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West Nile virus is a mosquito-borne flavivirus that is primarily maintained in nature in a mosquito-bird-mosquito transmission cycle. Mammals, including humans and horses, are incidentally infected through biting by mosquitoes infected with West Nile virus. Since 1994, West Nile virus outbreaks have occurred with a high incidence of severe disease in humans and horses. In the USA, West Nile virus was first detected in 1999 in New York City and has since spread to 39 states in humans. The virus has resulted in over 4161 known human cases and at least 277 human deaths. Surveillance techniques employing nucleic acid-based assays have played an essential role in monitoring the spread of West Nile virus and are displacing the former gold standard cell culture-based assays. In this article we review the current techniques for diagnosis of West Nile virus, focusing on RNA detection, and suggest a number of new directions for genetic diagnosis of West Nile virus.