Evaluating Fecal Indicator and Pathogen Relationships in Sewage Impacted Surface Waters to Blend with Reclaimed Water for Potable Reuse in North Carolina.

Surface waters used for drinking water supply often receive upstream wastewater effluent inputs, resulting in de facto wastewater reuse for drinking water and recreation. As populations grow, demands on water supplies increase. As this trend continues, it creates the need to understand the risks associated with such reuse. In North Carolina, potable reuse has been proposed as a combination of at least 80% surface water with up to 20% tertiary-treated, dual-disinfected, reclaimed wastewater, which is then stored for 5 days and further treated using conventional drinking water treatment methods. The state of North Carolina has set standards for both intake surface water and for the reclaimed water produced by wastewater utilities, using indicator microorganisms to measure compliance. The goal of this study was to quantify fecal indicator microorganisms, specifically E. coli, coliphages, and C. perfringens as well as key pathogens, specifically Salmonella spp. bacteria, adenoviruses, noroviruses, and the protozoan parasites Cryptosporidium and Giardia, in two types of water representing potential candidates for potable reuse in North Carolina, (1) run of river surface water and (2) sewage-impacted surface waters, with the purpose of determining if there are predictive relationships between these two microorganism groups that support microbial indicator reliability.

UV Inactivation of SARS-CoV-2 across the UVC Spectrum: KrCl* Excimer, Mercury-Vapor, and Light-Emitting-Diode (LED) Sources.

Effective disinfection technology to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can help reduce viral transmission during the ongoing COVID-19 global pandemic and in the future. UV devices emitting UVC irradiation (200 to 280 nm) have proven to be effective for virus disinfection, but limited information is available for SARS-CoV-2 due to the safety requirements of testing, which is limited to biosafety level 3 (BSL3) laboratories. In this study, inactivation of SARS-CoV-2 in thin-film buffered aqueous solution (pH 7.4) was determined across UVC irradiation wavelengths of 222 to 282 nm from krypton chloride (KrCl*) excimers, a low-pressure mercury-vapor lamp, and two UVC light-emitting diodes. Our results show that all tested UVC devices can effectively inactivate SARS-CoV-2, among which the KrCl* excimer had the best disinfection performance (i.e., highest inactivation rate). The inactivation rate constants of SARS-CoV-2 across wavelengths are similar to those for murine hepatitis virus (MHV) from our previous investigation, suggesting that MHV can serve as a reliable surrogate of SARS-CoV-2 with a lower BSL requirement (BSL2) during UV disinfection tests. This study provides fundamental information on UVC's action on SARS-CoV-2 and guidance for achieving reliable disinfection performance with UVC devices. IMPORTANCE UV light is an effective tool to help stem the spread of respiratory viruses and protect public health in commercial, public, transportation, and health care settings. For effective use of UV, there is a need to determine the efficiency of different UV wavelengths in killing pathogens, specifically SARS-CoV-2, to support efforts to control the ongoing COVID-19 global pandemic and future coronavirus-caused respiratory virus pandemics. We found that SARS-CoV-2 can be inactivated effectively using a broad range of UVC wavelengths, and 222 nm provided the best disinfection performance. Interestingly, 222-nm irradiation has been found to be safe for human exposure up to thresholds that are beyond those effective for inactivating viruses. Therefore, applying UV light from KrCl* excimers in public spaces can effectively help reduce viral aerosol or surface-based transmissions.

Rapid Detection of Escherichia coli in Water Using Sample Concentration and Optimized Enzymatic Hydrolysis of Chromogenic Substrates.

