Grantee Research Project Results
Final Report: Detection and Occurrence of Human Caliciviruses in Drinking Water
EPA Grant Number: R826837Title: Detection and Occurrence of Human Caliciviruses in Drinking Water
Investigators: Sobsey, Mark D.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1999 through December 31, 2000
Project Amount: $296,980
RFA: Drinking Water (1998) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
Norwalklike viruses (NLVs), or Noroviruses as they are now called, are important agents of waterborne disease in the United States and elsewhere. They are on the U.S. Environmental Protection Agency (EPA) Contaminant Candidate List (CCL) for drinking water. Because inadequate information is known about NLVs in water, including their presence and control in drinking water supplies, effective methods are needed for their detection. This project was directed at the development of improved methods to detect NLVs in water. The general objectives of this research project were to: (1) develop improved methods to recover, concentrate, and purify NLV caliciviruses of both genogroups from water; (2) develop new and improved reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe (OP) materials and methods to amplify and detect the recovered, concentrated, and purified NLVs; and (3) further evaluate these methods by applying them to the detection of field NLVs in environmental sewage and water samples, and thereby determine NLVs occurrence in representative samples of sewage, and raw and finished waters from both surface and ground sources.
Summary/Accomplishments (Outputs/Outcomes):
Substantial achievements were accomplished to meet the goals and objectives of this study in developing new and improved methods to recover and detect NLVs in water. Improved methods were developed for recovery of NLVs in water employing filter adsorption-elution methods, followed by further concentration using polyethylene glycol (PEG) precipitation. The resulting chlorinated polyethylene (PEC) precipitates were then resuspended in a small fluid volume for RNA extraction and detection by RT-PCR. New and improved methods were developed to extract NLV RNA from water sample concentrates and detect the extracted NLV RNA by conventional and real-time RT-PCR. For conventional RT-PCR, improved primers and RT-PCR amplification conditions were developed for the widely used polymerase regions of the NLV genome, and a new and “generic” internal control template was developed for inclusion in RT-PCR reactions to monitor for RT-PCR amplification efficiency and possible inhibition of amplification. In addition, new primers and conditions were developed for rapid and sensitive real-time RT-PCR amplification and detection of NLVs. We applied the new and improved methods for RT-PCR amplification to dilute stool (fecal) samples of genetically different NLVs and to samples of water seeded with the prototype Norwalk Virus (NV) (strain 8FIIa). The new and improved methods were found to be effective, as further detailed below. However, due to lack of time and resources, these new methods were not applied to field samples of water as originally proposed. Attempts were made by an empirical approach to culture NV in cell cultures using several different cell lines and culture conditions. Based on quantitation of inoculated cell culture by RT-PCR, there was no evidence of successful cultivation of NV.
Improved Methods to Recover, Concentrate, and Purify NV From Water. Building on the results of previous studies, it was shown that NV in seeded tap water and surface efficiently adsorbed to Virosorb 1MDS electropositive adsorbent filters (the EPA-approved filter for virus concentration from water) at pH levels of 8.5 or lower. Because the standard beef extract solution used to elute adsorbed viruses from these filters can interfere with virus detection by RT-PCR amplification, alternative chemically defined elution solutions containing 0.1 to 0.5M amino acids with or without low (0.01 to 0.1 percent) concentrations of nonionic detergents were evaluated for elution of NV adsorbed to electropositive 1MDS filters. The results of this study indicate that a simple, well-defined elution solution composed of 0.5 or 0.25 M L-lysine, and the detergent Triton X-100, at 0.1 percent concentration was an effective alternative to elution solutions containing beef extract. No significant differences in NV recovery were measured between the lysine- and beef extract-based elution solution when virus RNA was heat-released and RT-PCR amplified from eluent concentrates of tap water experiments. To detect low levels of viruses in water, viruses in adsorbent filter eluates have to be further concentrated to smaller sample volumes prior to analysis.
