Search

Viruses are transmissible via their interaction with contact surfaces of food containers or tools. This study evaluated the recoveries of MS2 coliphage, a virus surrogate, from polypropylene (PP), polyvinyl chloride (PVC), polyethylene (PE), and glass (borosilicate and soda lime), as influenced by the surface chemistry and topography. MS2 (5-6 logs) in PBS with 1% TSB was inoculated onto each of 9 different surfaces, 24-h cold-incubated, and recovery was quantified by infectivity. The order of MS2 recovery efficiency from smooth surfaces was PP > PE ≥ soda lime glass, which classified into 3 ANOVA groups, p = 0.05. The MS2 recovery ratios of smooth vs. rough surfaces were 1.4-1.5. Atomic force microscopy revealed 21-nm diam pinholes (<28-nm of MS2 size) in the borosilicate glass. The lowest and highest MS2 recoveries among the 9 surfaces were demonstrated by the hole-bearing borosilicate glass (34 ± 8%) and smooth PP (69 ± 14%) respectively. Generally greater MS2 recovery was obtained from smooth PP and PE surfaces compared to glass, but topographic alterations (pinholes or increased roughness) decreased recovery possibly by trapping the viruses., (Copyright © 2019. Published by Elsevier Ltd.)

Outbreaks of viral gastroenteritis are often connected to contaminated drinking water. The assessment of the water quality relies on the cultivation of indicator bacteria, and little is known of the fate of viruses in groundwater, especially in arctic regions. In Iceland, the groundwater temperature is between 3 and 6°C. The aim of this study was to determine virus inactivation at low temperature in a groundwater microcosm and in a borehole in a postglacial lava field. Two phage species that are commonly used as surrogates for norovirus were used, MS2 and PhiX174. Dialysis bags were used for the samples, and a device was constructed to hold many samples at a time and protect the samples in the borehole. No significant decrease of infective PhiX174 phages in the borehole or of the MS2 phages in the microcosm was observed. A slightly significant decrease of PhiX174 in the microcosm was noticed, with one log reduction time of 476 days. On the other hand, a significant reduction in MS2 was found in the field test, where the time needed for 90% reduction was 12·5 days. The results showed that an infective virus can exist in groundwater for months or years in arctic regions and a great difference may exist between results from microcosm and field tests. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that arctic regions are highly sensitive to virus contamination as an infective virus may exist in groundwater for years at low temperature. The results also show that the virus inactivation observed in field tests may differ considerably from the inactivation observed in laboratory microcosms. The results emphasize the importance of large protection zones around drinking water intakes as well as good wastewater treatment so that the likelihood of faecal contamination of groundwater is reduced., (© 2020 The Society for Applied Microbiology.)

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

New disinfection procedures are being developed and proposed for use in drinking-water production. Authorising their use requires an effective test strategy that can simulate conditions in practice. For this purpose, we developed a test rig working in a flow-through mode similar to the disinfection procedures in waterworks, but under tightly defined conditions, including very short contact times. To quantify the influence of DOC, temperature and pH on the efficacy of two standard disinfectants, chlorine and chlorine dioxide, simulated use tests were systematically performed. This test rig enabled quantitative comparison of the reduction of four test organisms, two viruses and two bacteria, in response to disinfection. Chlorine was substantially more effective against Enterococcus faecium than chlorine dioxide whereas the latter was more effective against the bacteriophage MS2, especially at pH values of >7.5 at which chlorine efficacies already decline. Contrary to expectation, bacteria were not generally reduced more quickly than viruses. Overall, the results confirm a high efficacy of chlorine and chlorine dioxide, validating them as standard disinfectants for assessing the efficacy of new disinfectants. Furthermore, these data demonstrate that the test rig is an appropriate tool for testing new disinfectants as well as disinfection procedures., (Copyright © 2018. Published by Elsevier GmbH.)

A continuous-flow UV reactor operating at 254nm wave-length was used to investigate inactivation of microorganisms including bacteriophage in coconut water, a highly opaque liquid food. UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three bacteria (E. coli ATCC 25922, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in buffer and coconut water were investigated (D 10 values ranging from 2.82 to 4.54mJ·cm -2 ). A series of known UV-C doses were delivered to the samples. Inactivation levels of all organisms were linearly proportional to UV-C dose (r 2 >0.97). At the highest dose of 30mJ·cm -2 , the three pathogenic organisms were inactivated by >5 log 10 (p<0.05). Results clearly demonstrated that UV-C irradiation effectively inactivated bacteriophage and pathogenic microbes in coconut water. The inactivation kinetics of microorganisms were best described by log linear model with a low root mean square error (RMSE) and high coefficient of determination (r 2 >0.97). Models for predicting log reduction as a function of UV-C irradiation dose were found to be significant (p<0.05) with low RMSE and high r 2 . The irradiated coconut water showed no cytotoxic effects on normal human intestinal cells and normal mouse liver cells. Overall, these results indicated that UV-C treatment did not generate cytotoxic compounds in the coconut water. This study clearly demonstrated that high levels of inactivation of pathogens can be achieved in coconut water, and suggested potential method for UV-C treatment of other liquid foods., Industrial Relevance: This research paper provides scientific evidence of the potential benefits of UV-C irradiation in inactivating bacterial and viral surrogates at commercially relevant doses of 0-120mJ·cm -2 . The irradiated coconut water showed no cytotoxic effects on normal intestinal and healthy mice liver cells. UV-C irradiation is an attractive food preservation technology and offers opportunities for horticultural and food processing industries to meet the growing demand from consumers for healthier and safe food products. This study would provide technical support for commercialization of UV-C treatment of beverages., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

One key assumption impacting data quality in viral inactivation studies is that reduction estimates are not altered by the virus seeding process. However, seeding viruses often involves the inadvertent addition of co-constituents such as cell culture components or additives used during preparation steps which can impact viral reduction estimates by inducing non-representative oxidant demand in disinfection studies and fouling in membrane assessments. The objective of this study was therefore to characterize a mammalian norovirus surrogate, murine norovirus (MNV), and bacteriophage MS2 at sequential stages of viral purification and to quantify their potential contribution to artificial oxidant demand and non-representative membrane fouling. Our results demonstrate that seeding solvent extracted and 0.1 micron filtered MNV to ~10 5 PFU/mL in an experimental water matrix will result in additional total organic carbon (TOC) and 30 min chlorine demand of 39.2 mg/L and 53.5 mg/L as Cl 2 , respectively. Performing sucrose cushion purification on the MNV stock prior to seeding reduces the impacts of TOC and chlorine demand to 1.6 and 0.15 mg/L as Cl 2 , respectively. The findings for MNV are likely relevant for other mammalian viruses propagated in serum-based media. Thus, advanced purification of mammalian virus stocks by sucrose cushion purification (or equivalent density-based separation approach) is warranted prior to seeding in water treatment assessments. Studies employing bacteriophage MS2 as a surrogate virus may not need virus purification, since seeding MS2 at a concentration of ~10 6 PFU/mL will introduce only ~1 mg/L of TOC and ~1 mg/L as Cl 2 of chlorine demand to experimental water matrices.

