Applied and Environmental Microbiology, Jun 1, 1982
The simian rotavirus SAl was used to develop a simple, reliable, and efficient method to concentr... more The simian rotavirus SAl was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA 1I adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter- bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SAl was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SAl serum which would detect the simian, human, bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SAl1. The buoyant density of the sewage isolates in CsCI gradients was 1.36 g/ cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 + 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.
Experimental apparatus showing solution application, sample extraction, and pressure measurement ... more Experimental apparatus showing solution application, sample extraction, and pressure measurement methods. Only one of the six pairs of sampler and . manometer tubing is shown 30 2a The relative virus concentrations versus pore volumes and fitted breakthrough curves at 0.20 m. (C/C0), is the apparent steady-state value. The markers are labeled by transport experiment number and column letter. The curves were constructed from Eq. [4] 45 2b. The relative virus concentrations versus pore volumes and fitted breakthrough curves at 0.40 m. 46 2c. The relative virus concentrations versus pore volumes and fitted breakthrough curves at 0.80 m. 47 2d. The relative virus concentrations versus pore volumes and fitted breakthrough curves at 1.05 m. 48 3. The relative virus concentration profiles under saturated flow (TE6E) at four sampling times. The average linear velocity was 0.24 m d-1 50 4. The relative virus concentration profile under unsaturated flow (TE3 and TE9) from day 3 (one pore volume at 1.05 m) to day 9, and fitted curve using Eq. [4] and [5], where u = 4.62 d-1 , z is depth (m), y = 0.37 m d-1 , G = 3 m-1 , and L = 0.05 m . . . 51 5. The relative virus concentrations versus pore volumes at the 1.05 m depth and fitted breakthrough curves in transport experiment 10. (C/C0), is the apparent steady-state value. The curves were constructed from Eq. [4]. 55 6. The relative virus concentrations versus pore volumes at the 1.05 m depth and fitted breakthrough curves in transport experiment 12. (C/Co) s is the apparent steady-state value. The curves were constructed from Eq. [4]. 57 7. The relative virus concentration profile with depth from TE10 and TE12 on day 7 (1.8-1.9 pore volumes). The fitted curves were constructed from Eq. [4] and [5], where z is depth (m), y = 0.27 m 61 , G = 3 m-1 , L = 0.05 m, and the organic u, = 0.81 d-1 and the leached u1 = 3.1 di 59 Under unsaturated flow conditions enhanced removal of this virus occurs, and the removed virus are apparently inactivated. Organic matter reduced the removal of virus during transport by unsaturated flow. Virus concentrations reached and maintained a steady-state, exponentially-declining profile with depth. TE3: Long term test with ponding.
Applied and Environmental Microbiology, Jul 1, 2003
Ct values, the concentration of free chlorine multiplied by time of contact with virus, were dete... more Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroformextracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5°C than at 15°C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15°C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5°C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.
Little information regarding the effectiveness of UV radiation on the inactivation of calicivirus... more Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm 2 , respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm 2 in treated groundwater. A dose of 103 mJ/cm 2 was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.
Applied and Environmental Microbiology, May 1, 2000
Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg... more Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.
Applied and Environmental Microbiology, Nov 1, 1994
N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell cultur... more N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCI, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 102 PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCI, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.
The purpose of this study was to provide a clearer understanding of virus adsorption, focusing sp... more The purpose of this study was to provide a clearer understanding of virus adsorption, focusing specifically on the role of electrostatic interactions between virus particles and adsorbent surfaces. The adsorption of poliovirus 1, reovirus types 1 and 3, and coliphages MS-2 and T2 to colloidal silica synthetically modified to carry either positive or negative surface charge was evaluated. Adsorption experiments were performed by combining virus and silica in 0.1-ionic-strength buffers of pH 4.0, 6.4, and 8.5. Samples agitated for specified adsorption periods were centrifuged to pellet adsorbent particles plus adsorbed virus, and the supernatants were assayed for unadsorbed virus. All viruses adsorbed exclusively to negatively charged silica at pH values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all viruses adsorbed exclusively to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge is negative. Viruses in near-isoelectric state adsorbed to all types of silica, albeit to a lesser degree.