Viruses in lysine-Triton X-100 and beef extract eluates were further concentrated by precipitation with 8 percent PEG after adjusting to pH 7.2, adding 0.3N NaCl and holding at 4°C for several hours or overnight. Resulting PEG precipitates were extracted for NV RNA, which was then RT-PCR amplified. This PEG precipitation method gave high (>75 percent) recovery efficiencies of NVs in adsorbent filter eluates. When this improved filter adsorption-elution and PEG precipitation method was tested in replicate experiments using tap water seeded with approximately 103 NVs, the lysine-based eluent was found to yield significantly greater recoveries of NVs than 3 percent beef extract, 0.05M glycine (pH 9.5). Average recoveries were 66 percent with lysine-Triton X-100 elution and 15 percent with beef extract elution. Data from replicate filtration-elution experiments with seeded surface water also indicated that the lysine-based elution solution achieved similar or greater recoveries of NVs compared to the beef extract-based elution solution. Average recoveries were 32 percent using lysine-Triton X-100 for elution and 24 percent using beef extract for elution. However, it was noted that inhibition of RT-PCR was observed as false negatives for experiments, where the 0.5M lysine-Triton X-100 elution solution was used and assayed directly. Use of a lower molarity of lysine (0.25M) for elution was found to minimize this inhibition, while also achieving similar or higher NV recoveries versus beef extract eluents.
New and Improved Methods to Extract and Recover Viral RNA and Remove Sample-Related Inhibitors. Efficient and sensitive RT-PCR for NLVs and other viruses requires the removal or inactivation of inhibitors present in sample concentrates. Initially, we compared NLV recovery by three different RNA extraction methods: (1) heat release; (2) lysis, followed by precipitation of viral RNA (UltraSpec); and (3) microspin columns (QIAamp viral RNA mini a kit, Qiagen). The extraction methods were applied to a panel of stool samples positive for NLVs by RT-PCR using the "Ando" GI and GII primers or the RIVM (National Institute of Public Health and the Environment, Bilthoven, NL) JV12/13 primers. The NLV panel consisted of 7 genogroup I strains and 17 genogroup II strains, including the prototype strains of NV (8FIIa) and Snow Mountain Virus (SMV). When applied to these NLVs, detection limits were best extracting with QIAamp microspin columns, which use guanidinium thiocyanate to lyse viruses and extract RNA. The microspin columns were the method of choice to prepare RNA extracts for further investigations in this study. To better overcome sample-related inhibition of RT-PCR, and to increase sample extraction, volume methods for improved RNA extraction on mini and midi columns were developed and evaluated. A lysis buffer was developed that allows multiple loading of columns for RNA extraction, facilitating ready processing of up to 4 mL of water concentrates on a midi column. Analysis of samples with the real-time and standard RT-PCR methods has demonstrated the ability of the method to overcome sample inhibitors and provide sensitive detection of viral RNA. Using midi columns designed for 1.0 mL samples, the method provides efficient RNA recovery of RNA from 4.0 mL samples of seeded concentrate obtained from water samples by PEG precipitation. The method employs specially formulated lysis buffer that is added in equal volume to the water concentrate (1:1 ratio) instead of the commercial lysis buffer:liquid sample ratio of 4:1. This allows a larger volume of sample to be loaded on the columns for RNA purification. The developed lysis buffer contains a high concentration of guanidine thiocyanate (about 50 percent w/v) and can be used with commercially available mini and midi columns (Qiagen). This buffer successfully extracted low levels of viral RNA from water sample concentrates seeded with NV as well as hepatitis A virus and coxsackievirus B3.
Three methods were evaluated for RNA reconcentration after elution from the columns. In one method, 1/10th volume of 5 M ammonium acetate, 2.5 volumes of cold ethanol, and 1 µl of glycogen (method 1) or 1 µl of glycoblue (method 2) was added, vortexed briefly, and kept at -20°C for 1 hour or 30 minutes (both were tested simultaneously). After centrifugation at 14,000 rpm for 15 minutes at 4°C, samples were left to air dry in the laminar flow hood and finally suspended in 45 µl nuclease free water. In a third method, 1/10th volume of 5 M ammonium acetate, an equal amount of isopropanol, and 1 µl of glycogen (20mg/mL) was added, vortexed briefly, and kept at -20°C for 1 hour or 30 minutes. After centrifugation at 14,000 rpm for 15 minutes at 4°C, the pellet was washed once with 500 µl of 75 percent cold ethanol, and centrifuged at 14,000 rpm for 5 minutes at 4°C. Pellets were air dried and suspended in nuclease free water. The resuspension volume can be as little as 5 µl so that the entire viral RNA from a 4.0 mL sample can be evaluated in one RT-PCR assay. Efficient recovery of RNA after column purification was obtained with all three alcohol precipitation methods. The glycoblue method was chosen for use because it gives good recovery and is easy to visualize.