This study examined the inactivation of human norovirus (HuNoV) GI.1 and GII.4 by chlorine under conditions mimicking sewage treatment. Using a porcine gastric mucin-magnetic bead (PGM-MB) assay, no statistically significant loss in HuNoV binding (inactivation) was observed for secondary effluent treatments of ≤25 ppm total chlorine; for both strains, 50 and 100 ppm treatments resulted in ≤0.8-log 10 unit and ≥3.9-log 10 unit reductions, respectively. Treatments of 10, 25, 50, and 100 ppm chlorine inactivated 0.31, 1.35, >5, and >5 log 10 units, respectively, of the norovirus indicator MS2 bacteriophage. Evaluation of treatment time indicated that the vast majority of MS2 and HuNoV inactivation occurred in the first 5 min for 0.2-μm-filtered, prechlorinated secondary effluent. Free chlorine measurements of secondary effluent seeded with MS2 and HuNoV demonstrated substantial oxidative burdens. With 25, 50, and 100 ppm treatments, free chlorine levels after 5 min of exposure ranged from 0.21 to 0.58 ppm, from 0.28 to 16.7 ppm, and from 11.6 to 53 ppm, respectively. At chlorine treatment levels of >50 ppm, statistically significant differences were observed between reductions for PGM-MB-bound HuNoV (potentially infectious) particles and those for unbound (noninfectious) HuNoV particles or total norovirus particles. While results suggested that MS2 and HuNoV (measured as PGM-MB binding) behave similarly, although not identically, both have limited susceptibility to chlorine treatments of ≤25 ppm total chlorine. Since sewage treatment is performed at ≤25 ppm total chlorine, targeting free chlorine levels of 0.5 to 1.0 ppm, these results suggest that traditional chlorine-based sewage treatment does not inactivate HuNoV efficiently. IMPORTANCE HuNoV is ubiquitous in sewage. A receptor binding assay was used to assess inactivation of HuNoV by chlorine-based sewage treatment, given that the virus cannot be routinely propagated in vitro Results reported here indicate that chlorine treatment of sewage is not effective for inactivating HuNoV unless chlorine levels are above those routinely used for sewage treatment., (Copyright © 2017 American Society for Microbiology.)

Evidence has shown that environmental surfaces play an important role in the transmission of nosocomial pathogens. Deploying antimicrobial surfaces in hospital wards could reduce the role environmental surfaces play as reservoirs for pathogens. Herein we show a significant reduction in viable counts of Staphylococcus epidermidis, Saccharomyces cerevisiae, and MS2 Bacteriophage after light treatment of a medical grade silicone incorporating crystal violet, methylene blue and 2 nm gold nanoparticles. Furthermore, a migration assay demonstrated that in the presence of light, growth of the fungus-like organism Pythium ultimum and the filamentous fungus Botrytis cinerea was inhibited. Atomic Force Microscopy showed significant alterations to the surface of S. epidermidis, and electron microscopy showed cellular aggregates connected by discrete surface linkages. We have therefore demonstrated that the embedded surface has a broad antimicrobial activity under white light and that the surface treatment causes bacterial envelope damage and cell aggregation.

Surfactant Modified Zeolite (SMZ) represents a versatile, cost-effective permeable reactive material, capable of treating multiple classes of contaminants. The potential for HDTMA-Br, a cationic surfactant commonly used to modify zeolite, to desorb from the zeolite surface has been identified as a potential issue for the ongoing use of SMZ in water remediation contexts. This paper investigates the toxicity of HDTMA-Br towards enteric virus surrogates, F-RNA bacteriophage MS2 and E. coli, Bacillus subtilis, and soil microflora. The concentration of surfactant desorbing from SMZ was quantified through a bioassay using E. coli. Results showed HDTMA-Br concentrations of ≥10 -5 M were toxic to MS2, ≥10 -4 M were toxic to E. coli and ≥10 -6 M were toxic to B. subtilis. No toxic relationship was established between HDTMA-Br and soil microflora. Desorption of ≥10 -4 M of HDTMA-Br was shown for the two SMZ samples under the mixing conditions used. Effects of this surfactant on total soil microflora were ambiguous since no toxic relationship could be established, however, HDTMA-Br, at concentrations desorbing from SMZ, were shown to impact the soil bacterium B. subtilis. Further research is required to determine the effect of this surfactant on microbial populations and species diversity in soils., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill ( i nsensitivity to Ll ysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJ P330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJ P330Q mutant was recessive and could be suppressed by Lo vercomes d na J ( L odj ) alleles selected for restoration of lysis. All three L odj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the L odj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. IMPORTANCE Small single-stranded nucleic acid lytic phages ( Microviridae and Leviviridae ) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions., (Copyright © 2017 American Society for Microbiology.)

In order to assure the microbial safety of drinking water, UVC-LED treatment has emerged as a possible technology to replace the use of conventional low pressure (LP) mercury vapor UV lamps. In this investigation, inactivation of Human Enteric Virus (HuEV) surrogates with UVC-LEDs was investigated in a water disinfection system, and kinetic model equations were applied to depict the surviving infectivities of the viruses. MS2, Qβ, and ΦX 174 bacteriophages were inoculated into sterile distilled water (DW) and irradiated with UVC-LED printed circuit boards (PCBs) (266nm and 279nm) or conventional LP lamps. Infectivities of bacteriophages were effectively reduced by up to 7-log after 9mJ/cm 2 treatment for MS2 and Qβ, and 1mJ/cm 2 for ΦX 174. UVC-LEDs showed a superior viral inactivation effect compared to conventional LP lamps at the same dose (1mJ/cm 2 ). Non-log linear plot patterns were observed, so that Weibull, Biphasic, Log linear-tail, and Weibull-tail model equations were used to fit the virus survival curves. For MS2 and Qβ, Weibull and Biphasic models fit well with R 2 values approximately equal to 0.97-0.99, and the Weibull-tail equation accurately described survival of ΦX 174. The level of UV-susceptibility among coliphages measured by the inactivation rate constant, k, was statistically different (ΦX 174 (ssDNA)>MS2, Qβ (ssRNA)), and indicated that sensitivity to UV was attributed to viral genetic material., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

