Inline-tandem purification of viruses from cell lysate by agarose-based chromatography.

An efficient chromatography-based virus purification method has been developed and validated for the non-pathogenic infectious virus PRD1. Compared to the conventional method that consists of relatively time-consuming and labour-intensive precipitation and density gradient ultracentrifugation steps, the method developed here is performed in a single flow using tandem-coupled anion exchange and size exclusion chromatography (AIEX-SEC) columns. This inline approach helps to minimize the loss of virus in the process and streamlines time consumption, since no physical transfer of the sample is required between purification steps. In the development process, sample feed composition, dynamic binding capacity and elution conditions for the AIEX resin as well as different exclusion limits for SEC resins were optimized to achieve maximal yield of pure infectious viruses. Utilizing this new approach, a high-quality virus sample was produced from a lysate feed in 320 min with a total yield of 13 mg purified particles per litre of cell lysate, constituting a 3.5-fold yield increase as compared to the conventional method, without compromising the high specific infectivity of the product (6 × 1012 to 7 × 1012 pfu/mg of protein). The yield of infectious viruses of the lysate feed was 54%. The easy scalability of chromatography-based methods provide a direct route to industrial usage without any significant changes needed to be made to the purification regime. This is especially interesting as the method has high potential to be used for purification of various viruses and nanoparticles, including adenovirus.

Pathogen and Surrogate Survival in Relation to Fecal Indicator Bacteria in Freshwater Mesocosms.

The microbial quality of agricultural water for fresh produce production is determined by the presence of the fecal indicator bacterium (FIB) Escherichia coli, despite poor correlations with pathogen presence. Additional FIB, such as enterococci, have been utilized for assessing water quality. The study objective was to determine the survival times (first time to detect zero or censored) of FIB (E. coli and enterococci), surrogates (Listeria innocua, Listeria seeligeri, Salmonella enterica serovar Typhimurium, and PRD1), and pathogens (four strains each of pathogenic E. coli and Listeria monocytogenes and five Salmonella serovars) simultaneously inoculated in freshwater mesocosms exposed to diel and seasonal variations. Six separate mesocosm experiments were conducted for ≤28 days each season, with samples (sediment/water) collected each day for the first 7 days and weekly thereafter. Microorganisms survived significantly longer in sediment than in water (hazard ratio [HR] for water/sediment is 2.2; 95% confidence interval [CI], 1.79 to 2.71). Also, FIB E. coli survived significantly longer than FIB enterococcus (HR for enterococci/E. coli is 12.9 [95% CI, 8.18 to 20.37]) after adjusting for the sediment/water and lake/river effects. Differences in the area under the curve (calculated from log CFU or PFU over time) were used to assess pathogen and surrogate survival in relation to FIB. Despite sample type (sediment/water) and seasonal influences, survival rates of pathogenic Salmonella serovars were similar to those of FIB E. coli, and survival rates of L. monocytogenes and pathogenic E. coli were similar to those of FIB enterococci. Further investigation of microbial survival in water and sediment is needed to determine which surrogates are best suited to assess pathogen survival in agricultural water used in irrigation water for fresh produce. IMPORTANCE Contamination of fresh produce via agricultural water is well established. This research demonstrates that survival of fecal indicator bacteria, pathogenic microorganisms, and other bacterial and viral surrogates in freshwater differs by sample type (sediment/water) and season. Our work highlights potential risks associated with pathogen accumulation and survival in sediment and the possibility for resuspension and contamination of agricultural water used in fresh produce production. Specifically, a greater microbial persistence in sediments than in water over time was observed, along with differences in survival among microorganisms in relation to the fecal indicator bacteria E. coli and enterococci. Previous studies compared data among microbial groups in different environments. Conversely, fecal indicator bacteria, surrogates, and pathogenic microorganisms were assessed within the same water and sediment mesocosms in the present study during four seasons, better representing the agricultural aquatic environment. These data should be considered when agricultural microbial water quality criteria in fresh produce operations are being determined.

Transport of bacteriophage MS2 and PRD1 in saturated dune sand under suboxic conditions.