Overall, the results from these studies show that lysine-Triton X-100 elution solutions are effective alternatives to beef extract elution solution for recovering low levels of NVs in tap water and surface water samples, that PEG precipitation is effective for further concentration of NVs in adsorbent filter eluates, and that optimized chemical extraction methods for NV RNA allow for efficient virus detection by RT-PCR amplification. For molecular detection of NLVs and other waterborne pathogens, utilities can process samples through the steps of PEG precipitation or even RNA extraction and then send the samples to another lab for molecular analysis.
Development and Evaluation of an Internal NLV-RNA Control Template for RT-PCR Amplification. Virus concentrates from water and other samples may still contain RT-PCR inhibitors, despite advanced RNA extraction and purification methods. To address the presence of RT-PCR inhibitors in RNA extracts of samples, a new internal RNA control (IC-7) was developed and evaluated. The new RNA internal standard was constructed using a 73-mer c-DNA primer that included a 12-nt region downstream of the previously published RIVM JV13 primer, followed by the RIVM JV13 primer sequence, a 51-base deletion, a 20-base internal control-specific sequence, and an additional 20 bases of virus-specific sequence. This primer was used in a RT-PCR mixture with JV12 and RNA of a NLV stool sample that belonged to the Bristol genotype generating a 300-bp RT-PCR product. After cloning into a cloning vector, followed by transforming cells, clones with the proper orientation were identified by sequencing. After linearizing by cutting with Spe I restriction endonuclease, internal control NLV RNA (called IC-7) was synthesized with T7 polymerase. Plasmid DNA was removed following 2 subsequent digestions with Rnase-free Dnase, and after phenol-chloroform extraction, RNA was collected by precipitation with isopropanol, resuspended in water, quantitated by spectroscopy at 260 nm, and stored in aliquots at -70°C.
When amplified with the JV12/13 primer pair, a 288-bp product was generated. The IC-7 amplicon (288 bp) is shorter in length than the NLV amplicons (327 bp), which allows physical separation of the two amplicons after gel electrophoresis. When serial tenfold dilutions of different NLV RNAs were amplified in the presence of 100 genomic copies of internal RNA, the endpoint titers of the NLV strains were the same with and without internal control present. These results indicate that the internal control does not inhibit RT-PCR detection of low levels of NLVs. Hence, this control RNA template can be amplified with the same sensitivity using standard RT-PCR primer pairs in the polymerase region of NLV genomes, resulting in a somewhat smaller PCR product that can be readily distinguished from a true NLV amplicon on the basis of its smaller size. When NLV RNA is present in a sample, the shorter length of IC-7 RT-PCR product allows visible separation from the NLV-specific amplicons after gel electrophoresis. The presence of 100 copies of the internal control does not alter the amplification of the NLV target, low levels of NLVs are amplified in its presence, and the internal control amplicon is readily distinguishable on the basis of size from the NLV amplicon.
Improved RT-PCR Primers and RT-PCR Amplification Methods and Conditions to Amplify and Detect NLVs. Numerous RT-PCR primer sets, mostly targeting the polymerase region, have been described for broad detection of NLVs by RT-PCR. However, no one set of NLV primers consistently detects all strains of NLVs, and many of these primer sets have not been applied to the detection of low levels of NLVs, as would be found in water and other environmental samples. To achieve the study goal of developing standardized protocols for RT-PCR detection of low levels of all human NLVs in water and other environmental samples, we attempted to identify or develop a sensitive but broadly reactive primer set to detect low levels of human NLVs in water without compromising specificity. Based on the database of NLV sequences, a new generic primer pair (designated RegA/MJV12) was designed that targets the same polymerase gene region of NLVs as the original JV12/JV13 primer pair, but uses degenerate nucleotides at certain positions of the primers to allow broader reactivity. The RegA/MJV12 primer pair was tested with a range of NLV RNA samples (from stool specimens), some of which initially tested negative with other primer pairs. RT-PCR products of the appropriate size were generated for all NLV samples, and the product yields were consistently higher than those of other primer pairs, thus indicating an improved detection limit.
