Evaluation of the suitability of a plant virus, pepper mild mottle virus, as a surrogate of human enteric viruses for assessment of the efficacy of coagulation-rapid sand filtration to remove those viruses.

Here, we evaluated the removal of three representative human enteric viruses - adenovirus (AdV) type 40, coxsackievirus (CV) B5, and hepatitis A virus (HAV) IB - and one surrogate of human caliciviruses - murine norovirus (MNV) type 1 - by coagulation-rapid sand filtration, using water samples from eight water sources for drinking water treatment plants in Japan. The removal ratios of a plant virus (pepper mild mottle virus; PMMoV) and two bacteriophages (MS2 and φX174) were compared with the removal ratios of human enteric viruses to assess the suitability of PMMoV, MS2, and φX174 as surrogates for human enteric viruses. The removal ratios of AdV, CV, HAV, and MNV, evaluated via the real-time polymerase chain reaction (PCR) method, were 0.8-2.5-log10 when commercially available polyaluminum chloride (PACl, basicity 1.5) and virgin silica sand were used as the coagulant and filter medium, respectively. The type of coagulant affected the virus removal efficiency, but the age of silica sand used in the rapid sand filtration did not. Coagulation-rapid sand filtration with non-sulfated, high-basicity PACls (basicity 2.1 or 2.5) removed viruses more efficiently than the other aluminum-based coagulants. The removal ratios of MS2 were sometimes higher than those of the three human enteric viruses and MNV, whereas the removal ratios of φX174 tended to be smaller than those of the three human enteric viruses and MNV. In contrast, the removal ratios of PMMoV were similar to and strongly correlated with those of the three human enteric viruses and MNV. Thus, PMMoV appears to be a suitable surrogate for human enteric viruses for the assessment of the efficacy of coagulation-rapid sand filtration to remove viruses.

Investigation of enteric adenovirus and poliovirus removal by coagulation processes and suitability of bacteriophages MS2 and φX174 as surrogates for those viruses.

We evaluated the removal of enteric adenovirus (AdV) type 40 and poliovirus (PV) type 1 by coagulation, using water samples from 13 water sources for drinking water treatment plants in Japan. The behaviors of two widely accepted enteric virus surrogates, bacteriophages MS2 and φX174, were compared with the behaviors of AdV and PV. Coagulation with polyaluminum chloride (PACl, basicity 1.5) removed AdV and PV from virus-spiked source waters: the infectious AdV and PV removal ratios evaluated by means of a plaque-forming-unit method were 0.1-1.4-log10 and 0.5-2.4-log10, respectively. A nonsulfated high-basicity PACl (basicity 2.1) removed infectious AdV and PV more efficiently than did other commercially available PACls (basicity 1.5-2.1), alum, and ferric chloride. The MS2 removal ratios tended to be larger than those of AdV and PV, partly because of differences in the hydrophobicities of the virus particles and the sensitivity of the virus to the virucidal activity of PACl; the differences in removal ratios were not due to differences in the surface charges of the virus particles. MS2, which was more hydrophobic than the other viruses, was inactivated during coagulation with PACl. Therefore, MS2 does not appear to be an appropriate surrogate for AdV and PV during coagulation. In contrast, because φX174, like AdV and PV, was not inactivated during coagulation, and because the hydrophobicity of φX174 was similar to or somewhat lower than the hydrophobicities of AdV and PV, the φX174 removal ratios tended to be similar to or somewhat smaller than those of the enteric viruses. Therefore, φX174 is a potential conservative surrogate for AdV and PV during coagulation. In summary, the surface hydrophobicity of virus particles and the sensitivity of the virus to the virucidal activity of the coagulant are probably important determinants of the efficiency of virus removal during coagulation.

The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices.