Soil passage of (pretreated) surface water to remove pathogenic microorganisms is a highly efficient process under oxic conditions, reducing microorganism concentrations about 8 log10 within tens of meters. However, under anoxic conditions, it has been shown that removal of microorganisms can be limited very much. Setback distances for adequate protection of natural groundwater may, therefore, be too short if anoxic conditions apply. Because removal of microorganisms under suboxic conditions is unknown, this research investigated removal of bacteriophage MS2 and PRD1 by soil passage under suboxic conditions at field scale. At the field location (dune area), one injection well and six monitoring wells were installed at different depths along three suboxic flow lines, where oxygen concentrations ranged from 0.4 to 1.7 mg/l and nitrate concentrations ranged from 13 to 16 mg/L. PRD1 and MS2 were injected directly at the corresponding depths and their removal in each flow line was determined. The highest bacteriophage removal was observed in the top layer, with about 9 log removal of MS2, and 7 log removal of PRD1 after 16 meters of aquifer transport. Less removal was observed at 12 m below surface, probably due to a higher groundwater velocity in this coarser grained layer. MS2 was removed more effectively than PRD1 under all conditions. Due to short travel times, inactivation of the phages was limited and the reported log removal was mainly associated with attachment of phages to the aquifer matrix. This study shows that attachment of MS2 and PRD1 is similar for oxic and suboxic sandy aquifers, and, therefore, setback distances used for sandy aquifers under oxic and suboxic conditions provide a similar level of safety. Sticking efficiency and the attachment rate coefficient, as measures for virus attachment, were evaluated as a function of the physico-chemical conditions.

Asymmetric flow field flow fractionation methods for virus purification.

Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells.

Investigation of E. coli and Virus Reductions Using Replicate, Bench-Scale Biosand Filter Columns and Two Filter Media.

The biosand filter (BSF) is an intermittently operated, household-scale slow sand filter for which little data are available on the effect of sand composition on treatment performance. Therefore, bench-scale columns were prepared according to the then-current (2006-2007) guidance on BSF design and run in parallel to conduct two microbial challenge experiments of eight-week duration. Triplicate columns were loaded with Accusand silica or crushed granite to compare virus and E. coli reduction performance. Bench-scale experiments provided confirmation that increased schmutzdecke growth, as indicated by decline in filtration rate, is the primary factor causing increased E. coli reductions of up to 5-log10. However, reductions of challenge viruses improved only modestly with increased schmutzdecke growth. Filter media type (Accusand silica vs. crushed granite) did not influence reduction of E. coli bacteria. The granite media without backwashing yielded superior virus reductions when compared to Accusand. However, for columns in which the granite media was first backwashed (to yield a more consistent distribution of grains and remove the finest size fraction), virus reductions were not significantly greater than in columns with Accusand media. It was postulated that a decline in surface area with backwashing decreased the sites and surface area available for virus sorption and/or biofilm growth and thus decreased the extent of virus reduction. Additionally, backwashing caused preferential flow paths and deviation from plug flow; backwashing is not part of standard BSF field preparation and is not recommended for BSF column studies. Overall, virus reductions were modest and did not meet the 5- or 3-log10 World Health Organization performance targets.

Effect of dissolved calcium on the removal of bacteriophage PRD1 during soil passage: the role of double-layer interactions.

The objective of this work was to investigate and obtain quantitative relations for the effects of Ca(2+) concentration on virus removal in saturated soil and to compare the experimental findings with predictions of the DLVO theory. In order to do so, a systematic study was performed with a range of calcium concentrations corresponding to natural field conditions. Experiments were conducted in a 50-cm column with clean quartz sand under saturated conditions. Inflow solutions were prepared by adding CaCl(2,) NaCl and NaHCO(3) to de-ionized water. Values of pH and ionic strength were fixed at 7 and 10mM, respectively. Bacteriophage PRD1 was used as a conservative model virus for virus removal. The samples were assayed using the plaque forming technique. Attachment, detachment and inactivation rate coefficients were determined from fitting breakthrough curves. Attachment rate coefficients were found to increase with increasing calcium concentration. Results were used to calculate sticking efficiency, for which an empirical formula as a function of Ca(2+) was developed. Numerical solutions of the Poisson-Boltzmann equation were obtained to evaluate the effect of Ca(2+) on the double-layer interactions between quartz and PRD1. Based on these results, the DLVO interaction energies were calculated. It turned out that the experimental findings cannot be explained with the distance profiles of the DLVO interaction. The discrepancy between theory and experiment can be attributed to underestimation of the van der Waals interactions, chemisorption of Ca(2+) onto the surfaces, or by factors affecting the double-layer interactions, which are not included in the Poisson-Boltzmann equation. When abruptly changing from inflow solution containing Ca(2+) to a Ca(2+)-free solution, pronounced mobilization of viruses was observed. This indicates virus removal is not irreversible and that chemical perturbations of the groundwater can cause a burst of released viruses.