MS2 inactivation by UV irradiance was investigated with the focus on how the disinfection efficacy is influenced by bacteriophage MS2 aggregation and adsorption to particles in solutions with different compositions. Kaolinite and Microcystis aeruginosa were used as model inorganic and organic particles, respectively. In the absence of model particles, MS2 aggregates formed in either 1mM NaCl at pH=3 or 50-200mM ionic strength CaCl 2 solutions at pH=7 led to a decrease in the MS2 inactivation efficacy because the virions located inside the aggregate were protected from the UV irradiation. In the presence of kaolinite and Microcystis aeruginosa, MS2 adsorbed onto the particles in either 1mM NaCl at pH=3 or 50-200mM CaCl 2 solutions at pH=7. In contrast to MS2 aggregates formed without the presence of particles, more MS2 virions adsorbed on these particles were exposed to UV irradiation to allow an increase in MS2 inactivation. In either 1mM NaCl at pH from 4 to 8 or 2-200mM NaCl solutions at pH=7, the absence of MS2 aggregation and adsorption onto the model particles explained why MS2 inactivation was not influenced by pH, ionic strength, and the presence of model particles in these conditions. The influence of virus adsorption and aggregation on the UV disinfection efficiency found in this research suggests the necessity of accounting for particles and cation composition in virus inactivation for drinking water., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: State-of-the-art bioaerosol samplers have poor collection efficiencies for ultrafine virus aerosols. This work evaluated the performance of a novel growth tube collector (GTC), which utilizes laminar-flow water-based condensation to facilitate particle growth, for the collection of airborne MS2 viruses., Methods and Results: Fine aerosols (<500 nm) containing MS2 coliphage were generated from a Collison nebulizer, conditioned by a dilution dryer and collected by a GTC and a BioSampler. The GTC effectively condensed water vapour onto the virus particles, creating droplets 2-5 μm in diameter, which facilitated collection. Comparison of particle counts upstream and downstream revealed that the GTC collected >93% of the inlet virus particles, whereas the BioSampler's efficiency was about 10%. Viable counts of the GTC-collected viruses were also one order of magnitude higher than those of the BioSampler (P = 0·003)., Conclusion: The efficiency of the GTC for the viable collection of MS2 viruses exceeds that of industry standard instrument, the BioSampler, by a factor of 10-100., Significance and Impact of the Study: This study reveals that the GTC is an effective collector of viable MS2 aerosols, and concludes the instrument will be an effective tool for studying viable virus aerosols and the inhalation risks posed by airborne viruses., (© 2016 The Society for Applied Microbiology.)

Enteric viruses are a major problem in the food industry, especially as human noroviruses are the leading cause of nonbacterial gastroenteritis. Chitosan is known to be effective against some enteric viral surrogates, but more detailed studies are needed to determine the precise application variables. The main objective of this work was to determine the effect of increasing chitosan concentration (0.7-1.5% w/v) on the cultivable enteric viral surrogates, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and bacteriophages (MS2 and phiX174) at 37 °C. Two chitosans (53 and 222 kDa) were dissolved in water (53 kDa) or 1% acetic acid (222 KDa) at 0.7-1.5%, and were then mixed with each virus to obtain a titer of ~5 log plaque-forming units (PFU)/mL. These mixtures were incubated for 3 h at 37 °C. Controls included untreated viruses in phosphate-buffered saline and viruses were enumerated by plaque assays. The 53 kDa chitosan at the concentrations tested reduced FCV-F9, MNV-1, MS2, and phi X174 by 2.6-2.9, 0.1-0.4, 2.6-2.8, and 0.7-0.9 log PFU/mL, respectively, while reduction by 222 kDa chitosan was 2.2-2.4, 0.8-1.0, 2.6-5.2, and 0.5-0.8 log PFU/mL, respectively. The 222 kDa chitosan at 1 and 0.7% w/v in acetic acid (pH 4.5) caused the greatest reductions of MS2 by 5.2 logs and 2.6 logs, respectively. Overall, chitosan treatments showed the greatest reduction of MS2, followed by FCV-F9, phi X174, and MNV-1. These two chitosans may contribute to the reduction of enteric viruses at the concentrations tested but would require use of other hurdles to eliminate food borne viruses.

This study determined whether a hand sanitizing wipe can reduce virus transmission in households, and could reduce the probability of infection by rhinovirus and rotavirus. Bacteriophage MS-2 (a marker virus) was used to assess viral transmission in five households having at least two children of ages 2-18. Hands of one female adult were inoculated with ~10(8) PFU MS-2 bacteriophages in each home, and after 8 h, hands of all family members and select fomites were sampled to determine baseline contamination without intervention. This sequence was repeated with the intervention, where all family members were instructed to use a quaternary ammonium compound-based sanitizing wipe at least once per day. A significant reduction of virus after the intervention occurred on inoculated hands (95.3%; p = 0.0039), all fomites combined (74.5%; p < 0.005), and non-inoculated hands and fomites combined (73.5%; p < 0.005). However, viral reduction on non-inoculated hands was not significant, likely due to small sample size. Using rhinovirus and rotavirus as models it was estimated that infection risk was reduced by ~30 to 89% with the use of sanitizing wipes once per day depending on the starting concentration of these viruses on hands of susceptible individuals. Therefore, using a hand sanitizing wipe can significantly reduce viral transmission and risk of illness in homes. Previous studies have shown other hand hygiene interventions, such as alcohol-based hand sanitizers, are even more effective for reducing risk of illness in homes; however the sanitizing wipe used in this study is appropriate to use for microbial reduction.

Aims: To avoid interference by water-iodine disinfection chemistry and measure directly the effect of iodine, captured from a triiodide complex bound to a filter medium, on viability of penetrating viral particles., Methods and Results: Aerosols of MS2 coli phage were passed through control P100 or iodinated High-Efficiency Particulate Air media, collected in plastic bags, incubated for 0-10 min, collected in an impinger containing thiosulphate to consume all unreacted iodine, plated and enumerated. Comparison of viable counts demonstrated antimicrobial activity with an apparent half-life for devitalization in tens of seconds; rate of kill decreased at low humidity and free iodine was captured by the bags., Conclusions: The results support the mechanism of near-contact capture earlier proposed; however, the disinfection chemistry in the aerosol phase is very slow on the time scale of inhalation., Significance and Impact of the Study: This study shows that disinfection by filter-bound iodine in the aerosol phase is too slow to be clinically significant in individual respiratory protection, but that it might be of benefit to limit airborne transmission of infections in enclosed areas., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Simple, low-cost household interventions are known to be effective in lowering the incidence of waterborne diseases in developing countries. However, high costs along with operational and maintenance issues have prevented the successful adoption of these interventions among the affected communities. To address these limitations, a cost-effective, gravity-driven water purification cartridge has been developed by employing the synergistic disinfection action of low concentrations of silver and chlorine on bacteria and viruses. The silver and chlorine treatment components within the cartridge have been developed using inexpensive materials and integrated with a life indicator and auto-shut-off-mechanism within a compact form factor. The antibacterial as well as antiviral performance of the cartridge was tested by using ground water spiked with Escherichia coli and MS2 bacteriophage. The results show that, although individually, the silver and chlorine treatment systems were unable to inactivate the test strains, the integrated cartridge inactivates both bacteria as well as viruses up to the log reduction requirement of the USEPA guide standard for microbiological water purifiers over its designated life of 2,000 liters.

