Discovery of a unique novel clade of mosquito-associated bunyaviruses

Abstract

Bunyaviruses are the largest known family of RNA viruses, infecting vertebrates, insects, and plants. Here we isolated three novel bunyaviruses from mosquitoes sampled in Côte d'Ivoire, Ghana, and Uganda. The viruses define a highly diversified monophyletic sister clade to all members of the genus Orthobunyavirus and are virtually equidistant to orthobunyaviruses and tospoviruses. Maximal amino acid identities between homologous putative proteins of the novel group and orthobunyaviruses ranged between 12 and 25%. The type isolates, tentatively named Herbert virus (HEBV), Taï virus (TAIV), and Kibale virus (KIBV), comprised genomes with L, M, and S segments of about 7.4 kb, 2.7 kb, and 1.1 kb, respectively. HEBV, TAIV, and KIBV encode the shortest bunyavirus M segments known and did not seem to encode NSs and NSm proteins but contained an elongated L segment with an ∼500-nucleotide (nt) insertion that shows no identity to other bunyaviruses. The viruses replicated to high titers in insect cells but did not replicate in vertebrate cells. The enveloped virions were 90 to 110 nm in diameter and budded at cellular membranes with morphological features typical of the Golgi complex. Viral RNA recovered from infected cells showed 5'-terminal nontemplated sequences of 9 to 22 nt, suggestive of cap snatching during mRNA synthesis, as described for other bunyaviruses. Northern blotting identified RNA species of full and reduced lengths, suggested upon analogy with other bunyaviruses to constitute antigenomic-sense cRNA and transcript mRNAs, respectively. Functional studies will be necessary to determine if this group of viruses constitutes a novel genus in the bunyavirus family.

Fig 1

Growth of HEBV and KIBV. (A) C6/36 and U4.4 cells were infected with HEBV and KIBV at MOIs of 0.1 and 0.01, respectively. The genome copy numbers per milliliter in cell culture supernatants were measured by RT-PCR for 5 days. (B) Vertebrate cells were infected with HEBV at the indicated MOIs, and five blind passages at 37°C were performed. The genome copy numbers per milliliter in cell culture supernatants were measured by RT-PCR at 7 days postinfection (solid bars) and at the fifth passage (dashed bars). (C) Cells were infected and passaged as described above for panel B but were incubated at 33°C. Supernatants of the same cell lines infected at different MOIs were pooled, and genome copy numbers were measured by RT-PCR.

Fig 2

Maturation and morphology of HEBV. Shown are ultrathin sections of C6/36 cells infected with HEBV (A and B) and negative-stained ultracentrifuged virions of HEBV (C). Budding arcs are indicated by black arrows, annular spherical particles are indicated by white arrowheads, and dense spherical particles are indicated by black arrowheads. Abbreviations: Nu, nucleus; Mi, mitochondria; Go, Golgi apparatus. Bars = 500 nm (A) and 100 nm (B and C).

Fig 3

Schematic view of the genome organization of HEBV, TAIV, and KIBV. Open reading frames are shown as light yellow boxes, mRNAs are indicated by black arrows, and nontemplate sequences at the 5′ terminus are symbolized by red boxes. Predicted proteins are shown as light blue boxes. Northern blot probes are shown as dark yellow boxes, putative transmembrane domains (hydrophobic regions) are marked by green boxes, glycosylation sites are marked by triangles, the unique region in the RdRp gene is indicated by a light gray box, the endonuclease domain is indicated by a dark gray box, the putative signal peptide is indicated by a blue box, the Gn zinc finger motif is indicated by an orange box, and the Gc fusion peptide is indicated by a dashed box. Genome positions and predicted molecular protein masses are indicated.