Monolithic ion exchange chromatographic methods for virus purification.

We report an ion exchange chromatographic purification method powerful for preparation of virus particles with ultrapure quality. The technology is based on large pore size monolithic anion exchangers, quaternary amine (QA) and diethyl aminoethyl (DEAE). These were applied to membrane-containing icosahedral bacteriophage PRD1, which bound specifically to both matrices. Virus particles eluted from the columns retained their infectivity, and were homogenous with high specific infectivity. The yields of infectious particles were up to 80%. Purified particles were recovered at high concentrations, approximately 5mg/ml, sufficient for virological, biochemical and structural analyses. We also tested the applicability of the monolithic anion exchange purification on a filamentous bacteriophage phi05_2302. Monolithic ion exchange chromatography is easily scalable and can be combined with other preparative virus purification methods.

Virus attenuation by microbial mechanisms during the idle time of a household slow sand filter.

The biosand filter (BSF) is a household slow sand filter that is operated intermittently such that an idle time of typically 18-22 h occurs in between daily charges of water. Virus attenuation during the idle time was investigated over repeated daily filtration cycles to capture the effect of media aging that encompasses processes occurring throughout the filter depth rather than restricted to the schmutzdecke at the media surface. A threshold aging period of about one to two weeks was required before virus attenuation began. The observed rates of MS2 and PRD-1 reduction were first-order and reached maxima of 0.061- and 0.053-log per hr, respectively, over seven-to-ten weeks. Suppression of microbial activity by sodium azide eliminated virus reduction during the idle time thus indicating that the operative media aging process was microbially mediated. The mechanism of virus reduction was not modification of media surfaces by physical/chemical or microbial processes. Instead, it appears that the activity of the microbial community within the filter is responsible. The most likely biological pathways are production of microbial exoproducts such as proteolytic enzymes or grazing of bacteria and higher microorganisms on virus particles. Implications of these findings for BSF design and operation and their relevance to other biological filtration technologies are discussed.

Optimal preparation and purification of PRD1-like bacteriophages for use in environmental fate and transport studies.

Bacteriophages are bacterial viruses with unique characteristics that make them excellent surrogates for mammalian pathogenic viruses in environmental studies. Simple and reliable methodologies for isolation, detection, characterization and enumeration of somatic and F-specific bacteriophage are available in the literature. Limited information or methods are available for producing high-titer purified phage suspensions for studying microbial transport and survival in natural and engineered environments. This deficiency arises because most research on the production of high-titer phage suspensions was completed over half a century ago and more recent advances on these methods have not been compiled in a single publication. We present a review of the available methods and new data on the propagation, concentration and purification of two bacteriophage host systems (somatic PRD1/Salmonella thyphimurium and F-specific PR772/Escherichia coli) that are commonly utilized in laboratory and field-scale assessments of subsurface microbial transport and survival. The focus of the present study is to recommend the approach(es) that will ensure maximum bacteriophage yields while optimizing suspension purification (i.e. avoiding modification of surface charge of the phage capsids and/or inadvertent introduction of dissolved organic matter to the study system).

Discrimination of bacteria and bacteriophages by Raman spectroscopy and surface-enhanced Raman spectroscopy.

Detection of pathogenic organisms in the environment presents several challenges due to the high cost and long times typically required for identification and quantification. Polymerase chain reaction (PCR) based methods are often hindered by the presence of polymerase inhibiting compounds and so direct methods of quantification that do not require enrichment or amplification are being sought. This work presents an analysis of pathogen detection using Raman spectroscopy to identify and quantify microorganisms without drying. Confocal Raman measurements of the bacterium Escherichia coli and of two bacteriophages, MS2 and PRD1, were analyzed for characteristic peaks and to estimate detection limits using traditional Raman and surface-enhanced Raman spectroscopy (SERS). MS2, PRD1, and E. coli produced differentiable Raman spectra with approximate detection limits for PRD1 and E. coli of 10(9) pfu/mL and 10(6) cells/mL, respectively. These high detection concentration limits are partly due to the small sampling volume of the confocal system but translate to quantification of as little as 100 bacteriophages to generate a reliable spectral signal. SERS increased signal intensity 10(3) fold and presented peaks that were visible using 2-second acquisitions; however, peak locations and intensities were variable, as typical with SERS. These results demonstrate that Raman spectroscopy and SERS have potential as a pathogen monitoring platform.