Laboratory studies of virus aerosols have been criticized for generating airborne viruses from artificial nebulizer suspensions (e.g., cell culture media), which do not mimic the natural release of viruses (e.g., from human saliva). The objectives of this study were to determine the effect of human saliva on the infectivity and survival of airborne virus and to compare it with those of artificial saliva and cell culture medium. A stock of MS2 bacteriophage was diluted in one of three nebulizer suspensions, aerosolized, size selected (100 to 450 nm) using a differential mobility analyzer, and collected onto gelatin filters. Uranine was used as a particle tracer. The resulting particle size distribution was measured using a scanning mobility particle sizer. The amounts of infectious virus, total virus, and fluorescence in the collected samples were determined by infectivity assays, quantitative reverse transcription-PCR (RT-PCR), and spectrofluorometry, respectively. For all nebulizer suspensions, the virus content generally followed a particle volume distribution rather than a number distribution. The survival of airborne MS2 was independent of particle size but was strongly affected by the type of nebulizer suspension. Human saliva was found to be much less protective than cell culture medium (i.e., 3% tryptic soy broth) and artificial saliva. These results indicate the need for caution when extrapolating laboratory results, which often use artificial nebulizer suspensions. To better assess the risk of airborne transmission of viral diseases in real-life situations, the use of natural suspensions such as saliva or respiratory mucus is recommended.

Objective: This study compared the performance of two high-volume bioaerosol air samplers for viable virus to an accepted standard low-volume sampler. In typical bioaerosol emergency response scenarios, highvolume sampling is essential for the low infective concentrations and large air volumes involved., Design: Two high-volume air samplers (XMX/2LMIL and DFU-1000) were evaluated alongside a lowvolume sample (BioSampler). Low and high concentrations (9.3-93.2 agent containing particles per liter of air [ACPLA]) of male-specific coliphage 2 (MS2) virus were released into a 12 m3 aerosol test chamber and collected using the air samplers. The collection media from the samplers were then processed and viable virus was assessed via plaque assay., Setting: Aerosol test chamber., Subjects, Participants: None., Interventions: Collection media and flow rate were modified for the XMX/2L-MIL sampler for viable analysis., Main Outcome Measures: Concentration estimates in units of plaque forming units per liter of air (PFU/liter) assessed by the samplers as compared to the levels inside the chamber as evaluated with a slit to agar plate in units of ACPLA. Comparison was made via one-way analysis of variance., Results: Both the XMX/2L-MIL and DFU-1000 achieved collection effectiveness equal to or greater than the low-volume air sampler for the evaluated MS2 concentrations. The XMX/2L-MIL reliably collected quantifiable low concentrations of MS2, but the DFU-1000 was unable to do so., Conclusions: For emergency response to suspected bioaerosols, the evaluated high-volume samplers are as effective as the standard low-flow sampler and should be considered in conducting a health risk assessment. If low concentrations are expected, then high-flow samplers using liquid collection are preferred.

Plaque analysis allows the determination of phage titer and multiplicity of infection. Yet, this overnight assay provides only endpoint results, ignoring kinetic aspects. We introduce an alternative high-throughput and rapid method for kinetic analysis of lytic coliphage activity. Escherichia coli was infected with serial dilutions of MS2 coliphage, and bacterial growth was monitored using a multi-well plate reader providing within hours the equivalent data as obtained overnight. Additional information is yielded, including phage replication rate, progeny size per cycle, and viral propagation during bacterial growth. This method offers further insights into physicochemical mechanisms of lytic coliphage infection and temporal control. It also provides a virus-host interaction acumen., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Viral disinfection kinetics have been studied in depth, but the molecular-level inactivation mechanisms are not understood. Consequently, it is difficult to predict the disinfection behavior of nonculturable viruses, even when related, culturable viruses are available. The objective of this work was to determine how small differences in the composition of the viral genome and proteins impact disinfection. To this end, we investigated the inactivation of three related bacteriophages (MS2, fr, and GA) by UV254, singlet oxygen ((1)O2), free chlorine (FC), and chlorine dioxide (ClO2). Genome damage was quantified by PCR, and protein damage was assessed by quantitative matrix-assisted laser desorption ionization (MALDI) mass spectrometry. ClO2 caused great variability in the inactivation kinetics between viruses and was the only treatment that did not induce genome damage. The inactivation kinetics were similar for all viruses when treated with disinfectants possessing a genome-damaging component (FC, (1)O2, and UV254). On the protein level, UV254 subtly damaged MS2 and fr capsid proteins, whereas GA's capsid remained intact. (1)O2 oxidized a methionine residue in MS2 but did not affect the other two viruses. In contrast, FC and ClO2 rapidly degraded the capsid proteins of all three viruses. Protein composition alone could not explain the observed degradation trends; instead, molecular dynamics simulations indicated that degradation is dictated by the solvent-accessible surface area of individual amino acids. Finally, despite the similarities of the three viruses investigated, their mode of inactivation by a single disinfectant varied. This explains why closely related viruses can exhibit drastically different inactivation kinetics.

Enteric viruses monitoring in surface waters requires the concentration of viruses before detection assays. The aim of this study was to evaluate different methods in terms of recovery efficiencies of bacteriophage PP7 of Pseudomonas aeruginosa, measured by real-time PCR, using it as a viral control process in water analysis. Different nucleic acid extraction methods (silica-guanidinium thiocyanate, a commercial kit (Qiagen Viral RNA Kit) and phenol-chloroform with alcohol precipitation) exhibited very low recovery efficiencies (0.08-4.18 %), being the most efficient the commercial kit used for subsequent experiments. To evaluate the efficiency of three concentration methods, PBS (as model for clean water) and water samples from rivers were seeded to reach high (HC, 10(6) pfu ml(-1)) and low concentrations (LC, 10(4) pfu ml(-1)) of PP7. Tangential ultrafiltration proved to be more efficient (50.36 ± 12.91, 17.21 ± 9.22 and 12.58 ± 2.35 % for HC in PBS and two river samples, respectively) than adsorption-elution with negatively charged membranes (1.00 ± 1.34, 2.79 ± 2.62 and 0.05 ± 0.08 % for HC in PBS and two river samples, respectively) and polyethylene glycol precipitation (15.95 ± 7.43, 4.01 ± 1.12 and 3.91 ± 0.54 %, for HC in PBS and two river samples, respectively), being 3.2-50.4 times more efficient than the others for PBS and 2.7-252 times for river samples. Efficiencies also depended on the initial virus concentration and aqueous matrixes composition. In consequence, the incorporation of an internal standard like PP7 along the process is useful as a control of the water concentration procedure, the nucleic acid extraction, the presence of inhibitors and the variability of the recovery among replicas, and for the calculation of the sample limit of detection. Thus, the use of a process control, as presented here, is crucial for the accurate quantification of viral contamination.