Methods for rapid detection of fecal indicator bacteria in water are important to ensure that water is safe for drinking, bathing, recreation, fishing and shellfish harvesting. In this study, we tested experimental conditions for bacterial hydrolysis of two promising enzymatic substrates, 5-Bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc) and Resorufin ß-D-glucuronide (REG), and optimized parameters such as temperature and pH to determine conditions for rapid reactions. We then innovated a membrane filter-based approach to facilitate more rapid enzyme-based detection of Escherichia coli in water based on the combination of an initial concentration step and optimized test conditions. For this approach, a water sample (10‒100 mL) is filtered through a 0.45-µm pore size filter with a diameter of 4 or 13 mm. After filtration, a newly designed rapid detection broth is added containing the enzymatic inducer Methyl-beta-D-Glucuronide sodium (MetGlu) and the substrate REG or X-Gluc. After a few (1‒7) hours of incubation at 35 °C, the filter shows pink color (for REG-containing broth) or green color (for X-Gluc containing broth) if E. coli is present. The study provides insights and approaches towards developing a simple, fast, and low-cost method to detect fecal indicator bacteria in water.

Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples.

Human enteric adenoviruses (HAdVs) are commonly detected in waters contaminated with human fecal material and persistent in the environment. Detecting infectious enteric HAdVs is limited by the difficulty of growing them in cell cultures. Recently, an improved cell line (293 CMV) has been described, which enhanced the propagation of enteric HAdVs (Kim et al., 2010. Appl. Environ. Microbiol. 76, 2509-2516). The present study evaluated the transactivated 293 CMV cell line for detecting enteric HAdVs from field samples, which is an important step in demonstrating the usefulness of the improved cell line for water monitoring programs. Field samples consisted of the following: concentrated sewage samples (from 1L) collected from three different wastewater treatment plants (WWTPs) and concentrated raw source water samples (from 20L) collected from six water treatment plants (WTPs). Infectious HAdVs were detected using a combined cell culture/mRNA RT-PCR assay. Concentrated samples were assayed, in parallel, using the standard (STD) G293 and 293 CMV cell lines. Viral replication was determined by measuring viral mRNA and viral DNA levels during infection. Infectious HAdVs were successfully detected from environmental samples using the new transactivated and standard cell lines. Infectivity assays of concentrated sewage samples demonstrated higher viral mRNA expression (p=0.02) and viral DNA concentrations (p=0.02) in the transactivated 293 CMV than in the G293 cell line. Although not statistically significant, infectious HAdVs were detected in more raw water samples using the 293 CMV cells (8 of 18) than in the STD G293 cells (4 of 18). However, when results of the source water samples were pooled, the number of flasks positive using the 293 CMV cells was significantly greater than those using the G293 cells (p=0.01). Overall, the results of the present study demonstrate the effectiveness of the new transactivated 293 CMV cell line for improved propagation and detection of HAdVs from environmental samples.

Human viruses and viral indicators in marine water at two recreational beaches in Southern California, USA.

Waterborne enteric viruses may pose disease risks to bather health but occurrence of these viruses has been difficult to characterize at recreational beaches. The aim of this study was to evaluate water for human virus occurrence at two Southern California recreational beaches with a history of beach closures. Human enteric viruses (adenovirus and norovirus) and viral indicators (F+ and somatic coliphages) were measured in water samples over a 4-month period from Avalon Beach, Catalina Island (n = 324) and Doheny Beach, Orange County (n = 112). Human viruses were concentrated from 40 L samples and detected by nested reverse transcriptase polymerase chain reaction (PCR). Detection frequencies at Doheny Beach were 25.5% (adenovirus) and 22.3% (norovirus), and at Avalon Beach were 9.3% (adenovirus) and 0.7% (norovirus). Positive associations between adenoviruses and fecal coliforms were observed at Doheny (p = 0.02) and Avalon (p = 0.01) Beaches. Human viruses were present at both beaches at higher frequencies than previously detected in the region, suggesting that the virus detection methods presented here may better measure potential health risks to bathers. These virus recovery, concentration, and molecular detection methods are advancing practices so that analysis of enteric viruses can become more effective and routine for recreational water quality monitoring.

Persistence of viral genomes after autoclaving.