Field scale quantification of microbial transport from bovine faeces under simulated rainfall events.

The dispersion and transport of Cryptosporidium parvum oocysts, Escherichia coli and PRD1 bacteriophage seeded into artificial bovine faecal pats was studied during simulated rainfall events. Experimental soil plots were divided in two, one sub-plot with bare soil and the other with natural vegetation. Simulated rainfall events of 55 mm.h(-1) for 30 min were then applied to the soil plots. Each experimental treatment was performed in duplicate and consisted of three sequential artificial rainfall events ('Runs'): a control run (no faecal pats); a fresh faecal pat run (fresh faecal pats); and an aged faecal pat run (one week aged faecal pats). Transportation efficiency increased with decreasing size of the microorganism studied; Cryptosporidium oocysts were the least mobile followed by E. coli and then PRD1 phage. Rainfall events mobilised 0.5 to 0.9% of the Cryptosporidium oocysts, 1.3-1.4% of E. coli bacteria, and 0.03-0.6% of PRD1 bacteriophages from the fresh faecal pats and transported them a distance of 10 m across the bare soil sub-plots. Subsequent rainfall events applied to aged faecal pats only mobilised 0.01-0.06% of the original Cryptosporidium oocyst load, between 0.04 and 15% of the E. coli load and 0.0006-0.06% of PRD1 bacteriophages, respectively.

Transport of coliphage PRD1 in a surface flow constructed wetland.

A tracer study was conducted in a 3-ha surface flow constructed wetland to analyze transport performance of PRD1, an enteric virus model. The convection-dispersion equation (CDE), including a first-order reaction model, adequately simulated transport performance of PRD1 in the wetland under an average hydraulic loading rate of 82 mm/d. Convective velocity (v) and longitudinal dispersion coefficient (D) were estimated by modeling a conservative tracer (bromide) pulse through the wetland. Both PRD1 and bromide were simultaneously added to the entering secondary treated wastewater effluent. The mass of bromide and PRD1 recovered was 76 and 16%, respectively. The PRD1 decay rate was calculated to be 0.3/day. The findings of this study suggest that the CDE model and analytical moment equations represent a suitable option to characterize virus transport performance in surface flow constructed wetlands.

Removal and inactivation of Cryptosporidium and microbial indicators by a quaternary ammonium chloride (QAC)-treated zeolite in pilot filters.

A set of pilot filters packed with Zeolite filter media treated with a quaternary ammonium chloride (QAC) were evaluated to verify the proof of concept of their combined antimicrobial capabilities. Escherichia coli was removed and inactivated the most (2.83 log), followed by MS-2 (2.75 log), Klebsiella terriena (2.21 log), PRD-1 (1.95 log), Chlorella vulgaris (1.92 log), and Cryptosporidium parvum oocysts (1.78 log). Especially, inactivation of C. parvum oocysts (1.19 log) was higher than physical removal (0.54 log). The data suggest that QAC-treated Zeolite in the pilot filters has microbial inactivation capabilities and may have useful applications in other types of filter media.

Effect of goethite coating and humic acid on the transport of bacteriophage PRD1 in columns of saturated sand.

The transport of bacteriophage PRD1, a model virus, was studied in columns containing sediment mixtures of quartz sand with goethite-coated sand and using various solutions consisting of monovalent and divalent salts and humic acid (HA). Without HA and in the absence of sand, the inactivation rate of PRD1 was found to be as low as 0.014 day(-1) (at 5+/-3 degrees C), but in the presence of HA it was much lower (0.0009 day(-1)), indicating that HA helps PRD1 to survive. When the fraction of goethite in the sediment was increased, the removal of PRD1 also increased. However, in the presence of HA, C/C0 values of PRD1 increased by as much as 5 log units, thereby almost completely eliminating the effect of addition of goethite. The sticking efficiency was not linearly dependent on the amount of goethite added to the quartz sand; this is apparently due to surface charge heterogeneity of PRD1. Our results imply that, in the presence of dissolved organic matter (DOM), viruses can be transported for long distances thanks to two effects: attachment is poor because DOM has occupied favourable sites for attachment and inactivation of virus may have decreased. This conclusion justifies making conservative assumptions about the attachment of viruses when calculating protection zones for groundwater wells.

