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Evaluation of a Microarray for Genotyping Noroviruses
Citation:
FOUT, G. AND N. BRINKMAN. Evaluation of a Microarray for Genotyping Noroviruses. Presented at Third International Calcivirus Conference, Cancun, MEXICO, November 10 - 13, 2007.
Impact/Purpose:
Purpose: This task is an evaluation of the VFAR approach using noroviruses. Noroviruses are the group of human caliciviruses that have caused many waterborne outbreaks. They are grouped into two major genogroups with a number of subtypes in each genogroup. Subtypes differ in their virulence properties, and genogroup II, subtype 4 strains have been reported to cause the more virulent outbreaks occurring worldwide during 2002-3. In this task, the genome regions that allow the grouping of waterborne viruses into subtypes will be identified. A microarray assay will be developed from these regions and, when necessary, microarray data will be supplemented by DNA sequencing. The data obtained from these assays will be compared with virulence data as it becomes available. If successful, this approach will be used to identify and type other waterborne viruses. Objective: This task directly supports the 2003 Drinking Water Research Program Multi-Year Plan's long term goal 2 to “develop new data, innovative tools and improved technologies to support decision making by the Office of Water on the Contaminate Candidate List and other regulatory issues, and implementation of rules by states, local authorities and water utilities” under GRPA Goal 2 (Clean and Safe Water). The overarching objective is to provide Agency scientists, risk assessors, and regulators as well as to individuals outside of the Agency that work in the field of drinking water quality with an evaluation of the use of the VFAR approach for determining which human enteric viruses should be on future CCL lists.
Description:
Noroviruses that infect humans are divided into three genogroups based upon their sequence diversity. Of these, genogroups I and II have been identified as leading causes of waterborne disease outbreaks worldwide and are frequently found in rivers and lakes that serve as drinking water sources. To develop a better understanding of the risk posed by these organisms, a more rapid procedure for identifying and genotyping noroviruses in the environment was investigated. Currently, noroviruses are usually detected in water samples using reverse transcriptase-polymerase chain reaction (RT-PCR) based assays. Although sequencing of RT-PCR amplicons is the gold standard for genotyping these viruses, RT-PCR assays of water concentrates often produce a range of non-specific amplicons in a single reaction. This usually necessitates the cloning of products prior to sequencing. To determine whether genotyping could be performed more rapidly and without the need for cloning, we evaluated the use of a generic microarray format using multiple strain specific probes that would be able to hybridize to region B of the RNA polymerase gene. With this method, a single base extension (SBE) reaction was used to label genotype-specific probes that had annealed to a matching virus sequence. This labeling was done in the presence of single and multiple norovirus templates. These probes were then hybridized to an Affymetrix GenFlex Tag Array and the labeled probes were detected. The specificity of this approach was examined by designing probes with and without nucleotide mismatches. Our results show that the perfect-matched tag-probes were specifically labeled in SBE reactions for all strains examined. In addition, most of the mismatched tag-probes were not labeled and those that were resulted in specific fingerprints that could be used to subtype strains. We also showed that norovirus seeded into source water concentrates can be genotyped. These results demonstrate the utility of the GenFlex Tag Array for genotyping noroviruses from water samples. This approach also could be adapted to include the simultaneous identification of multiple waterborne pathogens.