Isolation of a new species of flavivirus and a novel strain of Culex flavivirus (Flaviviridae), from a natural mosquito population in Uganda.

The genus Flavivirus, which contains approximately 70 single-stranded, positive-sense RNA viruses, represents a unique model for studying the evolution of vector-borne disease, as it includes viruses that are mosquito-borne, tick-borne or have no known vector. Both theoretical work and field studies suggest the existence of a large number of undiscovered flaviviruses. Recently, the first isolation of cell fusing agent virus (CFAV) was reported from a natural mosquito population in Puerto Rico, and sequences related to CFAV have been discovered in mosquitoes from Thailand. CFAV had previously been isolated from a mosquito cell line in 1975 and represented the only known ‘insect-only’ flavivirus, appearing to replicate in insect cells alone. A second member of the ‘insect-only’ group, Kamiti River virus (KRV), was isolated from Kenyan mosquitoes in 2003. A third tentative member of the ‘insect-only’ group, Culex flavivirus (CxFV), was first isolated in 2007 from Japan and further strains have subsequently been reported from the Americas. We report the discovery, isolation and characterization of two novel ‘insect-only’ flaviviruses from Entebbe, Uganda: a novel lineage tentatively designated Nakiwogo virus (NAKV) and a new strain of CxFV. The individual mosquitoes from which these strains were isolated, identified retrospectively by using a reference molecular phylogeny generated using voucher specimens from the region, were Mansonia africana nigerrima and Culex quinquefasciatus, respectively. This represents the first isolation, to our knowledge, of a novel insect-only flavivirus from a Mansonia species and the first isolation of a strain of CxFV from Africa. The GenBank/EMBL/DDBJ accession numbers for the COI gene sequences from mosquito species obtained during this study are GQ165759 [GenBank] –GQ165807 [GenBank] , that for Culex flavivirus Uganda is GQ165808 [GenBank] , that for Nakiwogo virus is GQ165809 [GenBank] and that for cell fusing agent virus strain Rio Piedras is GQ165810. Three supplementary tables listing primer sequences used, flaviviral abbreviation codes and accession numbers, and mosquito accession numbers and Barcode of Life Datasystem (BOLD) reference numbers are available with the online version of this paper