Human noroviruses (HNoV) have been implicated in gastrointestinal outbreaks associated with fresh produce, juices, and ready-to-eat foods. In order to determine the risk of HNoV transmission by contaminated blueberry juice, survival characteristics of cultivable HNoV surrogates (murine norovirus, MNV-1; feline calicivirus, FCV-F9; and bacteriophage MS2) in blueberry juice (pH = 2.77) after 0, 1, 2, 7, 14, and 21 days at refrigeration temperatures (4°C) were studied. High-pressure homogenization (HPH) was studied as a novel processing method for noroviral surrogate inactivation in blueberry juice. Blueberry juice or phosphate-buffered saline (PBS; pH 7.2 as control) was inoculated with each virus, stored over 21 days at 4°C or subjected to HPH, and plaque assayed. FCV-F9 (∼5 log(10) PFU/mL) was undetectable after 1 day in blueberry juice at 4°C. MNV-1 (∼4 log(10) PFU/ml) showed minimal reduction (1 log(10) PFU/mL) after 14 days, with greater reduction (1.95 log(10) PFU/mL; p < 0.05) after 21 days in blueberry juice at 4°C. Bacteriophage MS2 (∼6 log(10) PFU/mL) showed significant reduction (1.93 log(10) PFU/mL; p < 0.05) after 2 days and was undetectable after 7 days in blueberry juice at 4°C. FCV-F9 remained viable in PBS for up to 21 days (2.28 log(10) PFU/mL reduction), while MNV-1 and MS2 survived after 21 days (1.08 and 0.56 log(10) PFU/mL reduction, respectively). Intriguingly, FCV-F9 and bacteriophage MS2 showed reduction after minimal homogenization pressures in blueberry juice (pH = 2.77), possibly due to the combination of juice pH, juice components, and mechanical effects. MNV-1 in blueberry juice was only slightly reduced at 250 (0.33 log(10) PFU/mL) and 300 MPa (0.71 log(10) PFU/mL). Virus surrogate survival in blueberry juice at 4°C correlates well with the ease of HNoV transmission via juices. HPH for viral inactivation in juices is dependent on virus type, and higher homogenization pressures may be needed for MNV-1 inactivation.

We have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to the Levivirus group and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found in Levivirus and Allolevivirus predate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making the Levivirus-like genome organization ancestral and the Allolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.

Aims: The ability to distinguish between viable and/or infectious micro-organisms and inactivated cells is extremely important for correctly performing microbial risk assessments. In this study, we evaluated whether propidium monoazide (PMA)-qPCR could distinguish between viable and nonviable bacteria and viruses., Methods and Results: A PMA-qPCR combined assay was applied to viable and inactivated bacteria (Escherichia coli and Bacillus subtilis) and viruses (MS2 and murine norovirus [MNV]). PMA, a DNA-intercalating agent, in combination with PCR was better able to distinguish between viable and nonviable bacteria and viruses than conventional PCR., Conclusions: These results suggest that a combined PMA-qPCR assay can be used to measure the viability of bacterial cells and bacteriophage MS2, but not MNV., Significance and Impact of the Study: PMA-qPCR could potentially be used to measure the viability of some micro-organisms, including virus. However, a thorough evaluation should be performed prior to measuring the viability of micro-organisms by PMA-qPCR in a quantitative way., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Effective sanitization is important in viral epizootic outbreaks to avoid further spread of the pathogen. This study examined thermal inactivation as a sanitizing treatment for manure inoculated with highly pathogenic avian influenza virus H7N1 and bacteriophages MS2 and 6. Rapid inactivation of highly pathogenic avian influenza virus H7N1 was achieved at both mesophilic (35°C) and thermophilic (45 and 55°C) temperatures. Similar inactivation rates were observed for bacteriophage 6, while bacteriophage MS2 proved too thermoresistant to be considered a valuable indicator organism for avian influenza virus during thermal treatments. Guidelines for treatment of litter in the event of emergency composting can be formulated based on the inactivation rates obtained in the study.

We investigated the resistance of murine norovirus (MNV) and coliphage MS2, a culturable human norovirus surrogate, to temperature, salt, and pH. Virus inactivation was measured by plaque, real-time TaqMan reverse transcription (RT) PCR, and long-template RT-PCR assays. Both MNV and MS2 were rapidly inactivated at temperatures above 60°C. Similarly, MNV tolerated low salt concentrations (0.3% NaCl) to a greater degree than high salt concentrations (3.3 to 6.3% NaCl). MNV was relatively resistant to strong acidic conditions (pH 2) and was more tolerant of slightly acidic (pH 4) or neutral (pH 7) conditions. In contrast, MS2 was resistant to high salinity. Overall, temperature had a greater effect on infectivity than salt or low pH. Additionally, temperature and low pH had a synergistic effect on MNV infectivity. Both real-time and long-template RT-PCR assays significantly underestimated the inactivation by temperature, salt, and pH. The inactivation kinetics of both MNV and MS2 under various environmental conditions gave a good fit by the Weibull model (R² > 0.9). This study suggests both the capacity of infectious human norovirus to persist in the face of various environmental conditions and its sensitivity to high temperatures, which may provide a mechanism of protection against this virus.

Free chlorine as hypochlorite is recommended to decontaminate fecally contaminated surfaces to control human norovirus (NoV). We evaluated the efficacy of sodium hypochlorite to decontaminate GII.4 NoV and three surrogates of human NoVs, feline calicivirus (FCV), murine norovirus (MNV), and coliphage MS2, on a fecally soiled stainless steel surface. Reduction of infectivity of FCV, MNV, and MS2 was measured by plaque assay and the decline of genomic copy numbers of GII.4 NoV by reverse transcriptase-polymerase chain reaction. Sodium hypochlorite solution at 5000 ppm could inactivate FCV by 3 log(10) plaque forming units after approximately 1.9 minutes of contact time, but required longer exposure times of 3.2 and 4.5 minutes to reduce MNV and MS2 by 3 log(10), respectively. However, detection of viral RNA by reverse transcriptase-polymerase chain reaction assay may not be reliable to estimate the effectiveness of sodium hypochlorite against human NoV. Of three NoV surrogates, FCV is not the most resistant of the virus tested for inactivation by hypochlorite and thus is not the worst-case model for estimating NoV inactivation. Although the use of 5000 ppm of hypochlorite for fecally soiled surfaces is effective, it may require longer exposure times of ≥3 minutes to control NoVs. Surface precleaning before hypochlorite disinfection is recommended to initially reduce the fecal organic load for better virus inactivation and should be a part of the environmental hygiene response measures during an NoV outbreak or where NoV fecal contamination of environmental surfaces is likely or suspected to be present.

