Grantee Research Project Results
1999 Progress 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 Period Covered by this Report: 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:
The general objectives of this study are to: (1) develop improved methods to recover, concentrate, and purify Norwalk-like caliciviruses (NLVs) 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.Progress Summary:
Work is in progress on the two initial goals of this study: the development of improved recovery methods for NLVs in water and the detection of the recovered NLVs by improved RT-PCR materials and methods.
In initial studies, we confirmed our previous encouraging results for several different amino acid eluants applied to the recovery (elution) of adsorbed Norwalk virus (NV) from the positively charged filter (Virosorb 1MDS) used to concentrate viruses from water. NV, as stool from infected volunteers, was purified by organic solvent extraction, and assayed by RT-PCR using NV Pol primers (DeLeon, et al., 1992). Stock virus titer was about 107 PCR units/ml, and test waters were seeded to give concentrations of 100,000 or less RT-PCR units/ml. Lysine, glycine, and arginine eluants were demonstrated to give efficient recoveries of NV from seeded tap water samples under a range of pH conditions. Furthermore, these amino acid eluants were demonstrated to be less inhibitory than beef extract-containing eluants to the detection of NV by RT-PCR protocols similar to those used previously (De Leon, et al., 1992; Schwab, et al., 1995; 1996). The compatibility of lysine, glycine, and arginine eluants with RT-PCR detection of NV from water was confirmed. The eluants were most compatible with RT-PCR when used at pH 9.5. At pH 8.5, glycine was the most compatible eluant. Eluants were least compatible with RT-PCR at pH 7.0. A detection limit of 10 PCR units/10 µl sample was confirmed from our previous studies using the RNA release methods and RT-PCR protocols previously employed. To improve the elution efficiency of NV adsorbed to positively charged filters from water, the amino acid eluants were further tested with several non-ionic detergents as additives. Initially, several non-ionic detergents, such as Tween 20, Tween 80, NP-40, and Laureth-12, were individually tested at three different concentrations (1, 0.1, and 0.01 percent) for RT-PCR compatibility. All the candidate detergents resulted in one log10 decrease in the detection limit by RT-PCR. However, the detergent concentrations and NV detection limits were not consistently correlated. Overall, detergents performed best when used at a concentration of 0.01 percent.
Molecular detection is the only sensitive method now available for detection of NLVs in water and other environmental samples, however, a standardized method does not exist. To date, at least 10 primer sets, mostly targeting the polymerase region, have been described for broad detection of NLVs by RT-PCR. However, no one set of NLV primer sets has consistently detected 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. This study has as a goal the development of a standardized protocol for RT-PCR detection of low levels of all human NLVs in water and other environmental samples. Therefore, a task of this study is to evaluate several primer sets, in order to determine which primer set (or sets) is most sensitive, robust, and gives broadest detection of low levels of human NLVs in water without compromising specificity.
During the first year of the project, we developed a framework for comparative evaluation of several published, third generation NLV primer sets, including degenerate primers, inosine containing primers, primer cocktails, and traditional broad-spectrum primers. This framework makes use of two commonly used, but somewhat different, RT-PCR chemistries and amplification protocols (Moe lab, ML, and Sobsey lab, SL), with the cycling protocol adapted to the appropriate annealing temperature for each individual primer set. For both systems, direct 3' priming was used in the heat-release RT step instead of random priming. The framework was developed to compare different RT-PCR primers and overcome inconsistent and atypical PCR conditions, which prevent a meaningful literature-based comparison of the primer sets. Hence, each primer set is being experimentally evaluated for breadth of detection, sensitivity of detection, and consistency of amplification on the same representative panel of NLV clinical isolates.
Initial results from RT-PCR amplification of the various, genetically representative NLVs indicate that primer set JV12/JV13 is more sensitive, and suffered less inhibition than the GI/GII primer set. The initial panel of isolates included 10 stool samples previously characterized by the GI/GII primer set and one previously uncharacterized stool sample from a recent outbreak. The previously characterized samples had been stored at 4?C and some had apparently lost titer. This made the levels of NLV in these samples more realistically representative of the lower NLV levels present in water and sewage than in fresh stool specimens. The GI/GII primer set was able to amplify only 1 sample of the 11 using the ML protocol, but 6 of the 11 NLVs using the SL protocol. The JV12/JV13 primer set was able to amplify 4 of the 11 samples with the ML protocol and 7 of the 11 with the SL protocol. Three samples were not amplified by either primer set with either protocol. In general, both primer sets showed similar sensitivity with the SL protocol, but the JV12/JV13 primer set was generally more sensitive with the ML protocol. Using the SL protocol, the GI/GII primer set amplified one sample that could not be amplified by the JV12/JV13 primer set, and the JV12/JV13 primer set amplified two samples that the GI/GII set did not. Additionally, two samples yielded amplicons with incorrect size (molecular weight) depending on the RT-PCR protocol used.
The majority of human NLVs are organized into a series of genetic clusters within two genogroups. Ten clusters were outlined by Noel, et al. at the CDC-Atlanta to include four clusters in Genotype I and six clusters in Genotype II. Phylogenetic analysis of the initial panel of NLV isolates tested indicates that at least four of the genetic clusters were represented in the initial experiments. In an effort to more completely evaluate the selected primers, phylogenetic analysis of outbreak samples available in appropriate quantities was done. Outbreak samples are represented by two letter designations and highlighted samples were included in the initial panel of isolates. Five of the isolates from the initial panel were obtained from a surveillance study and were not included in the phylogenetic analysis of outbreak samples. Relatedness of isolates is proportional to lateral distance between isolates. Hence, examination of the outbreak samples determined that representatives of most genetic clusters within both genogroups are available for inclusion in the panel of isolates for the next round of experiments on RT-PCR amplification of low NLV levels.
Future Activities:
We will use the developed concentration and purification methods for HuCV recovery, the selected primers for RT-PCR amplification, and the selected oligoprobes for hybridization to examine field samples of source and finished water for HuCVs. We will attempt to cultivate HuCVs in cell cultures using the prototype 8FIIa strain of Norwalk virus as a model. We will determine if virus propagation is occurring by measuring an increase in virus titer as detected by quantitative RT-PCR amplification on material harvested from serial passages in inoculated cell cultures.Journal Articles:
No journal articles submitted with this report: View all 11 publications for this projectSupplemental Keywords:
viruses, drinking water, human health., RFA, Health, Scientific Discipline, Toxics, Water, Contaminant Candidate List, Disease & Cumulative Effects, Biology, Drinking Water, 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.