Comparative analysis of bacterial viruses Bam35, infecting a gram-positive host, and PRD1, infecting gram-negative hosts, demonstrates a viral lineage.

Extra- and intracellular viruses in the biosphere outnumber their cellular hosts by at least one order of magnitude. How is this enormous domain of viruses organized? Sampling of the virosphere has been scarce and focused on viruses infecting humans, cultivated plants, and animals as well as those infecting well-studied bacteria. It has been relatively easy to cluster closely related viruses based on their genome sequences. However, it has been impossible to establish long-range evolutionary relationships as sequence homology diminishes. Recent advances in the evaluation of virus architecture by high-resolution structural analysis and elucidation of viral functions have allowed new opportunities for establishment of possible long-range phylogenic relationships-virus lineages. Here, we use a genomic approach to investigate a proposed virus lineage formed by bacteriophage PRD1, infecting gram-negative bacteria, and human adenovirus. The new member of this proposed lineage, bacteriophage Bam35, is morphologically indistinguishable from PRD1. It infects gram-positive hosts that evolutionarily separated from gram-negative bacteria more than one billion years ago. For example, it can be inferred from structural analysis of the coat protein sequence that the fold is very similar to that of PRD1. This and other observations made here support the idea that a common early ancestor for Bam35, PRD1, and adenoviruses existed.

Virus removal from wastewater in a multispecies subsurface-flow constructed wetland.

Virus removal was studied in a multispecies subsurface-flow constructed wetland. Tracer studies and a virus survival test were conducted using bromide and bacteriophage PRD1 that were simultaneously added into a 6-year-old gravel-filled wetland. The estimated dimensionless variance and the observed bromide breakthrough curve suggest a plug-flow reactor with some dispersion. Most of the PRD1 was removed during the first 4 days; however, the PRD1 background concentration was not reached by the end of the study. Average bacteriophage removal was 98.8%, whereas bromide mass recovery was 75%. The removal rate of PRD1 was estimated to be -1.17 d(-1); in contrast, its inactivation rate in situ for a 12.4-day period was -0.16 d(-1). Apparently, virus removal is governed by an initial irreversible attachment followed by a comparatively long inactivation period. This study suggests that a subsurface-flow wetland can decrease the virus load by approximately 99% with a 5.5-day detention time.

Comparative surface-to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage.

AIMS: To determine the transfer efficiency of micro-organisms from fomites to hands and the subsequent transfer from the fingertip to the lip. METHODS AND RESULTS: Volunteers hands were sampled after the normal usage of fomites seeded with a pooled culture of a Gram-positive bacterium (Micrococcus luteus), a Gram-negative bacterium (Serratia rubidea) and phage PRD-1 (Period A). Activities included wringing out a dishcloth/sponge, turning on/off a kitchen faucet, cutting up a carrot, making hamburger patties, holding a phone receiver, and removing laundry from the washing machine. Transfer efficiencies were 38.47% to 65.80% and 27.59% to 40.03% for the phone receiver and faucet, respectively. Transfer efficiencies from porous fomites were <0.01%. In most cases, M.luteus was transferred most efficiently, followed by phage PRD-1 and S. rubidea. When the volunteers' fingertips were inoculated with the pooled organisms and held to the lip area (Period B), transfer rates of 40.99%, 33.97%, and 33.90% occurred with M. luteus, S. rubidea, and PRD-1, respectively. CONCLUSIONS: The highest bacteral transfer rates from fomites to the hands were seen with the hard, non-porous surfaces. Even with low transfer rates, the numbers of bacteria transferred to the hands were still high (up to 10(6) cells). Transfer of bacteria from the fingertip to the lip is similar to that observed from hard surfaces to hands. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious doses of pathogens may be transferred to the mouth after handling an everyday contaminated household object.