A research study has been performed to determine the ultraviolet (UV) fluence-response of several target non-pathogenic microorganisms to UV light emitting diodes (UV-LEDs) by performing collimated beam tests. UV-LEDs do not contain toxic mercury, offer design flexibility due to their small size, and have a longer operational life than mercury lamps. Comsol Multiphysics was utilized to create an optimal UV-LED collimated beam design based on number and spacing of UV-LEDs and distance of the sample from the light source while minimizing the overall cost. The optimized UV-LED collimated beam apparatus and a low-pressure mercury lamp collimated beam apparatus were used to determine the UV fluence-response of three surrogate microorganisms (Escherichia coli, MS-2, T7) to 255 nm UV-LEDs, 275 nm UV-LEDs, and 254 nm low-pressure mercury lamps. Irradiation by low-pressure mercury lamps produced greater E. coli and MS-2 inactivation than 255 nm and 275 nm UV-LEDs and similar T7 inactivation to irradiation by 275 nm UV-LEDs. The 275 nm UV-LEDs produced more efficient T7 and E. coli inactivation than 255 nm UV-LEDs while both 255 nm and 275 nm UV-LEDs produced comparable microbial inactivation for MS-2. Differences may have been caused by a departure from the time-dose reciprocity law due to microbial repair mechanisms., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In an effort to validate the use of ozone for contaminant oxidation and disinfection in water reclamation, extensive pilot testing was performed with ozone/H(2)O(2) and biological activated carbon (BAC) at the Reno-Stead Water Reclamation Facility in Reno, Nevada. Three sets of samples were collected over a five-month period of continuous operation, and these samples were analyzed for a suite of trace organic contaminants (TOrCs), total estrogenicity, and several microbial surrogates, including the bacteriophage MS2, total and fecal coliforms, and Bacillus spores. Based on the high degree of microbial inactivation and contaminant destruction, this treatment train appears to be a viable alternative to the standard indirect potable reuse (IPR) configuration (i.e., membrane filtration, reverse osmosis, UV/H(2)O(2), and aquifer injection), particularly for inland applications where brine disposal is an issue. Several issues, including regrowth of coliform bacteria in the BAC process, must be addressed prior to full-scale implementation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Quantitative assessment of multiple sources to short-term variations in recreational water quality, as indexed by faecal indicator organism (FIO) concentrations, is becoming increasingly important with adoption of modern water quality standards and catchment-based water quality management requirements (e.g. the EU Water Framework Directive, Article 11 'Programmes of Measures' and the US Clean Water Act, 'Total Maximum Daily Loads'). This paper describes a study combining microbial tracers, intensive FIO measurement, open channel hydrology and molecular microbial source tracking (MST) to enhance understanding of recreational water quality at Amroth in southwest Wales, UK. Microbial tracers were released from four stream inputs during a moderate hydrograph event. Tracers from two local streams impacted simultaneously with a period of maximum FIO concentrations at the near-shore compliance monitoring site. Connection between these inputs and this site were rapid (9-33 min). Water quality impairment from a more remote stream input followed, 12.85 h after tracer release, sustaining FIO concentrations above desired compliance levels. MST analysis showed dominance of ruminant Bacteroidales genetic markers, associated with agricultural pollution. This integration of tracers and MST offers additional information on the movement and individual sources causing water quality impairment., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aims: The survival of Escherichia coli, Salmonella enterica serovar Typhimurium, Enterococcus faecalis and coliphage MS2 was studied in stored, fresh and diluted (1 : 1) human urine at 15 and 30 degrees C., Methods and Results: Survival rate was studied by the plate count method. All the organisms showed rapid inactivation in stored urine, but they survived better in diluted and fresh urine. The high pH level and temperature were the major factors found to influence the survival of the micro-organisms with the survival rate being higher at 15 degrees C than at 30 degrees C., Conclusions: The destruction of all micro-organisms in stored urine required <1 week at 30 degrees C. Thus, the storage of urine is a useful way to reduce the risk of contamination while using urine as a fertilizer., Significance and Impact of the Study: The urine fertilization is aimed for the developing countries and the high temperatures in these countries may hasten the destruction of micro-organisms in urine. On the contrary, a higher survival rate of these organisms in fresh and diluted urine is a public health concern because the dilution of urine with water is likely to happen during flushing.

Novel inactivation methods are needed to control the spread of foodborne viruses responsible for nonbacterial gastroenteritis worldwide. The advent of high-pressure homogenization combining high pressure, shear stress, and cavitation provides the opportunity to evaluate this technology for viral inactivation in fluid foods under continuous processing conditions. Our objective was to evaluate murine norovirus (MNV-1) and MS2 coliphage (single-stranded RNA) as human enteric virus surrogates for their susceptibility to a novel high-pressure homogenization process for application in commercial settings. Experiments were conducted in duplicate with MNV-1 and MS2 coliphage in phosphate-buffered saline, using homogenization pressures of 0, 100, 200, 250, and 300 MPa (the maximum achievable by the homogenizer), resulting in exposure temperatures of 24, 46, 63, 70, and 75 degrees C, respectively, for <2 s. Only homogenization pressures of 300 MPa at 75 degrees C showed inactivation of approximately 3 log PFU for MS2 from an initial approximately 6 log PFU. Also, MNV-1 showed inactivation of approximately 0.8 log PFU at 300 MPa. Further studies are warranted to validate this inactivation process, which can retain the sensory and nutritional value of fluid food and shows promise for application in industrial environments.

A study was conducted to examine the role of aquatic plants used in constructed wetlands on the survival of enteric bacteria and viruses. Four small-scale wetland systems, receiving fresh water and two other wetland systems, receiving secondary unchlorinated sewage were used in this study. Fresh water and secondary sewage without the presence of any aquatic plants were used as controls. Escherichia coli, Salmonella typhimurium, bacteriophage MS-2 and poliovirus were added to the waters collected from the wetlands and controls. The presence of aquatic plants significantly increased the die-off of both bacteria in fresh water and secondary sewage. No significant difference in the die-off of E. coli and S. typhimurium was observed in water from wetlands with different types of plants in freshwater. However, there was a significant difference in the die-off of E. coli in water with aquatic plants when sewage was used. The presence of the plants significantly increased the inactivation of MS-2 and poliovirus. Additional work on the survival of E. coli indicated that the plausible mechanism of bacterial die-off in constructed wetlands is through increased microbial competition or predation.

This study reports on the use of naturally occurring F-specific coliphages, as well as spiked MS-2 phage, to evaluate a land-based effluent treatment/reuse system and an effluent irrigation scheme. Both the natural phages and the spiked MS-2 phage indicated that the effluent treatment/reuse system (FILTER - Filtration and Irrigated cropping for Land Treatment and Effluent Reuse) achieved a reduction in phage levels over the treatment system by one to two log10. FILTER reduced natural F-specific phage numbers from around 10(3) to below 10(2) 100-ml(-1) and the spiked phage from 10(5) to around 10(4) 100-ml(-1) (incoming compared with outgoing water). In the effluent irrigation scheme, phage spiked into the holding ponds dropped from 10(6) to 10(2) 100-ml(-1) after 168 h (with no detectable levels of natural F-specific phage being found prior to spiking). Only low levels of the spiked phage (10(2) gm(-1)) could be recovered from soil irrigated with phage-spiked effluent (at 10(6) phage 100 ml(-1)) or from fruits (around 10(2) phage per fruit) that had direct contact with soil which had been freshly irrigated with the same phage-spiked effluent.

Laboratory column experiments were conducted to study the effects of anionic polyacrylamide (PAM) polymer and surfactant linear alkylbenzene sulfonate (LAS) on the movement of Escherichia coli and the FRNA phage MS-2. The study was designed to evaluate if PAM or PAM + LAS would enhance the mobility of human pathogens in tropical soils under unsaturated conditions. No breakthrough of phage was observed in a 10 cm column after passing 100 pore volumes of solution containing 1x10(8) plaque-forming units (PFU)/ml. In later experiments, after passing 10-20 pore volumes of influent containing 1x10(8)/ml MS-2 or E. coli through 15 cm columns, the soil was sliced and the organisms eluted. Phage moved slightly deeper in the polymer-treated column than in the control column. There was no measurable difference in the movement of E. coli in either polymer-treated or control columns. The properties of the soil (high amounts of metal oxides, kaolinitic clay), unsaturated flow conditions, and relatively high ionic strengths of the leaching solution attributed to significant retention of these indicators. The impacts of PAM and LAS on the mobility of E. coli or MS-2 phage in the chosen soils were not significant.

