Grantee Research Project Results
Final Report: PCR Based Detection of Cytopathogenic and Non-Cytopathogenic Viruses in Water
EPA Grant Number: R824756Title: PCR Based Detection of Cytopathogenic and Non-Cytopathogenic Viruses in Water
Investigators: Pepper, Ian L. , Reynolds, Kelly A. , Gerba, Charles P.
Institution: University of Arizona
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 1, 1997
Project Amount: $227,258
RFA: Human Health Risk Assessment (1995) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
Drinking water outbreaks continue to occur in the United States even when finished waters meet all regulated water quality parameters. The World Health Organization reports that 16.4 million deaths worldwide were due to infectious and parasitic diseases in 1993. Estimates of up to 80 percent of these are thought to be linked to contaminated water, equaling over 35,000 deaths a day. The overall aim of this project was to develop and apply advanced molecular biotechnology for the routine detection of infectious human viruses in environmental samples. Through support from the USEPA, our laboratory has developed the use of an integrated cell culture/PCR (ICC/PCR) methodology for rapid, sensitive, and specific detection of several types of human viruses that are either difficult or impossible to detect using conventional cell culture analysis alone. Methods were developed to detect noncytopathogenic viruses, those unable to produce visible infection and death to laboratory cell cultures, i.e. rotavirus and hepatitis A virus. Viruses that infect and replicate in host cells but are undetectable by cell culture analysis alone are of special concern since conventional survey of such populations may lead to false negative results.Although harmful human viruses have been previously documented in recreational, raw, and treated drinking water, and are known to pose a public health risk even in low numbers, little information is known regarding their presence and true illness impact. This is, in part, due to the fact that conventional cultural methodology is problematic, requiring long incubation times for determination of viability. Cell culture on environmental samples is further complicated by the presence of organic and inorganic materials, that are toxic to the cell. The standard cell culture method, for the detection of human pathogenic viruses in concentrated environmental samples, is expensive (ranging from $500-$2000 per sample) and time-consuming (requiring two weeks or more for confirmed results). Simpler methods of bacterial testing are often used to monitor overall water quality, however, bacterial indicators have proven ineffective for predicting the risk of viral infections. Indeed, direct surveillance of viruses in the population and environment is much needed, remembering that 90% of human viruses known today were completely unknown just 6 decades ago.
An alternative method for the detection of viruses in environmental samples is the polymerase chain reaction (PCR), an in-vitro, enzymatic method for amplifying target nucleic acids using specific oligonucleotide primers. Repeated cycles of PCR provide a 106-fold amplification of a single copy of target DNA within a few hours. The decreased time and cost, and increased sensitivity, allow the detection of low numbers of target DNA and RNA, however one cannot distinguish between amplification of infectious versus noninfectious viral sequences. In addition, PCR is complicated by the presence of inhibitory compounds and low equivalent sample volumes.
This study focused on a new concept for virus detection utilizing a biological amplification step followed by an enzymatic amplification step. This strategy combines both cultural and molecular techniques for rapid detection of viable human viruses, in large equivalent volume concentrates, without the limitations of toxicity or inhibition. The combined methodology provides the first reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens posing a significant threat to public health. ICC/PCR allows for detection of infectious viruses in days compared to the weeks necessary with cell culture alone. Another advantage to ICC/PCR is that no new method has been created, rather two, well known techniques are now being combined in a new way, enhancing the known strengths, while eliminating the known weaknesses of each method.
Recent experiments, using ICC/PCR and subsequent nucleic acid sequencing, showed that direct PCR and conventional cell culture alone yielded erroneous results with respect to viable virus presence in environmental and disinfected virus samples. Therefore, application of the ICC/PCR methodology has raised new questions as to the effectiveness of chemical disinfection and the conventional methods used to determine the elimination of viable viruses in disinfected waters. ICC/PCR enabled detection of viable viruses in cell culture lysates, following chlorine disinfection, that were not detectable with single passage cell culture analysis alone, suggesting the presence of chlorine resistant virus populations. The need remains to further evaluate the presence of these elusive virus populations using ICC/PCR methodology.
Specific project objectives included: 1) Optimization of ICC/PCR for routine monitoring of low numbers of infectious enteric viruses in water as an alternative to cell culture or PCR analysis. 2) Development of a method to overcome the problem of cell culture toxicity in environmental samples. 3) Development of a method to increase the equivalent volume examined by RT-PCR while avoiding an increase in inhibitory substances. 4) Determination of the effect of chlorine disinfection on virus detection by conventional culture, direct PCR, and ICC/PCR. 5) Comparison of the current RT-PCR and cell culture methodologies to the integrated cell culture/PCR methodology for detection of infectious enteroviruses in surface, groundwater and marine water samples. 6) Evaluation of multiple cell lines (BGM, FRhK, and MA104) for integrated cell culture/PCR detection of cytopathogenic and noncytopathogenic virus.