Applied and Environmental Microbiology, Sep 1, 2005
The results of this study confirm that adenoviruses are the most resistant enteric viruses to ina... more The results of this study confirm that adenoviruses are the most resistant enteric viruses to inactivation by UV light and that adenovirus 40 appears to be the most resistant. The effect of freeze-thawing and storage in water may affect the sensitivity of some adenoviruses to inactivation by UV light.
Applied and Environmental Microbiology, Apr 1, 1996
Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizi... more Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach. By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with >3 days by cell culture alone. The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds.
Applied and Environmental Microbiology, May 1, 1993
Standard methods for the detection of enteroviruses in environmental samples involve the use of c... more Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
Infiltration and runoff from manured agricultural fields can result in livestock pathogens reachi... more Infiltration and runoff from manured agricultural fields can result in livestock pathogens reaching groundwater and surface waters. Here, we measured the effectiveness of glass wool filters to simultaneously concentrate enteric viruses and bacteria of bovine origin from water. The recovery efficiencies were determined for bovine viral diarrhea virus types 1 and 2, bovine rotavirus group A, bovine coronavirus, poliovirus Sabin III, toxigenic Escherichia coli ,and Campylobacter jejuni seeded into water with three different turbidity levels (0.5, 215, and 447 NTU). Twenty liters of dechlorinated tap water (pH 7) were seeded with the test organisms, and then passed through a glass wool filter using a peristaltic pump (flow rate = 1 liter min -1 ). Retained organisms were eluted from the filters by passing beef extract-glycine buffer (pH 9.5) in the direction opposite of sample flow. Recovered organisms were enumerated by qPCR except for C. jejuni, which was quantified by culture. Mean recovery efficiencies ranged from 55 to 33 % for the bacteria and 58 to 16 % for the viruses. Using bootstrapping techniques combined with Analysis of Variance, recovery efficiencies were found to differ among the pathogen types tested at the two lowest turbidity levels; however, for a given pathogen type turbidity did not affect recovery except for C. jejuni. Glass wool filtration is a cost-effective method for concentrating several waterborne pathogens of bovine origin simultaneously, although recovery may be low for some specific taxa such as bovine viral diarrhea virus 1.
In addition to enteric viruses of fecal origin, emerging zoonotic viruses such as respiratory cor... more In addition to enteric viruses of fecal origin, emerging zoonotic viruses such as respiratory coronaviruses and influenza viruses may potentially be transmitted via contaminated foods. The goal of this study was to determine the recovery efficiencies and the survival of two respiratory viruses, namely, adenovirus 2 (Ad2) and coronavirus 229E (CoV229E), on fresh produce in comparison to the enteric poliovirus 1 (PV1). Adenovirus was recovered with efficiencies of 56.5, 31.8, and 34.8 % from lettuce, strawberries, and raspberries, respectively. Coronavirus was recovered from lettuce with an efficiency of 19.6 % yet could not be recovered from strawberries. Poliovirus was recovered with efficiencies of 76.7 % from lettuce, but only 0.06 % from strawberries. For comparison purposes, the survival of Ad2, CoV229E, and PV1 was determined for periods up to 10 days on produce. The enteric PV1 survived better than both respiratory viruses on lettuce and strawberries, with only B1.03 log 10 reductions after 10 days of storage at 4 °C compared to CoV229E not being recovered after 4 days on lettuce and reductions of 1.97 log 10 and 2.38 log 10 of Ad2 on lettuce and strawberries, respectively, after 10 days. Nevertheless, these respiratory viruses were able to survive for at least several days on produce. There is therefore the potential for transfer to the hands and subsequently to the mucosa via rubbing the eyes or nose. In addition, some respiratory coronaviruses (e.g., severe acute respiratory syndrome coronavirus) and adenoviruses are also capable of replication in the gut and there is thus some potential for acquisition through the consumption of contaminated produce.
Applied and Environmental Microbiology, Jul 1, 1979
This study was designed to determine the degree of adsorption of enteric viruses to marine sedime... more This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.