The collection of waterborne pathogen occurrence data often requires the concentration of microbes from large volumes of water due to the low number of microorganisms that are typically present in environmental and drinking waters. Hollow-fiber ultrafiltration (HFUF) has shown promise in the recovery of various microorganisms. This study has demonstrated that the HFUF primary concentration method is effective at recovering bacteriophage φX174, poliovirus, enterovirus 70, echovirus 7, coxsackievirus B4 and adenovirus 41 from large volumes of tap and river water with an average recovery of all viruses of 73.4% and 81.0%, respectively. This study also evaluated an effective secondary concentration method using celite for the recovery of bacteriophage and enteric viruses tested from HFUF concentrates of both matrices. Overall, the complete concentration method (HFUF primary concentration plus celite secondary concentration) resulted in a concentration factor of 3333 and average recoveries for all viruses from tap and river waters of 60.6% and 60.0%, respectively.

On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/µL, 1 GU/µL, and 5 × 10(3) GU/µL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

Transport and removal of bacteriophages MS2 and PhiX174 in steel slag-amended soils: column experiments and transport model analyses.

The aim of this study was to investigate the removal of bacteriophages MS2 and PhiX174 in soils amended with converter furnace steel slag. Column experiments were performed to examine the bacteriophage removal in slag-amended (slag content: 0%, 25%, and 50%) loam soils. For comparison, column experiments were also conducted with Escherichia coli. In addition, chloride (Cl) was used as a conservative tracer to determine transport characteristics. Results showed mass recoveries of Cl of 98.6 +/- 3.5%, indicating that the experiments were conducted successfully. The mass recovery of MS2 was 86.7% in no slag (100% soil), decreasing to 0% in slag contents of 25% and 50%. The mass recovery of PhiX174 decreased from 87.8% to 51.5% with increasing slag content from 0% to 50%. In the case of E. coli, the mass recoveries decreased from 47.0% to 10.5% with increasing slag content from 0% to 50%. In the transport models analyses, the HYDRUS-1D code was used to quantify the sorption parameters from breakthrough curves. For the 100% soil column, a one-site kinetic sorption model was fitted to the data, whereas a two-site kinetic sorption model was fitted for slag-amended (25% and 50% slag) soil data. Results demonstrate that the addition of steel slag to soil enhances the removal of bacteriophages due to the presence of FeO in the steel slag. However, CaO could not contribute to the bacteriophage removal in our experimental conditions because the effluent pH (7.7-8.9) in slag-amended (25% and 50% slag) soils was not high enough to promote the bacteriophage inactivation.

Adenovirus, MS2 and PhiX174 interactions with drinking water biofilms developed on PVC, cement and cast iron.

Biofilms colonizing pipe surfaces of drinking water distribution systems could provide habitat and shelter for pathogenic viruses present in the water phase. This study aims (i) to develop a method to detect viral particles present in a drinking water biofilm and (ii) to study viral interactions with drinking water biofilms. A pilot scale system was used to develop drinking water biofilms on 3 materials (7 cm(2) discs): PVC, cast iron and cement. Biofilms were inoculated with viral model including MS2, PhiX174 or adenovirus. Five techniques were tested to recover virus from biofilms. The most efficient uses beef extract and glycine at pH = 9. After sonication and centrifugation, the pH of the supernatant is neutralized prior to viral analysis. The calculated recovery rates varied from 29.3 to 74.6% depending on the virus (MS2 or PhiX174) and the material. Applying this protocol, the interactions of virus models (MS2 and adenovirus) with drinking water biofilms were compared. Our results show that adsorption of viruses to biofilms depends on their isoelectric points, the disc material and the hydrodynamic conditions. Applying hydrodynamic conditions similar to those existing in drinking water networks resulted in a viral adsorption corresponding to less than 1% of the initial viral load.

Evaluation of the efficiency of medical masks and the creation of new medical masks.