Fig 4

Phylogenetic relationships of HEBV, TAIV, and KIBV to representative members of the family Bunyaviridae. Phylogenies were investigated for the RdRp, Gn, Gc, and N proteins based on sizes of 364 aa, 140 aa, 622 aa, and 587 aa, respectively. Maximum likelihood (ML) analyses were performed on a gap-free alignment guided by the BLOSUM62 substitution matrix and using MAFFT (E-INS-I algorithm). Confidence testing was performed by 1,000 bootstrap replicates. Bars indicate evolutionary substitutions per position in the alignments. Smaller pictograms represent ML analyses of HEBV, TAIV, KIBV, all available orthobunyavirus, and tospovirus sequences based on sizes of 3,228 aa, 485 aa, 520 aa, and 331 aa for the RdRp, Gn, Gc, and N proteins, respectively. Abbreviations (and GenBank accession numbers for L, M, and S segments, respectively, in parentheses) are as follows: AGUV, Aguacate virus (accession numbers NC_015451, NC_015450, and NC_015452); AINOV, Aino virus (accession numbers NC_018465, NC_018459, and NC_018460); AKAV, Akabane virus (accession numbers NC_009894, NC_009895, and NC_009896); AMBV, Anhembi virus (accession numbers JN572062, JN572063, and JN572064); ANDV, Andes virus (accession numbers NC_003468, NC_003467, and NC_003466); BeNMV, bean necrotic mosaic virus (accession numbers NC_018070, NC_018072, and NC_018071); BUNV, Bunyamwera virus (accession numbers NC_001925, NC_001926, and NC_001927); CACV, Capsicum chlorosis virus (accession numbers NC_008302, NC_008303, and NC_008301); CDUV, Candiru virus (accession numbers NC_015374, NC_015373, and NC_015375); DOBV, Dobrava virus (accession numbers NC_005235, NC_005234, and NC_005233); GBNV, groundnut bud necrosis virus (accession numbers NC_003614, NC_003620, and NC_003619); GOLV, Gouléako virus (accession numbers HQ541738, HQ541737, and HQ541736); GRSV-TCSV, groundnut ringspot and tomato chlorotic spot virus reassortant (accession numbers NC_015469, NC_015468, and NC_015467); HEBV, Herbert virus (accession numbers JQ659256, JQ659257, and JQ659258); HTNV, Hantaan virus (accession numbers NC_005222, NC_005219, and NC_005218); HVZ10, Hantavirus Z10 virus (accession numbers NC_006435, NC_006437, and NC_006433); INSV, Impatiens necrotic spot virus (accession numbers NC_003625, NC_003616, and NC_003624); KIBV, Kibale virus (accession numbers KF590577, KF590576, and KF590575); LACV, La Crosse virus (accession numbers NC_004108, NC_004109, and NC_004110); LEAV, Leanyer virus (accession numbers HM627178, HM627176, and HM627177); MCAV, Macaua virus (accession numbers JN572068, JN572069, and JN572070); MYSV, melon yellow spot virus (accession numbers NC_008306, NC_008307, and NC_008300); OROV, Oropouche virus (accession numbers NC_005776, NC_005775, and NC_005777); PUUV, Puumala virus (accession numbers NC_005225, NC_005223, and NC_005224); RVFV, Rift Valley fever virus (accession numbers NC_014397, NC_014396, and NC_014395); SATV, Sathuperi virus (accession numbers NC_018461, NC_018466, and NC_018462); SBV, Schmallenberg virus (accession numbers JX853179, JX853180, and JX853181); SEOV, Seoul virus (accession numbers NC_005238, NC_005237, and NC_005236); SFSV, sandfly fever Sicilian virus (accession numbers NC_015412, NC_015411, and NC_015413); SFTSV, severe fever with thrombocytopenia syndrome virus (accession numbers NC_018136, NC_018138, and NC_018137); SHAV, Shamonda virus (accession numbers NC_018463, NC_018467, and NC_018464); SIMV, Simbuvirus (accession numbers NC_018476, NC_018478, and NC_018477); SNV, Sin Nombre virus (accession numbers NC_005217, NC_005215, and NC_005216); SORV, Sororoca virus (accession numbers JN572071, JN572072, and JN572073); TAIV, Taï virus (accession numbers KF590574, KF590573, and KF590572); TOSV, Toscana virus (accession numbers X68414, X89628, and X53794); TPMV, Thottapalayam virus (accession numbers NC_010707, NC_010708, and NC_010704); TSWV, tomato spotted wilt virus (accession numbers NC_002052, NC_002050, and NC_002051); TULV, Tula virus (accession numbers NC_005226, NC_005228, and NC_005227); TZSV, tomato zonate spot virus (accession numbers NC_010491, NC_010490, and NC_010489); UUKV, Uukuniemi virus (accession numbers NC_005214, NC_005220, and NC_005221); WSMOV, watermelon silver mottle virus (accession numbers NC_003832, NC_003841, and NC_003843); WYOV, Wyeomyia virus (accession numbers JN572080, JN572081, and JN572082).

Fig 5

Multiple-sequence alignments of conserved domains of HEBV, TAIV, KIBV, and other bunyaviruses. Alignments were performed by using the E-INS-I algorithm in MAFFT and manually edited. Numbers represent genome positions. Amino acids with 100% identity are highlighted in black, those with 75% identity are highlighted in dark gray, and those with 50% identity are highlighted in light gray. Gn zinc finger motifs are highlighted in black, and conserved basic residues are highlighted in dark gray.

Fig 5

Multiple-sequence alignments of conserved domains of HEBV, TAIV, KIBV, and other bunyaviruses. Alignments were performed by using the E-INS-I algorithm in MAFFT and manually edited. Numbers represent genome positions. Amino acids with 100% identity are highlighted in black, those with 75% identity are highlighted in dark gray, and those with 50% identity are highlighted in light gray. Gn zinc finger motifs are highlighted in black, and conserved basic residues are highlighted in dark gray.

Fig 6

Nontemplated sequences of mRNAs of HEBV and KIBV. Shown are 5′ genome termini of L, M, and S segment mRNAs of HEBV and KIBV. C6/36 cells were infected with HEBV and KIBV, and total RNA was extracted at 1 dpi. Genome termini were amplified by 5′ RACE-PCR, PCR products were cloned, and five random clones were analyzed. Nontemplate sequences (putative transcription primers obtained from host cell mRNAs) are marked by gray boxes. Conserved genome termini of HEBV and KIBV are shown in boldface type.

Fig 7

S segment replication and transcription products analyzed by Northern blotting. Viral RNA was isolated from HEBV- and KIBV-infected C6/36 cells at 2 dpi. RNA from noninfected C6/36 cells was used as a control. A DIG-labeled RNA was used as a size marker (M), with sizes given in nucleotides at the right. Positions of DIG-PCR probes are shown in Fig. 3.

Fig 8

SDS-PAGE analysis of HEBV major structural proteins. Particles were purified from cell culture supernatants of infected C6/36 cells by gradient ultracentrifugation. Proteins were stained with Coomassie blue R-250. Obtained MALDI-TOF data are shown below and LC-MS data are shown above the schematic view of proteins to the right.