Most previous studies of RT-PCR for detection of NLVs and other viruses in clinical, food, water, and other environmental samples have employed two-step two-tube RT-PCR formats. This format is time consuming and prone to nucleic acid contamination; often caused by the need to open the reaction tubes partway through the analysis. One-step RT-PCR assays are less laborious and more importantly, less prone to contamination, especially if employed with a "hot-start." Because of these advantages, we tested a commercially available one-step RT-PCR assay (Qiagen, also referred to as Q-solution) to detect NLVs. Tenfold serial dilutions of RNA from four different genotypes (NV, Southampton Virus, Lordsdale Virus, and SMV) were tested with both assays and RT-PCR products were analyzed on 2 percent agarose gels. The new one-step PCR system gave results for the different NLVs that were, on average, better than those for the conventional two-step PRC system, based on the amount of amplicon produced and the dilution endpoint of viral RNA detection. The one-step assay was further optimized by addition of Q-solution, an innovative PCR additive, for the amplification of templates that are gas chromatography (GC) rich or that have extensive secondary structure.
Most diagnostic RT-PCR assays for NLVs target regions of the RNA polymerase region of the genome, and although extremely useful for detection, the major capsid protein (VP1) is the reference method for establishing genotypes. In this study, we analyzed complete NV VP1 sequences (n = 100), including all known NV genotypes and determined a region (region D) that was most suitable to differentiate between genotypes. Within region D, we designed two genogroup-specific, broadly reactive, degenerate primer sets (GI and GII). In addition, nested primer pairs were developed for the application of this assay for environmental samples. The region D primers were evaluated in a single-tube, one-step RT-PCR assay using a panel of 81 (31 GI, 50 GII) NV strains from both outbreaks and sporadic cases including prototype NLVs (Norwalk, Southampton, Snow Mountain, Hawaii, Toronto, and Bristol Viruses). In total, 95 percent of the samples tested positive using the new region D primer sets. All other enteric viruses (rotavirus, astrovirus, adenovirus, poliovirus) tested negative. Viral RNA titers of several GI and GII strains were comparable with those obtained with strain-specific primers. Phylogenetic analysis of region D sequences (36 deduced amino acids for GI, 56 deduced amino acids for GII), revealed 19 clusters (7 within GI and 12 within GII) with strains having >85 percent amino acid identity. These include three new, genetically distinct clusters; two of which were unresolved using region A sequences. Phylogenetic analysis of the complete capsid gene (ORF2) sequences revealed identical grouping of strains and confirmed the newly identified clusters using region D. In conclusion, we developed and evaluated a broadly reactive RT-PCR assay for the detection and typing of GI and GII NLVs that has an improved performance over region A based RT-PCR assays for genetic classification of NLV strains. This new RT-PCR assay for NLVs will be useful for sensitive detection and genetic classification of NLVs in both clinical and environmental samples.
Oligoprobe Hybridization to Detect and Identify NLV Amplicons From RT-PCR Amplification. For conventional RT-PCR assays, the resulting DNA amplicons require confirmation as true positive results by either hybridization with an internal probe (Southern or dot blot) after gel electrophoresis, nucleotide sequencing, or perhaps other analytical methods. Southern hybridization is the most widely used method. In this study, we designed a set of four different 5'-biotin-labeled oligoprobes that, based on a multiple nucleic acid sequence alignment of at least four strains of each NLV genotype (if available) would allow us to detect the majority of the NLV strains. Because the GGI probe requires at least a 160-nt fragment upstream of the polymerase region primer, only amplicons from the JV12/13 and p289/p290 primer pair could be detected. The concentration of each individual probe was first optimized by dot-blot hybridization. Optimum amounts of probes were mixed and tested by hybridization at 42°C. RT-PCR products from all 10 NLV genotypes tested gave a clear signal by hybridization. To obtain a more specific identification of RT-PCR amplicons, we have further evaluated for environmental samples a previously described reverse line blot (RLB) hybridization system that detects nearly all of the different NLV genotypes. The RLB system successfully detected RT-PCR amplicons that were produced using the improved NLV primer pairs in the polymerase region of the genome. Therefore, either the broadly reactive set of four probes to detect all NLVs by conventional hybridization or the more specific set of RLB probes for detection of individual NLV genotypes can be applied to the identification of NLVs amplified from water and other environmental samples.