Aim: To measure the sanitizing effect of mesophilic (37 degrees C) anaerobic digestion in high ammonia concentrations produced in situ., Methods and Results: Indicator organisms and salmonella were transferred to small-scale anaerobic batch cultures and D-values were calculated. Batch cultures were started with material from two biogas processes operating at high (46 mmol l(-1)) and low (1.6 mmol l(-1)) ammonia concentration. D-values were shortened from c. 3 days to <1 day for the bacteria. MS2 had the same D-value (1.3 days) independent of ammonia concentration whereas PhiX174 and 28B were faster inactivated in the control (1.1 and 7.9 days) than in the high ammonia (8.9 and 39 days) batch cultures., Conclusion: Running biogas processes at high levels of ammonia shortens the time to meet EU regulation concerning reduction of salmonella and enterococci (5 log). Unless a minimum retention time of 2 days, post-treatment digestion is needed to achieve sufficient sanitation in continuous biogas processes., Significance and Impact of the Study: Running mesophilic biogas processes at high ammonia level produces residue with a high fertilizer value. With some stipulations concerning management parameters, such processes provide a method of bacterial sanitation without preceding pasteurization of the incoming organic waste.

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25 degrees C and 4 degrees C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25 degrees C (0.18 and 0.09 log(10)/day for FCV, 0.13 and 0.10 log(10)/day for PV, 0.12 and 0.06 log(10)/day for MS2, and 0.09 and 0.05 log(10)/day for MNV) but not significant at 4 degrees C. According to a multiple linear regression model, the NV NA reduction rates (0.04 +/- 0.01 log(10)/day) were not significantly different from the NA reduction rates of MS2 (0.05 +/- 0.03 log(10)/day) and MNV (0.04 +/- 0.03 log(10)/day) and were significantly different from those of FCV (0.08 +/- 0.03 log(10)/day) and PV (0.09 +/- 0.03 log(10)/day) at 25 degrees C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.

The prevalence of pathogenic microorganisms in seawater can result in waterborne and food borne outbreaks. This study was performed to determine the effect of sunlight and salinity on the die-off of Cryptosporidium parvum. Cryptosporidium parvum oocysts, Escherichia coli, and MS2 coliphage were seeded into tap water and seawater samples and then exposed to sunlight. The die-off of C. parvum in seawater, as measured by infectivity, was greater under sunlight (-3.08 log10) than under dark conditions (-1.31 log10). While, no significant difference was recorded in the die-off of C. parvum, under dark conditions, in tap water as compared to seawater (P < 0.05), indicating that the synergistic effect of salinity and sunlight was responsible for the enhanced die-off in seawater. The die-off of MS2 coliphage and E. coli was greater than that observed for C. parvum under all tested conditions. This indicates that these microorganisms cannot serve as indicators for the presence of C. parvum oocysts in seawaters. The results of the study suggest that C. parvum can persist as infectious oocysts for a long time in seawater and can thus pose a serious hazard by direct and indirect contact with humans.

An isothermal batch experiment was conducted in the laboratory to compare adsorption of bacteriophages MS2 and phiX174 onto 6 different soils (red loam soil, red clay soil, wushan soil, huangni soil, sandy fluvo-aquic soil and loamy fluvo-aquic soil) in China. Soils with sterilized or non-sterilized treatment were used. Relative coefficients of each numerical simulation of the isotherms using three models were evaluated. The results show that the properties of the soil and virus, and presence/absence of the soil autochthonous microorganisms have pronounced effect on the virus adsorption. Both MS2 and phiX174 are almost completely adsorbed by the red clay soil, but minimal adsorption is observed in the two fluvo-aquic soils. Adsorption of phiX174 to all the non-sterilized soils is generally much greater than that of MS2, while sterilization leads to opposite results. Freundlich and Langmuir isotherms are found to have better coefficients to simulate the apparent steady-state virus concentrations. Freundlich isotherm is capable of demonstrating the effect of virus concentration on adsorption behavior. Langmuir isotherms can be used to compare relative adsorption among treatments, while the present study suggests that maximum adsorption can not be calculated when using the Langmuir isotherms.

The performance of a duct-mounted air disinfection system, designed to reduce airborne pathogens in the hospital environment, was determined using a new testing methodology. The methodology places the equipment in a test duct, a microbial aerosol is generated and then sampled simultaneously before and after the test system. This allows a percentage efficiency value to be calculated. The air disinfection system is a novel chemical-coated filter and ultraviolet (UV) radiation air purification system, operating at a flow rate of 500 m(3)/h, against aerosols of MS2 phage and Mycobacterium vaccae (surrogates of viral and mycobactericidal pathogens). A three UV lamp system was effective against airborne phages, removing an average of 97.34% of the aerosolized challenge. With the UV component switched off, the average efficiency dropped to 61.46%. This demonstrates that the chemical-coated filter component plays a more significant role than the UV radiation in destroying phages. When six UV lamps were used, the system was able to remove mycobacteria with an efficiency exceeding 99.99%. This test methodology can be used to assess manufacturers' claims of efficacy of equipment against airborne micro-organisms in the hospital environment.

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.

A membrane bioreactor (MBR) may serve as a pre-disinfection or disinfection unit, in addition to its solid/liquid separation and biological conversion functions, to produce sewage effluent of high quality. This bench-scale pilot study focuses on investigating the performance of a submerged MBR in pathogen removal and the factors affecting the removal, using a 0.4-microm hollow-fiber membrane module submerged in an aeration tank and bacteriophage MS-2 as the indicator organism. Removal of the MS-2 phage was found to be contributed by physical filtration by the membrane itself, biomass activity in the aeration tank and bio-filtration achieved by the biofilm developed on the membrane surface. The membrane alone gave poor virus removal (0.4+/-0.1 log) but the overall removal increased substantially with the presence of biomass and the membrane-surface-attached biofilm. The contributions of the suspended biomass and attached biofilm to the phage removal are dependent on the inter-related parameters including the concentration of mixed liquor suspended solids (MLSS), the sludge retention time (SRT) and the food to mass (F/M) ratio. The correlations between effluent flux/trans-membrane pressure and virus removal give evidence that phage removal in the MBR is most likely susceptible to both biological and physical factors including the quantity and property of the biomass and the biofilm and the membrane pore size reduction.

Monitoring human enteric virus levels in domestic wastewater effluent is crucial to protecting human health. Occasionally, during intensive sampling, wastewater samples must be stored for later viral analysis. Little data exist regarding how enteric viruses survive during storage at different temperatures in secondary treated wastewater. During a field-scale study assessing pathogen removal performance by various onsite treatment technologies, the MS2 bacteriophage, an indicator of enteric viruses, was inoculated into septic tank (STE), sand filter, peat filter and constructed wetland (CW) effluents to determine virus decay at various storage temperatures. Virus stored at temperatures > or =10 degrees C and at -20 degrees C decayed nearly twice as fast as those stored at 4 degrees C or -80 degrees C. Decreased water quality decreased viral decay rates at 4 degrees C and -80 degrees C, with slowest decay occurring in STE and the fastest in sterile PBS and low pH peat effluent. In CW effluent after 8 days, less MS2 was inactivated when stored at 4 degrees C (20%) compared to -80 degrees C (58%); however, during extended storage (approximately 300 days), less MS2 was inactivated at -80 degrees C (75%) compared to 4 degrees C (93%). We recommend that viruses in wastewater be stored in the dark at 4 degrees C unless storage for >40 days is necessary.