The ability of autoclaving to degrade viral genomes was investigated by real-time PCR and real-time reverse-transcription (RT)-PCR. Several factors were considered: the nucleic acid composition of the virus (DNA or RNA), hydration state of the sample, and the duration of autoclaving. Viral genomes were damaged more easily under hydrated conditions compared to dry conditions. The genomes of RNA viruses, such as MS2 and norovirus degraded more readily than DNA virus (adenovirus). MS2 genome was the most vulnerable among those tested, with no amplification observed after 18min of autoclaving. Adenovirus genomes, on the other hand, were detected after autoclaving for 36min under hydrated or dry conditions. For norovirus, 18min of autoclaving under hydrated condition or 36min under dry conditions was enough to destroy noroviral genomes. For noroviral samples, 1.1% of noroviral gene segments were remained after autoclaving for 18min under dry conditions; however, when a two-step approach was used for the RT-PCR reaction with priming at the poly-A tail about 2552bp from the qPCR amplification site, the gene segment was not amplified after autoclaving for 18min. Thus, norovirus amplification observed after 18min of autoclaving in the dry sample is likely from less than full length genomic segments of norovirus RNA remaining in the sample.

Integrating quantitative PCR and Bayesian statistics in quantifying human adenoviruses in small volumes of source water.

Rapid quantification of viral pathogens in drinking and recreational water can help reduce waterborne disease risks. For this purpose, samples in small volume (e.g. 1L) are favored because of the convenience of collection, transportation and processing. However, the results of viral analysis are often subject to uncertainty. To overcome this limitation, we propose an approach that integrates Bayesian statistics, efficient concentration methods, and quantitative PCR (qPCR) to quantify viral pathogens in water. Using this approach, we quantified human adenoviruses (HAdVs) in eighteen samples of source water collected from six drinking water treatment plants. HAdVs were found in seven samples. In the other eleven samples, HAdVs were not detected by qPCR, but might have existed based on Bayesian inference. Our integrated approach that quantifies uncertainty provides a better understanding than conventional assessments of potential risks to public health, particularly in cases when pathogens may present a threat but cannot be detected by traditional methods.

An improved infectivity assay combining cell culture with real-time PCR for rapid quantification of human adenoviruses 41 and semi-quantification of human adenovirus in sewage.

A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 10(7) to 10(1) infectious units per assay (R(2) = 0.97) in samples analyzed 3-5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures (p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 10(2) to 10(3) mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10-20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples.

Uncertainty analysis of the recovery of hollow-fiber ultrafiltration for multiple microbe classes from water: a Bayesian approach.

In this study, we introduce a Bayesian approach to address uncertainty of microbial recoveries from hollow-fiber ultrafilters (HFUF) and to determine any sources of uncertainty. Microbial recoveries were measured under twenty conditions, including two types of water, two types of ultrafilters, and five types of microorganisms. The probability distributions of the recoveries were approximated using Bayesian statistics with Markov chain Monte Carlo sampling after integrating the likelihood function of the recovery data and prior information about the data. Then a variance-decomposition method was used for examining influential factors on microbial recovery by HFUF. The results revealed that HFUF efficiently recovered Escherichia coli KO11, E. coli O157:H7 and bacteriophage MS2, but recoveries for Bacillus atrophaeus spores and adenovirus 41 were markedly different between source and treated waters. The uncertainty analysis indicated that the probability distributions for recoveries had dissimilar patterns under different conditions. Among these test factors, the type of microorganisms and associated interaction effects had great impacts on the recovery. To sum up, the Bayesian approach to uncertainty analysis shows advantages in evaluating the recovery of HFUF by providing its full probability distribution.

Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters.