Real-Time RT-PCR to Detect and Quantify NLVs. A real-time RT-PCR method was developed to rapidly amplify and detect genogroup I NLVs using a broadly reactive primer pair and a TaqMan probe for nucleotide sequences within the RNA polymerase region of these viruses. Development of rapid detection of NV and other genogroup I (GI) viruses by TaqMan RT-PCR is useful because the standard RT-PCR and probe or sequencing detection methods for these viruses are time- and labor-intensive. TaqMan RT-PCR reactions were conducted in glass capillaries in a final volume of 10 µl. Nucleotides, RT-Taq DNA polymerase, and buffer were included in the LightCycler-RNA Hybridization probe kit (Roche Molecular Biochemicals). Template RNA (1.0 µL each) was added to 9 µL of a LightCycler-RNA (LightCycler-RNA Master Hybridization Probes kit; Roche Molecular Biochemicals), containing 250 nM each of a set of newly developed primers, designated JTMG1F-JTMG1R, 100 nM of a newly developed fluorogenic probe (JTMG1P), and 5 mM Mg2+. RT was conducted at 55°C for 30 minutes, followed by denaturation at 95°C for 45 seconds, followed by 40 cycles with 95°C denaturation for 5 seconds, 55°C annealing for 35 seconds, and extension at 72°C for 15 seconds. Fluorescence was acquired at the end of the 55°C annealing step. Specificity also was confirmed with subsequent agarose gel electrophoresis to visualize amplicons (96 bp in size) as DNA bands.
The new real-time system correctly identified all 22 genotype 1 NLV samples tested, representing a wide range of subgroups. The new system did not detect genogroup II NLVs or other human enteric viruses (hepatitis A virus and several representative enteroviruses). The developed real-time RT-PCR assay will be useful for direct detection of Genogroup I from clinical and environmental samples in less than 2 hours. Because the real-time RT-PCR detected as low as 40 copies of genome equivalent, it is well suited to the detection of NLVs in water and other environmental samples typically containing low numbers of viruses. The proposed TaqMan RT-PCR for detection of genogroup I can be coupled with another TaqMan RT-PCR for the detection of genogroup II simultaneously in a multiplex format. A genogroup II real-time RT-PCR system was not developed in this study because of inadequate time and other resources. The development of such a system is recommended as a priority for future research.
Attempted Cell Culture Infectivity Assay of NV and SMV. Culture of NV and SMV was attempted in 10 well-characterized cell lines: BGMK, FRHK4, CACO2, CHO, A-549, G-293, RD, L20B, MDCK, and HTC8. Cell lines grown to 90 percent confluence in T25 flasks were inoculated with 0.5 mL of chloroform extracted NV stock (about 10-100,000 RT-PCR units/mL) diluted 1:2 with maintenance medium. Viruses were allowed to adsorb for approximately 40 minutes at 37°C and then 8 mL of standard maintenance medium containing 2 percent fetal bovine serum was added to flasks. Cultures were incubated at 37°C and monitored daily for cytopathic effect (CPE). The virus inoculum was assayed for NV by endpoint RT-PCR using polymerase (POL) primers following inoculation of cell lines. No CPE was observed after 10 days, incubation was terminated, and cell culture extracts were analyzed by RT-PCR. There was no evidence of increased viral RNA titer.
The experiment was repeated with NV and SMV stocks of higher titer (107 and 106/mL, respectively, as determined by endpoint titration RT-PCR with POL and G2 primers, respectively). Eight of the previous 10 cell lines (excluding CHO and L20B) were inoculated with NV and SMV in parallel. Two methods of inoculation and up to two virus preparations (supernatant of stool suspension and chloroform-extracted stool) were evaluated in each cell line. The first method of inoculation was as before and cultures were incubated at 37°C for 10 days with daily checks for CPE. Each culture was serially passaged twice and an aliquot at each passage was archived at -20°C. As an alternative inoculation method, suspended cells of each type were inoculated with 0.5 mL of a chloroform-extracted stool suspension, diluted with maintenance medium, viruses were allowed to adsorb in suspension for 40 minutes at 37°C, and then suspensions were transferred to T25 flasks and incubated at 37°C for 7 days. Each flask was serially passaged and an aliquot archived at -20°C. Subsequent assays cell culture extract samples by RT-PCR gave no evidence of increased virus titer. Overall, there was a loss of RT-PCR detectable virus in the tissue culture inocula, which may have resulted in less virus inoculated into cell cultures than estimated from initial titers of virus stock. This loss of virus titer may have contributed to the negative results of the attempted cell-culture infectivity attempts. However, the results seem to suggest that NV and SMV stocks employed in these studies were not propagatable in cell cultures using the cell lines and culture conditions tested.
