relationship between diatoms and viruses in natural waters. To elucidate on this relationship, we assessed the temporal change in the abundance of diatoms and their viruses on the western coast of Japan from 2004 to 2009. Three species of diatoms were used as hosts to enumerate viruses in water and sediment sam- ples: Chaetoceros debilis, C. tenuissimus and C. salsugineum. Diatom viruses in the water column rapidly increased during C. debilis and C. tenuissimus blooms, maintaining a high abundance throughout the blooming period. These data suggest the potential importance of viruses in control- ling the population dynamics of diatoms in natural environments. The number of viruses in sediments fluctuated considerably, even during non-blooming periods, which suggests that virus-infected diatom cells were supplied to the sediments from water-column populations throughout the year. This process might be an important strategy to reduce the impact of viruses on diatom populations. Because diatom populations are thought to have an intimate relationship with their viruses in natural waters, further studies of diatom dynamics should include the effects of viral infections.

Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat

The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23–25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall.

Novel concentration method for the detection of norovirus and sapovirus from water using minute particles of amorphous calcium phosphate

A novel concentration method using minute particles of amorphous calcium phosphate (ACP) was developed for the detection of caliciviruses including norovirus and sapovirus, agents of human gastroenteritis, from water. In seeding experiments with feline calicivirus (FCV), ACP particles were able to adsorb efficiently the viruses in water, and the FCV-concentrated solution was obtained by dissolution of the virus-adsorbing ACP particles with citric acid after centrifugation. By quantitative real-time RT-PCR, the recovery efficiencies from 300 ml ultrapure water seeded with 10³, 10⁴ and >10⁵ copies of FCV were 48, 68 and >100 %, respectively. A comparative study showed that in the addition of viruses at <10⁵ copies, the recovery efficiency of our method was significantly higher (P<0.05) than that of the similar calcium flocculation-citrate dissolution method. Using our newly developed method, we successfully detected 2.1 x 10⁴ copies l⁻¹ of norovirus (each of genogroups I and II) and 5.4 x 10³ copies l⁻¹ of sapovirus (genogroups I, II, IV and V) from river water. The data suggest that our new viral concentration is a rapid, simple, cost efficient and high virus recovery method, and it can be used for routine monitoring of norovirus and sapovirus in water, especially environmental water.

Silver-modified iron oxide nanoparticle impregnated fiberglass for disinfection of bacteria and viruses in water

Recent work has shown that a new material system composed of iron oxide (Fe 2O 3) nanoparticles loaded onto a fiberglass support displays excellent antiviral properties against the model virus, MS2 phage, but is ineffective against bacteria, specifically Escherichia coli ( E. coli). To increase the antibacterial properties and still maintain antiviral activity, silver (Ag) nanoparticles were added to this system through an aqueous hydrothermal reduction process with 0.25 M silver nitrate (AgNO 3). A 0.05 mg/mL loading of the new Ag modified oligodynamic nanoparticle impregnated fiberglass system consisting of Fe 2O 3 (9.1 wt.%) and Ag (0.1 wt.%)/g-fiber, displayed robust antibacterial activity by achieving a 2 log removal of 10 6 CFU/mL E. coli in 1 min. The inactivation rate of the Ag-modified Fe 2O 3 impregnated fiberglass (FG-Fe 2O 3/Ag) system was comparable to previously reported Ag nanoparticle impregnated fiberglass. The FG-Fe 2O 3/Ag displayed excellent dynamic performance, effectively eliminating 10 6 E. coli CFU /mL with no bacterial breakthrough up to 250 bed volumes.

Evaluation of electropositive filtration for recovering norovirus in water

The virus adsorption-elution (VIRADEL) technique has been widely used in the recovery of various enteric viruses in water, and an electropositive filter such as 1 MDS has been commonly applied. However, effective methods of monitoring waterborne norovirus (NoV) have not yet been well characterized and optimized. Hence, in this study, the VIRADEL technique was evaluated and optimized for effectively detecting NoV in water by two commonly used electropositive filters (1MDS and NanoCeram filter). Various elution and concentration methods were evaluated by using both murine norovirus (MNV) and human NoV. Among the tested elution buffers, the most effective was 1.5% beef extract plus 0.01% Tween 80 for both 1MDS (67.5%) and NanoCeram (85.7%) microfilters. The recovery rate of GII-4 human NoV was higher by organic flocculation (86.6%) than by polyethylene glycol (PEG) precipitations (11.6~73.6%). When both 1MDS and NanoCeram filters were tested to detect NoV in surface and groundwater, the sensitivity of NoV recovered by these filters appeared to depend on the types and conditions of environmental water. The results of this study will help to set a standard of detection method for NoV in water.