Quantitative real-time PCR (qPCR) is used commonly to detect adenovirus (Ads) and norovirus (Nvs) in recreational waters. However, qPCR detection may be limited by interference from inhibitory substances found in recreational waters. In previous work, viruses in Avalon and Doheny Beach water samples were concentrated by electropositive cartridge filtration and PEG precipitation, and high inhibition was found in the samples when using qPCR for detection of Ads and Nvs. Therefore, different approaches were evaluated for removal or blocking of inhibitory compounds that affect qPCR. Avalon and Doheny concentrates were spiked with known amounts of Ads 41 and Nvs GII, and spiked deionized water was used as a positive control. Modifications included gel chromatography with columns of Sephadex G-200/Chelex 100, different sample volumes for nucleic acid extraction, organic solvent extraction, and nucleic acid precipitation. The efficiency of each treatment varied according to sampling location and virus type. The best option for improved Nvs detection by reverse transcription-qPCR was to reduce the sample volume for nucleic acid purification. The best option for improving Ads detection in both beach samples was Sephadex/Chelex spin column chromatography. Chloroform extraction only improved virus detection in Doheny Beach samples but not in Avalon Beach samples. Observed differences in effective treatments between viruses may be related to the different PCR targets, amplification conditions, and enzymes used in each assay, and differences between beaches may be related to differences in PCR inhibitory environmental compounds at each location. The results suggest that methods for detecting viruses from marine beaches, including treatments for the removal of PCR inhibitory compounds, should be optimized for each sampling site and probably for each virus of interest.

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

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

Simultaneous comparison of murine norovirus, feline calicivirus, coliphage MS2, and GII.4 norovirus to evaluate the efficacy of sodium hypochlorite against human norovirus on a fecally soiled stainless steel surface.

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.

Endotoxin levels at Swine farms using different waste treatment and management technologies.

Concentrated animal feeding operations (CAFOs) are a major source of airborne endotoxins, which are air pollutants that can cause adverse health effects to both on-site farmers and neighbors. Release of airborne endotoxins to the environment can be reduced using proper waste treatment and management technologies. In this study, the levels of endotoxins released from two swine CAFOs using conventional lagoon-sprayfield technology were compared to those of 15 farms using various alternative waste management technologies in North Carolina. Over a 2-year period, 236 endotoxin samples were collected from the 17 farm units and analyzed using the Limulus amebocyte lysate (LAL) test. Concentrations of airborne endotoxins near barn exhaust fans were significantly higher than at the upwind boundary of the farm and at other farm sites. For most of the study sites, mean concentrations of endotoxins at the downwind boundary of the farm were higher than those at the upwind boundary of the farm, indicating the release of endotoxins from swine CAFOs to the neighboring environment. Endotoxin levels were significantly associated with concentrations of airborne bacteria but not fungi. Environmental factors, such as temperature, relative humidity, and wind velocity, affected the levels of airborne endotoxins at the farms. Based on the ratios of airborne endotoxins in downwind and upwind samples from the farm units, at least five different alternative waste management technologies significantly reduced the release of endotoxins from swine CAFOs. These results suggest that swine CAFOs are important sources of airborne endotoxins, the levels of which can be reduced by applying more robust and effective waste management technologies.

Efficacy of hospital germicides against adenovirus 8, a common cause of epidemic keratoconjunctivitis in health care facilities.

The inactivation of virus-contaminated nonporous inanimate surfaces was investigated using adenovirus type 8, a common cause of epidemic keratoconjunctivitis. A 10-microl inoculum of adenovirus was placed onto each stainless steel disk (1-cm diameter), and the inoculum was allowed to air dry for 40 min. Twenty-one different germicides (including disinfectants and antiseptics) were selected for this study based on their current uses in health care. After a 1- or 5-minute exposure to 50 microl of the germicide, the virus-germicide test mixture was neutralized and assayed for infectivity. Using an efficacy criterion of a 3-log10 reduction in the titer of virus infectivity and regardless of the virus suspending medium (i.e., hard water, sterile water, and hard water with 5% fetal calf serum), only five disinfectants proved to be effective against the test virus at 1 min: 0.55% ortho-phthalaldehyde, 2.4% glutaraldehyde, 2.65% glutaraldehyde, approximately 6,000 ppm chlorine, and approximately 1,900 ppm chlorine. Four other disinfectants showed effectiveness under four of the five testing conditions: 70% ethanol, 65% ethanol with 0.63% quaternary ammonium compound, 79.6% ethanol with 0.1% quaternary ammonium compound, and 0.2% peracetic acid. Of the germicides suitable for use as an antiseptic, 70% ethanol achieved a 3-log10 reduction under four of the five test conditions. These results emphasize the need for proper selection of germicides for use in disinfecting noncritical surfaces and semicritical medical devices, such as applanation tonometers, in order to prevent outbreaks of epidemic keratoconjunctivitis.

UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR.

Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation. Adenovirus is a double-stranded DNA virus and produces messenger RNA (mRNA) during replication in host cells. The presence of viral mRNA in host cells is definitive evidence of infection. We recently developed a rapid and reliable cell culture-mRNA RT-PCR assay to detect and quantify adenovirus infectivity. Viral mRNA recovered from cell cultures 5-7 days after infection was purified on oligo-dT latex, treated with DNase, and amplified by RT-PCR using the primers specific for a conserved region of the hexon late mRNA transcript. Treatment of approximately 10(4) Ad41 with different doses of 254 nm germicidal UV radiation resulted in a dose-dependent loss of infectivity. As UV doses were increased from 75 to 200 mJ/cm2, virus survival decreased and no virus infectivity (measured by detectable mRNA) was found at a dose of 225 mJ/cm2 or higher. Our results using the cell culture mRNA RT-PCR assay indicate that Ad41 is more resistant to UV radiation than in a previous study using a conventional cell culture infectivity assay. Results were more similar to those found for Ad 40 using CPE as a measure of infectivity in another previous study.

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41.

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.

Rapid detection of infectious adenoviruses by mRNA real-time RT-PCR.

Adenoviruses are among the most persistent and ubiquitous viruses in water and wastewater, but some of them are difficult to detect due to non-cytopathogenicity and slow growth in cell cultures. A TaqMan real-time RT-PCR method in conjunction with cell culture infectivity was developed to rapidly detect mRNA produced by infectious adenoviruses in water samples. Only infectious adenoviruses were detected by this method, based on their ability to produce mRNA during replication in cell culture. The mRNA of Ad41 was detected as soon as 3 days after infection at levels as low as 5 infectious units (IU) per cell culture. In order to confirm that our methods detected only infectious viruses, 1-ml volumes of 10(4)IU of Ad41 were exposed to different free chlorine doses. No mRNA was detected in cells inoculated with Ad41 treated with the highest free chlorine dose of 100 mg min/l. However, mRNA of adenovirus was detected in cells inoculated with virus that was untreated or exposed to a lower free chlorine dose of 1 mg min/l. These results suggest that mRNA detection by real-time RT-PCR is a sensitive and specific method to detect low levels of infectious adenoviruses in water and other environmental media within 1-3 days.

Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR.

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.

Use of viral pathogens and indicators to differentiate between human and non-human fecal contamination in a microbial source tracking comparison study.

Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'.

UV disinfection of Giardia lamblia cysts in water.

The human and animal pathogen Giardia lamblia is a waterborne risk to public health because the cysts are ubiquitous and persistent in water and wastewater, not completely removed by physical-chemical treatment processes, and relatively resistant to chemical disinfection. Given the recently recognized efficacy of UV irradiation against Cryptosporidium parvum oocysts, the inactivation of G. lamblia cysts in buffered saline water at pH 7.3 and room temperature by near monochromatic (254 nm) UV irradiation from low-pressure mercury vapor lamps was determined using a "collimated beam" exposure system. Reduction of G. lamblia infectivity for gerbils was very rapid and extensive, reaching a detection limit of >4 log within a dose of 10 JM-2. The ability of UV-irradiated G. lamblia cysts to repair UV-induced damage following typical drinking water and wastewater doses of 160 and 400 JM(-2) was also investigated using experimental protocols typical for bacterial and eucaryotic DNA repair under both light and dark conditions. The infectivity reduction of G. lamblia cysts at these UV doses remained unchanged after exposure to repair conditions. Therefore, no phenotypic evidence of either light or dark repair of DNA damage caused by LP UV irradiation of cysts was observed at the UV doses tested. We conclude that UV disinfection at practical doses achieves appreciable (much greater than 4 log) inactivation of G. lamblia cysts in water with no evidence of DNA repair leading to infectivity reactivation.