Conclusions:
Overall, many of the objectives of this project were successfully fulfilled. New and improved methods were developed for efficient recovery, concentration, purification, and sensitive, specific RT-PCR detection of NLVs in source and treated drinking waters and other waters. New primers and new gene targets were developed for RT-PCR amplification by improved conventional and real-time RT-PCR methods. However, insufficient time and resources were available to test these newly developed methods in a wide range of environmental samples. Such testing is recommended to further validate these improved methods to detect human caliciviruses (NLVs; Noroviruses) in water and other environmental samples. Attempts to propagate NV and SMV in cell cultures were unsuccessful.
Technical Effectiveness, Economic Feasibility, and How the Research Provides Solutions to Environmental Problems and Benefits to the Environment and Human Health. This research has resulted in the development of new and improved methods to detect low levels of NLVs in water. The methods involve modifications of standard methods previously used to concentrate viruses from water. Because human NLVs are not cultivable, a number of different nucleic acid extraction, amplification, and detection methods by RT-PCR and hybridization were developed and evaluated. The methods are based mostly on commercially available kits and reagents, including the use of synthetic oligonucleotide primers and probes that can be purchased from commercial sources that routinely synthesize such reagents for their customers at reasonable cost. These new methods are now becoming more widely used to detect a variety of pathogens and other nucleic targets in clinical, food and environmental microbiology laboratories, including some water utility laboratories. Therefore, it now appears possible to implement the detection of NLVs in water for special studies, outbreak investigation, surveys, and other purposes. This could be achieved by having trained personnel perform virus recovery from water using modifications of the U.S. EPA Information Collection Request (ICR) methods previously used for virus surveys and then for the concentrated samples to be RNA extracted and analyzed for NLVs by conventional or real-time RT-PCR in laboratories already trained and experienced in these methods. The costs of these methods are no more and probably less than virus cultivation and immunological detection methods, making them economially feasible as well as technically possible.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 11 publications | 3 publications in selected types | All 3 journal articles |
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Anderson AD, Heryford AG, Sarisky JP, Higgins C, Monroe SS, Beard RS, Newport CM, Cashdollar JL, Fout GS, Robbins DE, Seys SA, Musgrave KJ, Medus C, Vinje J, Bresee JS, Mainzer HM, Glass RI. A waterborne outbreak of Norwalk-like virus among snowmobilers—Wyoming, 2001. Journal of Infectious Diseases 2003;187(2):303-306. |
R826837 (Final) |
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Vinje J, Hamidjaja RA, Sobsey MD. Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. Journal of Virological Methods 2004;116(2):109-117. |
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Vinje J, Oudejans SJG, Stewart JR, Sobsey MD, Long SC. Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization. Applied and Environmental Microbiology 2004;70(10):5996-6004. |
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Supplemental Keywords:
human caliciviruses, water, wastewater, detection, concentration, infectivity, reverse transcription-polymerase chain reaction, RT-PCR, gene probe, occurrence, caliciviruses, Hu-Cvs, Norwalk, Norwalk Virus, NLVs, enteric viruses, fecal contamination, gastroenteritis, gene probe, human calciviruses, microbial pathogens, microbial risk assessment, microbiology, norwalklike calciviruses, nucleic acid hybridization, oligoprobe, sewage, virology, virus cultivation, viruses., RFA, Scientific Discipline, Health, Toxics, Water, Contaminant Candidate List, Disease & Cumulative Effects, Drinking Water, Biology, Watersheds, enteric viruses, nucleic acid hybridization, microbiology, virology, norwalk-like calciviruses, microbial risk assessment, Norwalk, human calciviruses, virus cultivation, other - risk assessment, fecal contamination, viruses, RT-PCR, NLVs, microbial pathogens, calciviruses, Norwalk Virus, gastroenteritis, Hu-Cvs, gene probe, water quality, sewage, oligoprobeProgress 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.