Summary/Accomplishments (Outputs/Outcomes):
Rapid Virus Detection. The ICC/PCR methodology was much more rapid at detecting viable virus than conventional cell culture alone. With the desired detection limit of ICC/PCR defined at the conventional cell culture standard of approximately 1 pfu/flask, a poliovirus concentration of 2.8 pfu/flask was detected in 24 hours with ICC/PCR compared to 72 hours with conventional cell culture alone. This relationship was also observed at higher concentrations of poliovirus (28.0 pfu/flask). Theoretical virus concentrations below the conventional cell culture limit of 1 pfu/flask (0.28 pfu/flask), were detected by ICC/PCR within 24 hours compared to >14 days, or a secondary passage, with conventional cell culture alone. In fact, conventional cell culture sometimes required three passages (up to 6 weeks) for detection of viable viruses. Replicate flasks, used to sample total volumes greater than 1 ml, and longer incubation times aid in improving the detection efficiency to greater than 10-fold the detection sensitivity of conventional cell culture. Therefore, using multiple replicate flasks, virus levels of <1.0 pfu are detectable by ICC/PCR.Likewise, ICC/PCR detects HAV earlier than conventional cell culture at the target level of 1 pfu/flask, requiring only 3 days versus 14 days, respectively. Since the virus was noncytopathogenic, direct observation of virus infection of the cell monolayer was very difficult to detect. Only through the use of purified laboratory stocks, and well trained cell culture technicians, could the slight morphological changes be noted. Such infections from environmental sample concentrates would not be distinguishable from effects of environmental compounds naturally present. At higher concentrations of HAV (400 and 40 pfu/flask), ICC/PCR was 5 days faster than conventional cell culture, detecting viable HAV after only 72 hours of incubation.
The ICC/PCR methodology was also applied to a cytopathogenic and a noncytopathogenic strain of rotavirus, WA2 and SA11 respectively. Although, plaques could not be detected on MA104 continuous cell lines, a detection sensitivity of 0.28 plaque forming viruses was estimated, based on ATCC titers. Low concentrations (<1 infectious unit) of rotavirus WA2 were variably detected using ICC/PCR on days one to five of the cell culture assay. Longer incubation times yielded more consistent results in repeated studies and thus, a three day assay with 3-5 replicate flasks is recommended. Cytopathogenic strains of rotavirus, i.e. SA11, were found to be positive sooner than noncytopathogenic strains (<1 pfu in <24 h).
Data from this study shows that positive ICC/PCR detection is a function of: 1) initial virus inoculum; 2) incubation time; and 3) replicate flasks, at low concentration and/or short incubation times. Higher concentrations of poliovirus (>10 pfu/flask) may be detected in seven out of eight replicate flasks after only 5 hours of incubation. For 100% positive detection, at a poliovirus concentration of >10 pfu/flask, a minimum of 10 hours is required. For the target concentration of 1 pfu/flask inoculum, >20 hours is recommended. Similarly, higher levels of HAV (100 pfu/flask), may be detected as soon as 12 hours post-incubation but this fastidious virus requires a minimum of 2-3 replicate flasks to effectively evaluate it's presence or absence. Infectious HAV levels of 1 and 10 pfu/flask are detectable after 48 and 72 hours and require 2-3 replicate flasks, respectively. Therefore, for a given virus and a desired detection limit, one must evaluate the minimum incubation time and equivalent volume required to ensure reliable results using ICC/PCR.
Overcoming False Negative Results. Further evidence suggests that samples which were determined negative by conventional cell culture may not actually be negative for infectious viruses. In several instances, ICC/PCR was positive and conventional cell culture was negative until a third passage, approaching six weeks of incubation, was applied. Therefore the potential exists for a false negative environmental sample result based on conventional cell culture analysis. ICC/PCR overcame this false negative phenomenon. This condition was also repeatedly observed with laboratory studies using chlorine disinfected virus stocks. Viruses exposed to 0.5 mg/L of free chlorine were determined to be inactivated after only 2 minutes based on conventional cell culture assay with 2 weeks of incubation. Following 5 minutes of exposure to the disinfectant, ICC/PCR detected viable viruses in only 24 hours. Increased cell culture passage and incubation times beyond the initial 14 day assay did indeed confirm that viable viruses were still present.
In addition, sewage samples were collected and concentrated from 20 L into 30 ml volumes. The concentrates were diluted in 10-fold dilution series and examined by conventional cell culture, direct PCR, and ICC/PCR at various days of incubation. After three successive 10-fold dilutions, no viruses were detected by any method, but at the second 10-fold dilution, ICC/PCR positives were evident after five days of incubation, relative to a secondary passage, and >14 days with conventional cell culture alone. The most dramatic difference was seen with direct analysis of the sewage concentrate with ICC/PCR positives after 24 hours, compared to 10 days with conventional cell culture. Samples were falsely determined negative, at all dilutions, by direct PCR analysis despite the presence of viable viruses. False negative results were also seen with marine water concentrates harboring low levels of virus, due to inhibitory compounds and low equivalent sample volumes.