Source water quality effects on monochloramine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus

There is a need for more information regarding monochloramine disinfection efficacy for viruses in water. In this study, monochloramine disinfection efficacy was investigated for coxsackievirus B5 (CVB5), echovirus 11 (E11), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated ground water and two partially treated surface waters. Duplicate disinfection experiments were completed at pH 7 and 8 in source water at concentrations of 1 and 3 mg/L monochloramine at 5 and 15 °C. The Efficiency Factor Hom (EFH) model was used to calculate CT values (mg-min/L) required to achieve 2-, 3-, and 4-log 10 reductions in viral titers. In all water types, monochloramine disinfection was most effective for MNV, with 3-log 10 CT values at 5 °C ranging from 27 to 110. Monochloramine disinfection was least effective for HAdV2 and E11, depending on water type, with 3-log 10 CT values at 5 °C ranging from 1200 to 3300 and 810 to 2300, respectively. Overall, disinfection proceeded faster at 15 °C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but there was no indication that overall disinfection efficacy was enhanced or inhibited in any one water type. CT values for HAdV2 in two types of source water exceeded federal CT value recommendations in the US. The results of this study demonstrate that water quality impacts the inactivation of viruses and should be considered when developing chloramination plans.

No influence of oxygen levels on pathogenesis and virus shedding in Salmonid alphavirus (SAV)-challenged Atlantic salmon (Salmo salar L.)

BackgroundFor more than three decades, diseases caused by salmonid alphaviruses (SAV) have become a major problem of increasing economic importance in the European fish-farming industry. However, experimental infection trials with SAV result in low or no mortality i.e very different from most field outbreaks of pancreas disease (PD). This probably reflects the difficulties in reproducing complex biotic and abiotic field conditions in the laboratory. In this study we looked at the relationship between SAV-infection in salmon and sub-lethal environmental hypoxia as a result of reduced flow-through in tank systems.ResultsThe experiment demonstrated that constant reduced oxygen levels (60-65% oxygen saturation: 6.5-7.0 mg/L) did not significantly increase the severity or the progress of pancreas disease (PD). These conclusions are based upon assessments of a semi-quantitative histopathological lesion score system, morbidities/mortalities, and levels of SAV RNA in tissues and water (measured by 1 MDS electropositive virus filters and downstream real-time RT-PCR). Furthermore, we demonstrate that the fish population shed detectable levels of the virus into the surrounding water during viraemia; 4-13 days after i.p. infection, and prior to appearance of severe lesions in heart (21-35 dpi). After this period, viral RNA from SAV could not be detected in water samples although still present in tissues (gills and hearts) at lasting low levels. Lesions could be seen in exocrine pancreas at 7-21 days post infection, but no muscle lesions were seen.ConclusionsIn our study, experimentally induced hypoxia failed to explain the discrepancy between the severities reported from field outbreaks of SAV-disease and experimental infections. Reduction of oxygen levels to constant suboptimal levels had no effect on the severity of lesions caused by SAV-infection or the progress of the disease. Furthermore, we present a modified VIRADEL method which can be used to detect virus in water and to supplement experimental infection trials with information related to viral shedding. By using this method, we were able to demonstrate for the first time that shedding of SAV from the fish population into the surrounding water coincides with viraemia.

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

Long-Term Study on Tenacity of Avian Influenza Viruses in Water (Distilled Water, Normal Saline, and Surface Water) at Different Temperatures

Long-Term Study on Tenacity of Avian Influenza Viruses in Water (Distilled Water, Normal Saline, and Surface Water) at Different Temperatures

Long-Term Study on Tenacity of Avian Influenza Viruses in Water (Distilled Water, Normal Saline, and Surface Water) at Different Temperatures

The tenacity of three low pathogenicity avian influenza viruses (AIV; subtypes H4N6, H5N1, and H6N8) was tested at five different temperatures (-10, 0, 10, 20, and 30 C) in distilled water, normal saline, and surface water obtained from Lake Constance. Infectivity of AIV in the samples was quantified at regular intervals by end point titration on Madin-Darby canine kidney cells for a maximum period of 36 wk, and duplicate samples were tested each time. The results showed that the survival time of AIV in all of the water types was inversely proportional to storage temperature. All three viruses showed varying sensitivity to inactivation under each of the experimental conditions. Persistence of the viruses was the longest in distilled water, second longest in normal saline, and shortest in surface water. The virus-inoculated surface water remained infective for a few days at 30 and 20 C, a few weeks at 10 C, and for months at 0 and -10 C.