We conducted a series of experiments to compare the survival of Escherichia coli, feline calicivirus, and F-specific coliphage MS2 on lettuce and cabbage with and without disinfection. Inoculated produce was held at 4, 25, or 37 degrees C for 21 days or was treated with different concentrations of sodium bicarbonate, chlorine bleach, peroxyacetic acid, or hydrogen peroxide. Survival was measured by the decimal reduction value (time to 90% reduction in titer) and the change in log titers of the test organisms. A stronger correlation of survival measures was observed between feline calicivirus and MS2 than between E. coli and either of the viral agents at 25 and 37 degrees C. The maximum time to detection limit for MS2 at all temperatures was 9 days, whereas feline calicivirus was detected for a maximum of 14 days at 4 degrees C. In contrast, E. coli was detectable for 21 days at 4 and 25 degrees C and for 14 days at 37 degrees C. Significant increases in E. coli titer occurred within the first 5 days, but virus titers decreased steadily throughout the experiments. E. coli was also highly susceptible to all disinfectants except 1% sodium bicarbonate and 50 ppm chlorine bleach, whereas the viruses were resistant to all four disinfectants.

The Sm-like protein Hfq has been implicated in the regulation of sigmaS-dependent and sigmaS-independent genes in E. coli and in the regulation of virulence factors in both, Yersinia enterocolitica and Brucella abortus. Here, we have studied the effect of Hfq on virulence and stress response of Pseudomonas aeruginosa (PAO1). We have constructed a PAO1hfq- mutant and a PAO1hfq-rpoS- double mutant to permit distinction between direct and indirect effects of Hfq. When compared to the wild-type and the rpoS- strains, the hfq knock out strain showed a reduced growth rate and was unable to utilize glucose as a sole carbon source. Elastase activity was 80% reduced in the hfq- mutant when compared to the wild-type or the rpoS- strain, whereas alginate production seemed to be solely affected by sigmaS. The production of catalase and pyocyanin was shown to be affected in an additive manner by both, Hfq and sigmaS. Moreover, twitching and swarming mediated by typeIV pili was shown to be impaired in the hfq- mutant. When compared to PAO1 wild-type and the rpoS- mutant, the hfq- mutant decreased virulence in Galleria mellonella by a factor of 1 x 10(4) and 5 x 10(3), respectively. Likewise, when compared to wild-type, the PAO1hfq- mutant was significantly attenuated in virulence when administered intraperitoneally in mice. These results strongly suggest that Hfq is a global regulator of PAO1 virulence and stress response which is not exclusively due to its role in stimulating the synthesis of sigmaS.

The RNA F-specific coliphages, MS2 and Qbeta, have been used as virus indicators in water and wastewater studies. It is therefore useful to have a good understanding concerning the effects of environmental factors on their survival in order to choose an appropriate candidate for assessing microbial safety in relation to water quality management. The effects of pH and temperature on the survival of these two coliphages were investigated. MS2 survived better in acidic conditions than in an alkaline environment. In contrast, Qbeta had a better survival rate in alkaline conditions than in an acidic environment. The inactivation rates of both coliphages were lowest within the pH range 6-8 and the temperature range 5-35 degrees C. The inactivation rates of both coliphages increased when the pH was decreased to below 6 or increased to above 8. The inactivation rates of both coliphages increased with increasing temperature. Qbeta behaved peculiarly in extreme pH buffers, i.e. it was inactivated very rapidly initially when subjected to an extreme pH environment, although the inactivation rate subsequently decreased. In general, MS2 was a better indicator than Qbeta. However, within the pH range 6-9 and at temperatures not above 25 degrees C, either MS2 or Qbeta could be used as a viral indicator.

Aims: This study was undertaken to further understand the processes affecting the persistence of enteric viruses in groundwater., Methods and Results: Varying temperature, oxygen and nutrient levels were tested in the presence and absence of groundwater micro-organisms to determine which of the factors tested had dominant influence on the decay of Escherichia coli, the bacteriophage MS2, poliovirus and coxsackievirus. The results indicated that the most influential factor affecting the decay of the viruses and E. coli was the presence of groundwater micro-organisms. The results also implied that temperature, the presence of oxygen and nutrient levels indirectly influence viruses and E. coli decay by influencing the activity of the groundwater micro-organisms., Conclusions: E. coli and the viruses displayed maximum decay under aerobic conditions, at 28 degrees C without the addition of nutrients in the presence of groundwater micro-organisms., Significance and Impact of the Study: The results suggest that if the mode of action of the groundwater micro-organisms could be determined then the decay of viral pathogens in recharged waters may be more easily predicted.

A rapid, simple, and direct (RSD) assay of eluate from filter concentration was developed to enumerate low numbers of MS2 bacteriophage, used as a surrogate for enteric viruses, from samples collected from a prototype-sized water recycling system. The RSD assay utilized a 50-ml eluate volume in a modified single-layer assay, neutralizing eluate pH by buffered, double-strength agar. The RSD assay developed was simpler and minimized sample-handling steps compared with another published method. The RSD assay method showed greater sensitivity than the other published method for recovering phage from filter eluate while avoiding pH shifts, which can inactivate phage. Grant numbers: NAGW 2356.

Since temperature affects the inactivation rate of viruses in natural water systems, the aim of this study was to determine if a temperature shift could influence the structural integrity of model viruses. When crude lysates of MS-2 phage were seeded into groundwater microcosms and incubated at 27 degrees C, complete virus inactivation took place in eight days. The temperature was then shifted to 4 degrees C. Three days after the temperature shift, a two-log increase in virus titer (reactivation) occurred. However, when purified MS-2 lysates were added to groundwater microcosms, no reactivation was obtained. No reactivation of poliovirus took place when similar microcosm experiments were done. The sedimentation coefficients of MS-2 shifted from 80S to 58S, 48S, 37S, 32S, and 18S as inactivation proceeded in groundwater and distilled water controls. Similarly, the sedimentation coefficients of polioviruses changed from 156S to 142S, 135S, 117S, 105S, 95S, and 80 S as inactivation took place. There was no correlation between % virus inactivation and % decrease in virions with intact sedimentation coefficients, as reported earlier for poliovirus inactivated by chlorine. The results presented support our hypothesis that virus inactivation proceeds gradually, involving the rearrangement and (or) loss of capsomere components that may eventually lead to the ejection of nucleic acids. The intermediate particles generated as inactivation proceeds may be in a reversibly inactivated state, and may revert back to a fully infectious state when chemical components stabilize the virus particle.