A highly sensitive method for the detection and genotyping of West Nile virus by real-time PCR

In recent years, West Nile virus has been responsible for outbreaks in regions where it has not previously been found. Five genetic lineages with specific geographic distributions exist. Recent outbreaks of WNV associated with the introduction of lineage 1 strains into the western hemisphere, together with the emergence of lineage 2 WNV in Central Europe, has highlighted the potential for spread of pathogenic WNV strains beyond their expected geographical boundaries. Therefore, genotyping of WNV strains may have important applications in surveillance and epidemiology. We report here the development of a nested real-time PCR for the detection and genotyping of WNV strains by means of dissociation-curve analysis, using fluorescence resonance energy transfer (FRET) probe technology. Eight WNV strains, representing three lineages were tested and correctly genotyped at a detection limit of 0.07 viral genome copies/ml in one-step real-time RT-PCR or 7x10(-16) viral genome copies/ml in a nested real-time PCR. WNV could be identified and typed in serum and brain specimens from a human and horse with neurological disease. To our knowledge, this is the first assay designed for the simultaneous detection and genotyping of WNV by rapid, sensitive real-time PCR which may be implemented in diagnostic and epidemiology laboratories.