Viruses can be transmitted through and contaminate waters causing waterborne epidemics to humans and animals. There is insufficient understanding about how viruses survive in the environment, and to what extent this may differ between agents or in the co-presence of other micro-organisms. The limited data makes it difficult to determine the risks of viruses and this hinders the preparation of preventative plans against viral transmission through the waters. This project sought to establish a DNA microarraybased approach to detect and differentiate between viruses in environmental waters, to provide a sensitive, specific and rapid system for monitoring virus contamination. Such a system might provide data not only for improved predictions of the outbreak of diseases but may lead to the effective modelling ofvirus re-circulation through the environment. The Picornaviridae virus family was the focus of this project. 152 specific microarray probes were designed after using 'ClustalX' software to multiply align the respective virus sequences and conducting 'BLASTN' similarity searches to estimate their specificity. Standard and multiplex RT-PCR amplification of viral nucleic acids with direct incorporation of fluorescent Cy-dyes was combined with the DNA microarray hybridization technique to identify the virus composition of test and environmental samples. The microarray data was normalised and ranked using a range of statistical methods. After the development of appropriate detection criteria using pilot studies with known input virus samples the experimental and statistical process was applied to detection and identification of viruses within environmental samples. Following tests on a range of different techniques for RNA extraction, amplification and labelling the following optimal procedure was adopted: following the concentration of virus particles by acetone precipitation, RNA from the environmental samples was extracted using the 'QIAamp Viral RNA Mini Kit'; following olio-dT-primed cDNA synthesis, the 'Genomiphi V2 DNA Amplification Kit' was used to randomly amplify the cDNA; the DNA was then directly labelled by incorporating Cy-dyes in a PCR reaction with multiple virus-specific primers. A sewage sample was provided by the Reading HPA Environmental Virology Unit for testing in this project; they had identified a number of viruses in this sample by cell culture: Coxsackieviruses B2, B3, B4 and B5 and also detected some unknown isolates. The optimised microarray-based method developed in this project predicted the presence of the following viruses in the same sewage sample: Coxsackieviruses B4 and B3, Bovine Enterovirus, Poliovirus and Hepatitis A virus. Thtis, while some ofthe same viruses were detected by the microarray, a range of other viruses were also detected, using relatively stringent statistical thresholds. The microarray-based detection system appears to have broader specificity, and possibly sensitivity, than the cell culture-based approaches and importantly, is potentially able to direct non-cultivable and non-viable viruses in a water sample. These findings, coupled with the rapid nature of the technique, suggest that micorarrays, could, in the future, provide a superior alternative to cell culture-based methods for detection ofwaterborne viruses.
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