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

Abstract

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.

Fig. 1.

Mosquito collection sites in Uganda. Bar, 50 km.

Fig. 2.

Maximum-likelihood (ML) phylogenetic tree of the novel CxFV Uganda and NAKV strains and other members of the genus Flavivirus for the NS5 nucleotide dataset. Bootstrap values are shown for the main clades only. See Supplementary Table S2 for virus abbreviations and GenBank accession numbers. Identical topologies were obtained using both paup (including and excluding third-codon positions) and MrBayes for the insect-only flaviviruses. All horizontal branch lengths are drawn to scale; bar, 0.4 substitutions per site. The tree is midpoint-rooted for purposes of clarity only. Those flavivirus sequences that are published for the first time in the current study, namely NAKV, CxFV strain Uganda and CFAV strain Rio Piedras, are highlighted in grey.

Fig. 3.

Bayesian phylogeny of the ORF amino acid dataset of the genus Flavivirus, including novel strain CxFV Uganda and NAKV. Posterior probabilities (%) are shown for the main clades only. See Supplementary Table S2 for virus abbreviations and GenBank accession numbers. All horizontal branch lengths are drawn to scale; bar, 0.3 substitutions per site. The tree is midpoint-rooted for purposes of clarity only. Those flavivirus sequences that are published for the first time in the current study, namely NAKV, CxFV strain Uganda and CFAV strain Rio Piedras, are highlighted in grey.

Fig. 4.

Bayesian phylogeny of the E region insect-only amino acid dataset from the genus Flavivirus, including CxFV and NAKV. Country and year of isolation, strain and mosquito species are shown for each virus. Posterior probabilities (%) are shown for the main clades only. See Supplementary Table S2 for virus abbreviations and GenBank accession numbers. The topology of the E region nucleotide phylogeny, both including and excluding third-codon positions, was identical to that obtained by using amino acid data. All horizontal branch lengths are drawn to scale; bar, 0.2 substitutions per site. The tree is rooted by using KRV for the purpose of clarity only (midpoint rooting produces a similar topology). Those flavivirus sequences that are published for the first time in the current study, namely NAKV, CxFV strain Uganda and CFAV strain Rio Piedras, are highlighted in grey.

Fig. 5.

Electron micrographs of C6/36 cells infected with (a) CxFV Uganda and (b) NAKV. Bars, 100 nm.

Fig. 6.

ML phylogenetic tree of mosquitoes plus virus-positive samples from Uganda for the COI gene. Bootstrap values for main clades of >50 % only are shown for clarity. All horizontal branch lengths are drawn to scale; bars, 0.06 (a) or 0.03 (b) substitutions per site. V, Field-collected voucher specimen; H3E6, mosquito from which CxFV Uganda was isolated; H4A1 and H4D1, mosquitoes from which NAKV was isolated. All other sequences are from public databases as detailed in Supplementary Table S3. (a) ‘Fly’ region of COI, rooted using Chagasia. (b) ‘Barcode’ region of COI, Culex subset, rooted using Culiseta.