The effectiveness of medical masks in preventing respiratory infection was investigated by testing bacterial leakage, filtration efficiency, respiratory resistance and oxygen concentration of the enclosed space. Polypropylene (PP) fibres were treated with dimethyldioctadecylammonium bromide to impart a positive electrical charge capable of attracting bacteria. The fluffed PP fibres were used to make a polypropylene mask and to edge standard surgical and N-95 respirators to prevent leakage. A PP napkin was created by melting and blowing PP. The PP edging seal dramatically reduced bacterial leakage of standard masks and was more effective than adhesive paper tape edging in reducing respiratory resistance. Bacterial or viral filtration efficiency was almost 100% for the PP mask and the PP napkin. The specially designed PP mask with a synthetic adhesive at the edge of the mask may be more effective than the standard surgical mask and the N-95 respirator. The PP napkin is an important tool in preventing the spread of pathogens.

Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing.

Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.

ENU-induced mutagenesis at a single A: T base pair in transgenic mice containing phi X174.

Transgenic mice containing the bacteriophage phi X174 am3 as a chromosomally integrated and recoverable marker for in vivo mutation have been produced to measure spontaneous and induced substitutions at an A:T base pair among single copies. phi X174 was chosen for its small size (5 kb), unique sequence, and the opportunity to take advantage of previously reported in vitro data on mutation and repair; the am3 site provides sequence specificity in a reversion assay for mutation of an A:T base pair. Inbred C57Bl/6 mice have been made homozygous for approximately 100 copies of the the phage sequence without any apparent detrimental effects on the homozygous individuals. Recoveries of phage from mouse tissues are in the range of 1-5 x 10(7) PFU per micrograms mouse DNA; both recovery and mutation are independent of endogenous CpG methylation. Background mutation frequencies are 2-4 x 10(-7) among phage recovered from liver, brain, spleen, and kidney. Adult mice were treated with 200 mg/kg N-ethyl-N-nitrosourea, and phage were recovered at 2 and 14 days after treatment. At 2 days after treatment we observed a slight increase only among phage isolated from the brain of one mouse out of four. At 14 days after ENU treatment, there were significant increases in mutation frequencies among phage recovered from the liver (6 x) and spleen (10 x). These results demonstrate (1) response of a single A:T base pair to alkylation-induced mutation in a nonexpressed gene, (2) the role of cell proliferation in somatic mutagenesis, and (3) provide a model for a transgenic approach for study of site-specific mutagenesis in vivo in higher eukaryotes.

Viable bacteriophage in CO2 laser plume: aerodynamic size distribution.

The size of CO2 laser generated plume particles containing viable bacteriophage, phi X174, was determined with 2 models (10-830 and 15-500) of Andersen bioaerosol cascade impactors. Samples were collected during 100 one-second laser exposures (approximately 68 W/cm2) of a bacteriophage-agar substrate with and without a space-confining hood. The hood appeared to facilitate collection of the largest particles (P < 0.1). In addition, Andersen model 15-500 was a more efficient collector of the largest particles, a phenomenon which is likely a function of the dynamic nature of the laser plume as well as impactor design. We found that laser plume particles containing viable bacteriophage are very large, in one instance exhibiting a count median aerodynamic diameter (CMAD) and mass median aerodynamic diameter (MMAD) of 23 and 55 microns, respectively. Furthermore, the appearance of viable bacteriophage in these plume particles constitutes an extremely rare event. The limitations of cascade impactor design when used to analyze particles with high water concentrations generated in a laser plume are discussed.

In vitro production of viable bacteriophage in carbon dioxide and argon laser plumes.

Both CO2 and argon laser ablation of an agar substrate containing high titres of bacteriophage phi X174 create plumes which disperse viable phage particles. Irradiances at the beam impact site ranged from 73 to 215 W/cm2 for the CO2 laser and from 40 to 227 W/cm2 for the argon laser. To increase the absorption of argon laser radiation, oxidized hemoglobin was added to the target material. Plume-borne viable phage were observed to be associated with particles large enough to settle out from the plume within 100 mm of the beam impact site. The ratio of the number of dispersed viable phage to the number of viable phage potentially dispersible by a single, 1-second laser exposure was on the order of 10(-6) to 10(-5).