Municipal sewage sludge is a complex mixture of organic and inorganic compounds of biological and... more Municipal sewage sludge is a complex mixture of organic and inorganic compounds of biological and mineral origin that are removed from waste-
Applied and Environmental Microbiology, Jun 1, 1982
The simian rotavirus SAl was used to develop a simple, reliable, and efficient method to concentr... more The simian rotavirus SAl was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA 1I adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter- bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SAl was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SAl serum which would detect the simian, human, bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SAl1. The buoyant density of the sewage isolates in CsCI gradients was 1.36 g/ cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 + 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.
Experimental apparatus showing solution application, sample extraction, and pressure measurement ... more Experimental apparatus showing solution application, sample extraction, and pressure measurement methods. Only one of the six pairs of sampler and . manometer tubing is shown 30 2a The relative virus concentrations versus pore volumes and fitted breakthrough curves at 0.20 m. (C/C0), is the apparent steady-state value. The markers are labeled by transport experiment number and column letter. The curves were constructed from Eq. [4] 45 2b. The relative virus concentrations versus pore volumes and fitted breakthrough curves at 0.40 m. 46 2c. The relative virus concentrations versus pore volumes and fitted breakthrough curves at 0.80 m. 47 2d. The relative virus concentrations versus pore volumes and fitted breakthrough curves at 1.05 m. 48 3. The relative virus concentration profiles under saturated flow (TE6E) at four sampling times. The average linear velocity was 0.24 m d-1 50 4. The relative virus concentration profile under unsaturated flow (TE3 and TE9) from day 3 (one pore volume at 1.05 m) to day 9, and fitted curve using Eq. [4] and [5], where u = 4.62 d-1 , z is depth (m), y = 0.37 m d-1 , G = 3 m-1 , and L = 0.05 m . . . 51 5. The relative virus concentrations versus pore volumes at the 1.05 m depth and fitted breakthrough curves in transport experiment 10. (C/C0), is the apparent steady-state value. The curves were constructed from Eq. [4]. 55 6. The relative virus concentrations versus pore volumes at the 1.05 m depth and fitted breakthrough curves in transport experiment 12. (C/Co) s is the apparent steady-state value. The curves were constructed from Eq. [4]. 57 7. The relative virus concentration profile with depth from TE10 and TE12 on day 7 (1.8-1.9 pore volumes). The fitted curves were constructed from Eq. [4] and [5], where z is depth (m), y = 0.27 m 61 , G = 3 m-1 , L = 0.05 m, and the organic u, = 0.81 d-1 and the leached u1 = 3.1 di 59 Under unsaturated flow conditions enhanced removal of this virus occurs, and the removed virus are apparently inactivated. Organic matter reduced the removal of virus during transport by unsaturated flow. Virus concentrations reached and maintained a steady-state, exponentially-declining profile with depth. TE3: Long term test with ponding.
Applied and Environmental Microbiology, Jul 1, 2003
Ct values, the concentration of free chlorine multiplied by time of contact with virus, were dete... more Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroformextracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5°C than at 15°C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15°C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5°C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.
Little information regarding the effectiveness of UV radiation on the inactivation of calicivirus... more Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm 2 , respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm 2 in treated groundwater. A dose of 103 mJ/cm 2 was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.
Applied and Environmental Microbiology, May 1, 2000
Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg... more Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.
Applied and Environmental Microbiology, Nov 1, 1994
N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell cultur... more N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCI, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 102 PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCI, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.
The purpose of this study was to provide a clearer understanding of virus adsorption, focusing sp... more The purpose of this study was to provide a clearer understanding of virus adsorption, focusing specifically on the role of electrostatic interactions between virus particles and adsorbent surfaces. The adsorption of poliovirus 1, reovirus types 1 and 3, and coliphages MS-2 and T2 to colloidal silica synthetically modified to carry either positive or negative surface charge was evaluated. Adsorption experiments were performed by combining virus and silica in 0.1-ionic-strength buffers of pH 4.0, 6.4, and 8.5. Samples agitated for specified adsorption periods were centrifuged to pellet adsorbent particles plus adsorbed virus, and the supernatants were assayed for unadsorbed virus. All viruses adsorbed exclusively to negatively charged silica at pH values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all viruses adsorbed exclusively to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge is negative. Viruses in near-isoelectric state adsorbed to all types of silica, albeit to a lesser degree.
