Gouleako virus isolated from West African mosquitoes constitutes a proposed novel genus in the family Bunyaviridae

Abstract

The family Bunyaviridae is the most diversified family of RNA viruses. We describe a novel prototypic bunyavirus, tentatively named Gouléako virus, isolated from various mosquito species trapped in Côte d'Ivoire. The S segment comprised 1,087 nucleotides (nt), the M segment 3,188 nt, and the L segment 6,358 nt, constituting the shortest bunyavirus genome known so far. The virus had shorter genome termini than phleboviruses and showed no evidence of encoded NSs and NSm proteins. An uncharacterized 105-amino-acid (aa) putative open reading frame (ORF) was detected in the S segment. Genetic equidistance to other bunyaviruses (74 to 88% aa identity) and absence of serological cross-reactivity with phleboviruses suggested a proposed novel Bunyaviridae genus.

Fig. 1.

GOUV growth on insect cells, morphology, and genome characteristics. (a) Uninfected C6/36 cells. (b) C6/36 cells 4 days after infection with GOUV. (c) Negative staining electron microscopy of purified GOUV particles. Bar = 100 nm. (d) Numbers of GOUV genome copies per ml in cell culture supernatant of C6/36 cells infected with GOUV at MOIs of 0.1, 0.01, and 0.001 were measured by RT-PCR for 7 days. (e to g) Strategies used for full genome sequencing. The top panel shows the genome segments S (e), M (f), and L (g). Boxes represent open reading frames (ORFs) flanked by noncoding regions (NCR), which are indicated by lines. Coding directions are indicated as arrows. Glycoprotein precursor properties were identified by signalP-NN, TMHMM, and NetNGlyc 1.0 and are marked as follows: signal peptide, black box; Gn, light-gray box; Gc, dark-gray box; transmembrane domains (TMD), white boxes; and glycosylation sites, black triangles. Bars in the middle panel indicate genome fragments generated in initial random amplification reactions. The bottom panel shows specific PCRs used for genome walking. Oligonucleotide orientations and positions are marked by arrowheads.

Fig. 2.

Multiple sequence alignments of putative GOUV RNA-dependent RNA polymerase and glycoprotein precursor genes. (a) Alignment of GOUV and RdRp genes, third conserved motif. Premotif A and motifs A, B, C, D, and E are indicated. Amino acids conserved between GOUV and other bunyaviruses are marked in gray. Active sites corresponding to the PB1 protein of influenza virus (1) are highlighted by boxes. (b) Alignment of putative GOUV, UUKV, and SFTSV glycoprotein precursor proteins. Highly conserved amino residues are marked in black and conserved residues in gray. Abbreviations: BUNV, Bunyamwera virus; CCHV, Crimean-Congo hemorrhagic fever virus; DUGV, Dugbe virus; GOUV, Gouléako virus; HANV, Hantaan virus; LACV, La Crosse virus; PUUV, Puumala virus; RVFV, Rift Valley fever virus; SFNV, sandfly fever Naples virus; SFTSV, severe fever with thrombocytopenia syndrome virus; TOSV, Toscana virus; TSWV, tomato spotted wilt virus; UUKV, Uukuniemi virus; WSMV, watermelon silver mottle virus.

Fig. 3.

Relationship of GOUV to other bunyaviruses. Phylogenetic analyses including representative members of all Bunyaviridae genera were performed on a gap-free amino acid alignment guided by the BLOSUM62 substitution matrix, using the neighbor-joining (NJ) algorithm with a uniform-rates substitution model and confidence testing by 1,000 bootstrap replicates in MEGA version 5.0 (33). Maximum-likelihood (ML) analyses were performed with the Dayhoff substitution model and are shown in smaller scale on the right. Phylogenies were investigated for the RdRp (a), Gn (b), Gc (c), and N (d) protein genes. Bars indicate evolutionary substitutions per position in the alignment. (e) Distribution of pairwise amino acid sequence distances between putative RdRp proteins in the family Bunyaviridae. A distance matrix of pairwise identity values was calculated with MEGA 5.0 (33) for 28 L-segment sequences. For each range of identity values (x axis), the incidence in the matrix is plotted on the y axis. White bars indicate pairwise distances between viruses of same genera (intragenus), and black bars indicate pairwise distances between viruses of different genera (intergenus). Pairwise distances between Uukuniemi virus and main-group phleboviruses (sandfly fever group) are shaded in gray. Pairwise distances between Gouléako virus and phleboviruses are hatched, and ranges of pairwise distances between GOUV and orthobunya-, hanta-, nairo-, and tospoviruses are marked by horizontal bars. Horizontal lines indicate ranges of pairwise sequence distances within each of the five established genera of the family Bunyaviridae. CCHF virus, Crimean-Congo hemorrhagic fever virus.

Fig. 4.

Indirect immunofluorescence assay (IIFA) with GOUV and phleboviruses. GOUV-infected C6/36 cells were used to prepare slides for immunofluorescence assays. GOUV infection was confirmed by determination of infectious particles (5.0 × 10⁴ TCID₅₀/ml) and by measurement of virus RNA copies/ml (5.27 × 10¹¹/ml). GOUV-infected cells were tested with mouse anti-RVFV serum (a), mouse anti-RVFV nucleocapsid serum (b), human anti-sandfly fever virus serum (Euroimmun AG, Lübeck, Germany) (c), mouse anti-UUKV serum (9b) (d), and mouse anti-UUKV serum (8b) (e). Reactivity of all sera was confirmed on IFA slides spotted with EU14 cells infected with each respective virus. These slides were taken from the commercially available “Sandfly Fever Virus Mosaic 1” and “Phlebovirus Mosaic 1” detection kits (Euroimmun AG, Lübeck, Germany). These positive controls are shown as follows: mouse anti-RVFV serum (g), mouse anti-RVFV nucleocapsid serum (h), human anti-sandfly fever Cyprus virus (SFCV) serum (i1), human anti-sandfly fever Naples virus (SFNV) serum (i2), human anti-TOSV serum (i3), and human anti-SFSV serum (i4). Additional control experiments were done by incubation of 2 different mouse anti-UUKV sera (designated 8b and 9b) on IFA slides spotted with UUKV-infected BHK-21 cells, as shown in panels k and l. Experiments with negative controls were performed using uninfected C6/36 cells incubated with human anti-sandfly fever virus serum (f), uninfected EU14 cells incubated with human anti-sandfly fever virus serum (m), and uninfected BHK-21 cells incubated with mouse anti-UUKV serum (9b) (n). IFA detection of human and murine sera, respectively, was performed with an anti-human IgG conjugate (Euroimmun AG, Lübeck, Germany) and with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse serum (Sifin, Berlin, Germany). Cells were stained with DAPI (4′,6-diamidino-2-phenylindole). Bars, 20 μm (C6/36 cells) and 50 μm (EU14 and BHK-21 cells). All photographs were taken at equivalent exposure settings.