title: Real-Time RT-PCR Assays for Detection and Genotyping of West Nile Virus Lineages Circulating in Africa.
abstract: West Nile virus (WNV) is an emerging arbovirus, circulating worldwide between birds and mosquitoes, which impacts human and animal health. Since the mid-1990s, WNV outbreaks have emerged in Europe and America and represent currently the primary cause of encephalitis in the United States. WNV exhibits a great genetic diversity with at least eight different lineages circulating in the world, and four (1, 2, Koutango, and putative new) are present in Africa. These different WNV lineages are not readily differentiated by serology, and thus, rapid molecular tools are required for diagnostic. We developed here real-time RT-PCR assays for detection and genotyping of African WNV lineages. The specificity of the assays was tested using other flaviviruses circulating in Africa. The sensitivity was determined by testing serial 10-fold dilutions of viruses and RNA standards. The assays provided good specificity and sensitivity and the analytical detection limit was 10 copies/reaction. The RT-PCR assays allowed the detection and genotyping of all WNV isolates in culture medium, human serum, and vertebrate tissues, as well as in field and experimental mosquito samples. Comparing the ratios of genome copy number/infectious virion (plaque-forming units), our study finally revealed new insight on the replication of these different WNV lineages in mosquito cells. Our RT-PCR assays are the first ones allowing the genotyping of all WNV African variants, and this may have important applications in surveillance and epidemiology in Africa and also for monitoring of their emergence in Europe and other continents.
authors.affiliation: 1 Unité des Arbovirus et Virus de Fièvres Hémorragiques, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Gamou
authors.initials: G
authors.lastname: Fall
authors.affiliation: 1 Unité des Arbovirus et Virus de Fièvres Hémorragiques, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Martin
authors.initials: M
authors.lastname: Faye
authors.affiliation: 2 Institute of Aquaculture, University of Stirling , Stirling, Scotland.
authors.forename: Manfred
authors.initials: M
authors.lastname: Weidmann
authors.affiliation: 3 GenExpress GmbH , Berlin, Germany .
authors.forename: Marco
authors.initials: M
authors.lastname: Kaiser
authors.affiliation: 1 Unité des Arbovirus et Virus de Fièvres Hémorragiques, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Anne
authors.initials: A
authors.lastname: Dupressoir
authors.affiliation: 4 Unité d'Entomologie Médicale, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: El Hadj
authors.initials: EH
authors.lastname: Ndiaye
authors.affiliation: 4 Unité d'Entomologie Médicale, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Yamar
authors.initials: Y
authors.lastname: Ba
authors.affiliation: 4 Unité d'Entomologie Médicale, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Mawlouth
authors.initials: M
authors.lastname: Diallo
authors.affiliation: 1 Unité des Arbovirus et Virus de Fièvres Hémorragiques, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Ousmane
authors.initials: O
authors.lastname: Faye
authors.affiliation: 1 Unité des Arbovirus et Virus de Fièvres Hémorragiques, Institut Pasteur de Dakar , Dakar, Senegal .
authors.forename: Amadou Alpha
authors.initials: AA
authors.lastname: Sall
mesh_terms: D000330:Aedes; D000349:Africa; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005838:Genotype; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014902:West Nile virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D012367:RNA, Viral
keywords: African lineages; West Nile virus; genotyping; real-time RT-PCR