Overcoming False Positive ICC/PCR Results. Throughout this study, all replicate flasks were reported to be negative at time zero of incubation when tested using ICC/PCR. The time zero data point is an assurance of viable virus detection. For the time zero replicates, samples are placed on the cell monolayer and incubated for 1 hour to allow virus attachment to the cells. After the cells are covered with maintenance media, the assay is stopped, prior to production of progeny virus. Therefore, if all replicate flasks are negative at time zero and positive at time one, or greater, virus growth was necessary for positive ICC/PCR results, indicating the detection of viable viruses only. Our research shows that virus concentrations must be >28,000 pfu per flask inoculum to initiate an ICC/PCR positive result without the prerequisite growth phase. Since virus concentrations in raw sewage concentrates only approach 100-1000 pfu per flask inoculum, it is unlikely that any environmental sample would reach the maximum concentration of >28,000 pfu per flask of noninfectious virus that would lead to a false positive result.
Many water sample concentrates were tested from various environments throughout the course of this study, including stormwater run-off, ground water, surface water, sewage, sludge amended soil, and recreational marine water. In all environments, ICC/PCR proved to be a reliable method for determining the presence/absence of harmful human viruses and correlated with conventional cell culture, ultimately. Cell culture analysis alone was frequently complicated by toxic compounds naturally found in the sample concentrates. Fungal and bacterial populations in concentrated environmental samples were especially problematic, requiring repeat analysis and additional purification steps to isolate viruses alone. Such purification methodology leads to increased loss of virus concentration and limits the sensitivity of the cultural assay. ICC/PCR never required additional purification methodologies and therefore maximized the detection of virus populations.
Conclusions:
The ICC/PCR methodology allows for detection of infectious viruses in hours to days compared to the days to weeks necessary with cell culture alone, overcoming the major flaws of cell culture and direct PCR methodologies. Another advantage to ICC/PCR is that no new method has been created, rather two, well used techniques are now being combined in a new way, enhancing the known strengths, while eliminating the known weaknesses of each method.ICC/PCR capitalizes on the advantages of cell culture's large equivalent volume infectivity assay and the rapid, sensitive and specific nature of direct PCR, while overcoming each method's limitations of inhibition and toxicity. Inhibition is inherently overcome by diluting the inhibitory compounds with cell culture assay media while growing up the virus population present prior to PCR amplification. Therefore, a large population of viruses is being detected with low concentrations of inhibitors, eliminating the uncertainty of true versus false negative results with direct PCR alone. Toxic effects are minimized since the cultural assay can be stopped and viruses detected prior to cell death due to toxicity, eliminating the uncertainty of true versus false positive results with conventional cell culture alone. Cost of ICC/PCR analysis is difficult to evaluate since it is relatively inexpensive compared to conventional cell culture while relatively expensive compared to direct PCR. However, ICC/PCR is the best available method for rapid detection of viable noncytopathogenic and cytopathogenic viruses.
What does this new technology mean for environmental health, service, and regulatory agencies in the future? It demonstrates that a reliable method now exists for routine analysis of viruses in the environment. Previously, bacterial indicator organisms have been used to determine water quality with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method has not been available for direct virus testing, until the advent of ICC/PCR.
With improved, viable virus detection sensitivity and reduced assay times (ICC/PCR requires 24-72 hours versus 5-14 days, or more, with conventional cell culture), ICC/PCR is the future for effective environmental virus monitoring. Even with samples that are suitable for direct PCR amplification monitoring, having low inhibitory compounds and sufficiently high levels of target organisms, subsequent use of ICC/PCR would be useful to evaluate the viable nature of the target, with minimal cost and time involvement.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 19 publications | 3 publications in selected types | All 2 journal articles |
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Type | Citation | ||
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Reynolds KA, Gerba CP, Abbaszadegan M, Pepper IL. ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples. Canadian Journal of Microbiology 2001;47(2):153-157. doi:10.1139/CJM-47-2-153 |
R824756 (Final) |
not available |
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Reynolds KS, Gerba CP, Pepper IL. Rapid PCR-based monitoring of infectious enteroviruses in drinking water. Water Science and Technology, Volume 35, Issues 11-12, 1997, Pages 423-427. |
R824756 (Final) |
not available |
Supplemental Keywords:
water quality, virology, human pathogens., RFA, Health, Scientific Discipline, Water, Health Risk Assessment, Risk Assessments, Ecology and Ecosystems, Drinking Water, ecological risk assessment, enteric virus, waterborne disease, rapid detection, PCR, infectious organisms, microbial risk management, environmental sewage, cell cultureRelevant Websites:
Methodology soon to be included in the "MethodsFinder" database, Biosis, Philadelphia, PA (www.MethodsFinder.org ).Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.