Applied and Environmental Microbiology, Sep 1, 2005
The results of this study confirm that adenoviruses are the most resistant enteric viruses to ina... more The results of this study confirm that adenoviruses are the most resistant enteric viruses to inactivation by UV light and that adenovirus 40 appears to be the most resistant. The effect of freeze-thawing and storage in water may affect the sensitivity of some adenoviruses to inactivation by UV light.
Applied and Environmental Microbiology, Apr 1, 1996
Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizi... more Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach. By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with >3 days by cell culture alone. The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds.
Applied and Environmental Microbiology, May 1, 1993
Standard methods for the detection of enteroviruses in environmental samples involve the use of c... more Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
Infiltration and runoff from manured agricultural fields can result in livestock pathogens reachi... more Infiltration and runoff from manured agricultural fields can result in livestock pathogens reaching groundwater and surface waters. Here, we measured the effectiveness of glass wool filters to simultaneously concentrate enteric viruses and bacteria of bovine origin from water. The recovery efficiencies were determined for bovine viral diarrhea virus types 1 and 2, bovine rotavirus group A, bovine coronavirus, poliovirus Sabin III, toxigenic Escherichia coli ,and Campylobacter jejuni seeded into water with three different turbidity levels (0.5, 215, and 447 NTU). Twenty liters of dechlorinated tap water (pH 7) were seeded with the test organisms, and then passed through a glass wool filter using a peristaltic pump (flow rate = 1 liter min -1 ). Retained organisms were eluted from the filters by passing beef extract-glycine buffer (pH 9.5) in the direction opposite of sample flow. Recovered organisms were enumerated by qPCR except for C. jejuni, which was quantified by culture. Mean recovery efficiencies ranged from 55 to 33 % for the bacteria and 58 to 16 % for the viruses. Using bootstrapping techniques combined with Analysis of Variance, recovery efficiencies were found to differ among the pathogen types tested at the two lowest turbidity levels; however, for a given pathogen type turbidity did not affect recovery except for C. jejuni. Glass wool filtration is a cost-effective method for concentrating several waterborne pathogens of bovine origin simultaneously, although recovery may be low for some specific taxa such as bovine viral diarrhea virus 1.
In addition to enteric viruses of fecal origin, emerging zoonotic viruses such as respiratory cor... more In addition to enteric viruses of fecal origin, emerging zoonotic viruses such as respiratory coronaviruses and influenza viruses may potentially be transmitted via contaminated foods. The goal of this study was to determine the recovery efficiencies and the survival of two respiratory viruses, namely, adenovirus 2 (Ad2) and coronavirus 229E (CoV229E), on fresh produce in comparison to the enteric poliovirus 1 (PV1). Adenovirus was recovered with efficiencies of 56.5, 31.8, and 34.8 % from lettuce, strawberries, and raspberries, respectively. Coronavirus was recovered from lettuce with an efficiency of 19.6 % yet could not be recovered from strawberries. Poliovirus was recovered with efficiencies of 76.7 % from lettuce, but only 0.06 % from strawberries. For comparison purposes, the survival of Ad2, CoV229E, and PV1 was determined for periods up to 10 days on produce. The enteric PV1 survived better than both respiratory viruses on lettuce and strawberries, with only B1.03 log 10 reductions after 10 days of storage at 4 °C compared to CoV229E not being recovered after 4 days on lettuce and reductions of 1.97 log 10 and 2.38 log 10 of Ad2 on lettuce and strawberries, respectively, after 10 days. Nevertheless, these respiratory viruses were able to survive for at least several days on produce. There is therefore the potential for transfer to the hands and subsequently to the mucosa via rubbing the eyes or nose. In addition, some respiratory coronaviruses (e.g., severe acute respiratory syndrome coronavirus) and adenoviruses are also capable of replication in the gut and there is thus some potential for acquisition through the consumption of contaminated produce.
Applied and Environmental Microbiology, Jul 1, 1979
This study was designed to determine the degree of adsorption of enteric viruses to marine sedime... more This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.
Municipal sewage sludge is a complex mixture of organic and inorganic compounds of biological and... more Municipal sewage sludge is a complex mixture of organic and inorganic compounds of biological and mineral origin that are removed from waste-
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