title: Real time PCR assay for detection of all known lineages of West Nile virus.
abstract: West Nile virus (WNV) is one of the most widespread arbovirus and a large variety of WNV strains and lineages have been described. The molecular methods for the diagnosis of WNV target mainly lineages 1 and 2, which have caused outbreaks in humans, equines and birds. But the last few years new and putative WNV lineages of unknown pathogenicity have been described. Here we describe a new sensitive and specific real-time PCR assay for the detection and quantification of all the WNV lineages described until now. Primers and probe were designed in the 3'-untranslated region (3'-UTR) of the WNV genome and were designed to match all sequenced WNV strains perfectly. The sensitivity of the assay ranged from 1,5 to 15 copies per reaction depending on the WNV lineage tested. The method was validated for WNV diagnosis using different viral strains, human samples (cerebrospinal fluid, biopsies, serum and plasma) and mosquito pools. The assay did not amplify any other phylogenetically or symptomatically related viruses. All of the above make it a very suitable tool for the diagnosis of WNV and for surveillance studies.
authors.affiliation: Laboratory of Arboviruses and Imported Viral Diseases, National Centre for Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km. 2, 28220 Majadahonda, Madrid, Spain. Electronic address: a.vazquez@isciii.es.
authors.forename: Ana
authors.initials: A
authors.lastname: Vázquez
authors.affiliation: Laboratory of Arboviruses and Imported Viral Diseases, National Centre for Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km. 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Herrero
authors.affiliation: Laboratory of Arboviruses and Imported Viral Diseases, National Centre for Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km. 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Anabel
authors.initials: A
authors.lastname: Negredo
authors.affiliation: Laboratory of Arboviruses and Imported Viral Diseases, National Centre for Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km. 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Hernández
authors.affiliation: Laboratory of Arboviruses and Imported Viral Diseases, National Centre for Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km. 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: María Paz
authors.initials: MP
authors.lastname: Sánchez-Seco
authors.affiliation: Laboratory of Arboviruses and Imported Viral Diseases, National Centre for Microbiology, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km. 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Tenorio
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001717:Birds; D009033:Culicidae; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D006736:Horses; D006801:Humans; D015345:Oligonucleotide Probes / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014901:West Nile Fever / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D014902:West Nile virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D020413:3' Untranslated Regions; D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral
keywords: Diagnosis; Flavivirus; Lineages; Real-time PCR; Surveillance; West Nile virus