Grantee Research Project Results
2001 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, 2001 through December 31, 2002
Project Amount: $296,980
RFA: Drinking Water (1998) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall objectives of this research project 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 occurrence of NLVs in representative samples of sewage and raw and finished waters from both surface and ground sources.
Progress Summary:
During the research period, substantial progress was made in meeting Objectives 1 and 2 of this study, including the development of improved recovery methods for NLVs in water, and the detection of the recovered NLVs by improved RT-PCR materials and methods. Building on previous results, amino acid solutions were optimized to elute adsorbed Norwalk virus (NV) from the positively charged filter (Virosorb 1MDS) used to concentrate viruses from water. NV extracted from stools of infected volunteers was seeded into tap and surface waters. Of the amino acids tested, lysine at 0.5 M gave the most efficient NV recoveries from seeded tap water over a range of pH levels. Of the nonionic detergents tested, Triton X-100 at 0.1 percent in 0.5 M lysine best improved virus elution efficiency without greatly inhibiting RT-PCR detection. Viruses in lysine-Triton eluates were further concentrated by precipitation with 8 percent polyethylene glycol (PEG) after adjusting to pH 7.2, adding 0.3 N NaCl and holding at 4°C for several hours or overnight. Resulting PEG precipitates were extracted for NV RNA, which then was RT-PCR amplified. NV recovery was efficient after PEG precipitation from lysine-Triton X-100. Low levels of NV in seeded tap water were recovered by filter adsorption, lysine-Triton X-100 elution, and PEG precipitation with 58 percent efficiency, which was significantly higher (p = 0.002) than recovery using 3 percent beef extract-0.05 M glycine eluent (17 percent). In experiments on seeded surface water recovery efficiency by filter adsorption, lysine-Triton elution and PEG precipitation was 32 percent, compared to 24 percent when using beef extract glycine. These recoveries were not significantly different (p = 0.62). The 0.5 M lysine medium containing 0.1 percent Triton X-100 is an effective alternative to standard beef extract-glycine eluents for NV elution from the filters used to concentrate viruses from water. NV recoveries from seeded tap water and surface water were similar to, or greater than, recoveries achieved when using beef extract-based eluents. Lysine eluent is compatible with PEG precipitation of viruses. 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 laboratory for molecular analysis.
Because efficient and sensitive RT-PCR for NLVs and other viruses requires removal or inactivation of inhibitors present in sample concentrates, 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 kit, Qiagen). When applied to several Genogroup I and II NLVs, detection limits were best extracted using QUIAmp microspin columns, which lyses of viral RNA in guanidinium thiocyanate. The microspin columns were the method of choice to prepare RNA extracts for further investigation in this study.
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. 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.
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 objective 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 degenerate nucleotides were used at certain positions of the primers to allow broader reactivity. The RegA/MJV12 primer pair was tested with a range of NLV RNA samples, 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. Also, because one-step RT-PCR assays are less laborious and less prone to contamination, we evaluated a commercially available one-step RT-PCR assay (Qiagen) and optimized the primer concentrations and annealing temperature for NLV detection. The new one-step PCR system gave results for different NLVs that were on average better than those for the conventional two-step PCR system, based on the amount of amplicon produced and the dilution endpoint of detection.
Most RT-PCR assays of NLVs target the RNA polymerase region of the genome, which is not the best region for genotyping and molecular epidemiological analysis of these viruses. We designed two genogroup-specific broadly reactive degenerate primer sets (GI and GII) targeting the C-terminal fragment of the NLV VP1 capsid gene. In addition, nested primer pairs were developed for sensitive application of this assay to environmental samples. A panel of 81 (31 GI and 50 GII) NLV strains from both outbreaks and sporadic cases representing prototype NLVs was tested. The presence of NLV-RNA was confirmed by a polymerase-based RT-PCR. Sequence analysis was used to characterize the amplicons obtained. In total, 95 percent of the samples tested positive using the new VP1 capsid primer sets. All other enteric viruses tested negative in the assays. Viral RNA titers of four strains (2 GI, 2 GII) were comparable with those obtained with strain specific primers. Therefore, a broadly reactive RT-PCR assay targeting a C-terminal fragment of the VP1 gene was successfully developed for sensitive detection and genetic classification of NLVs in both clinical and environmental samples.
Overall, many of the objectives of this project have been fulfilled, although insufficient time was 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 in water and other environmental samples.
Future Activities:
Future activities for this research project will be focused solely on completing the final report, because research project time has expired and recommended further investigation, experimentation, and testing cannot be completed.
Journal Articles:
No journal articles submitted with this report: View all 11 publications for this projectSupplemental Keywords:
human caliciviruses, water, wastewater, detection, concentration, infectivity, reverse-transcription polymerase chain reaction, RT-PCR, gene probe, occurrence, toxics, water, biology, contaminant candidate list, disease and cumulative effects, drinking water, watersheds, caliciviruses, Hu-Cvs, Norwalk-like calicivirus, NLV, Norwalk, Norwalk virus, enteric viruses, fecal contamination, gastroenteritis, gene probe, microbial pathogens, microbial risk assessment, microbiology, nucleic acid hybridization, oligoprobe, sewage, virology, virus cultivation, viruses., 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.