Detection and Occurrence of Human Caliciviruses in Drinking Water

EPA Grant Number: R826837
Title: Detection and Occurrence of Human Caliciviruses in Drinking Water
Investigators: Sobsey, Mark D.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Nolt-Helms, Cynthia
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

Description:

The objectives of this study are: (1) to develop improved methods to recover, concentrate and purify human caliciviruses (HuCVs) from water, (2) to develop new and improved RT-PCR and oligoprobe (OP; gene probe) materials and methods to amplify and detect these HuCVs, (3) to apply these methods to the detection of field HuCVs in environmental sewage and water samples, thereby determining HuCV occurrence, and (4) to attempt to cultivate NV in differentiated intestinal and other cell cultures and develop a practical HuCV infectivity assay system.

Approach:

Methods will be further optimized and evaluated for HuCV concentration from water by adsorption to and elution from electropositive, microporous, Virosorb 1MDS filters, precipitation by PEG and possibly other agents, immunocapture using polyclonal antibodies (human serum immune globulin) bound to paramagnetic beads, chloroform or fluorocarbon extraction, and molecular exclusion by gel (Sephadex) chromatography and centrifugal ultrafiltration. Improved methods to amplify the nucleic acid of HuCVs concentrated from water will use specific primers and probes and conditions for RT-PCR amplification and nucleic acid hybridization that will be developed in this study. In general, RT-PCR amplification and non- radioactive oligoprobe hybridization of HuCVs will be done according to previously described procedures, but will involve the selection and testing of new and improved RT and PCR primers for amplification of genomic targets and new and improved non-radioactive oligoprobes for hybridization detection of the resulting amplicons. The methods developed in this study for concentration, purification, RT-PCR amplification and oligoprobe hybridization detection of HuCVs will applied to field samples of water. Initially, we will detect HuCVs in water samples implicated in HuCV gastroenteritis. Later, HuCV detection will be applied to geographically representative surface and ground waters of the United States. Some will be fecally contaminated from identifiable sources of sewage or other human fecal waste and other will be from relatively uncontaminated or "pristine" sources that are drinking waters or the protected raw (surface or ground) sources of drinking water supplies. Attempts will be made to cultivate Norwalk Virus in primary cell and organ cultures, diploid cell strains and continuous cell lines.

Expected Results:

This project will develop new methods to detect human caliciviruses and other enteric viruses in water that will be immediately applied to field samples of water as soon as they are developed. The research will produce one or more detailed protocols or stepwise procedures, including specification of the needed materials and full descriptions of methods, used to recover HuCVs as well as other enteric viruses in water and then detect them by RT-PCR amplification and oligoprobe hybridization. This project may also provide a method for propagation and infectivity assay of Norwalk Virus and possibly other HuCVs.

Publications and Presentations:

Publications have been submitted on this project: View all 11 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 3 journal articles for this project

Supplemental Keywords:

human caliciviruses, water, wastewater, detection, concentration, infectivity, RT-PCR, gene probe, occurrence., 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, oligoprobe

Progress and Final Reports:

  • 1999 Progress Report
  • Final Report