RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

Here, we expand upon this finding by describing a genetically distinct, segmented virus isolated from mosquitoes that also exhibits homology to viruses in the family Flaviviridae and that appears to be multicomponent (also termed multiparticle or multipartite;), with each genome segment separately packaged into virions. Although multicomponent genomes are relatively common among RNA viruses that infect plants and fungi, this method of genome organization has not previously been seen in animal viruses (). This virus has tentatively been designated Guaico Culex virus (GCXV), on the basis of the first collection location (near the Guaico community in Trinidad) and the genus of the mosquito that appears to serve as its primary host. Through phylogenetic analysis, we demonstrate that GCXV and JMTV both belong to a highly diverse clade of segmented (and likely multicomponent) viruses, which has recently been termed the Jingmenvirus group (). We also report the detection of a variant of JMTV in a red colobus monkey in Uganda, thus expanding the host range of Jingmenviruses to include primates and highlighting the potential relevance of these viruses to animal and human health.

Although great strides have been made in cataloging viral diversity, the evolutionary mechanisms that have generated this extraordinary variety of genomic organizations are still poorly understood. This is due in part to the extensive genetic divergence that exists between most viruses with different organizations (typically present in distinct families), thus preventing meaningful sequence-based evolutionary comparisons. Occasionally, though, more recent transitions are uncovered, and analysis of these events provides insight into the macroevolution of RNA viruses.reported just such a connection between segmented and unsegmented RNA viruses by describing a segmented virus (Jingmen tick virus [JMTV]) with sequence homology to the Flaviviridae, a large family of vertebrate and invertebrate viruses (including a number of important human pathogens, e.g., Zika, yellow fever, West Nile, dengue, Japanese encephalitis, and hepatitis C viruses) with unsegmented, positive-sense RNA genomes.

The diversity of genome organizations seen in RNA viruses is truly exceptional, surpassing that of any other group of organisms (). Differences are seen in the nature of the genetic material (single or double stranded, positive or negative sense, linear or circular), the number of genome segments (from 1–12), and the manner in which multi-segmented genomes are packaged (together or separately) (). These differences can have important functional implications for key processes such as gene expression, transmission, and genetic recombination. Genome segmentation, for example, can allow better control over gene expression by creating multiple distinct transcriptional units (); however, if these segments are separately packaged, a higher multiplicity of infection will be needed for successful transmission ().

Despite high levels of divergence, both the NS3 and NS5 phylogenies support the segmented Jingmenviruses as a monophyletic group (83%–99% bootstrap support), which is sister to the Tamana bat virus and the Flavivirus genus ( Figure 5 ). The Jingmenviruses collected from insect hosts form a well-supported (69%–100% bootstrap support) sub-clade within this group.

Maximum likelihood (ML) trees are shown with bootstrap support values from both the ML and neighbor-joining (NJ) trees: ML/NJ. The scale indicates the number of amino acid changes per site. See also Table S5

We sequenced one variant of JMTV (RC27) directly from plasma collected from a red colobus monkey in Uganda. A draft genome () was obtained with 70.6% to 98.1% coverage for each of the four genome segments that have been described for JMTV (). RC27 exhibited high similarity to isolates from ticks: 88%–92.6% nt identity across all four segments compared with SY84 from China () and Mogiana tick virus (MGTV) from Brazil (). In species-level phylogenetic analyses, the Chinese isolates () formed a distinct clade compared with the isolates from Uganda and Brazil ( Figure S6 ). Of note, RC27 contained a 450 nt deletion relative to SY84 and MGTV within the ORF of segment 2 (numbered according to). This deletion was observed in reads from metagenomic sequencing and confirmed by RT-PCR and Sanger sequencing. Attempts to isolate this virus on a variety of cell lines were unsuccessful.

Viral replication was detected in three mosquito cell lines and in intrathoracically inoculated adult female mosquitoes ( Figures S5 A and S5B). However, no replication was detected in tick-, sandfly-, or vertebrate-derived cells ( Figures S5 A and S5C), nor did the virus cause any observable illness in intracerebrally inoculated newborn mice, which is often a permissive environment for arbovirus replication. Virus was not detected in the larval progeny of infected mosquitoes, suggesting either the absence or a low occurrence of vertical transmission. No substantial mortality was observed in mosquitoes that survived inoculations during the 14-day viral growth curve, suggesting that GCXV is non-lethal in its mosquito hosts.

Purified GCXV particles (20%–70% sucrose gradient) were 30–35 nm in diameter, spherical, and enveloped ( Figure S4 A). Treatment with NP40 ablated infectivity, confirming the enveloped nature of the virus ( Figure 1 D). Multiple attempts to definitively identify viral particles within infected C6/36 cells using electron microscopy were unsuccessful; however, infected cells contained intracytosolic vacuoles loaded with vesicles 40–50 nm in diameter and dense particles ∼20 nm in diameter ( Figure S4 B). Vesicles of the same type were also observed at the cell surface. This is similar to the pathology previously observed for flavivirus infected C6/36 cells.

The three smallest segments exhibited no significant sequence similarity to known proteins. Five of the six predicted ORFs from segments 3 and 4 were detected in the proteogenomic analysis of viral particles ( Figure S3 A; Table S3 ). This suggests that these segments likely encode structural proteins; however, peptides from the putative NSP2 were also detected, so the purified sample may have had some NSP “contamination.” Although VP2 was not detected with the proteogenomic data, an analysis of synonymous nt conservation (), in the reading frame of VP1, supports the existence of a functional VP2 ORF ( Figure S3 B). Segments 3 and 4 both exhibited evidence for −1 ribosomal frameshifting. In both cases this included (1) overlapping ORFs in the expected orientation, (2) a known slippery heptanucleotide sequence at the end of the first ORF ( Figure 1 C), (3) predicted secondary structure just after the slippery sequence ( Figures S3 C and S3D), and (4) the detection of peptide sequences within the second participating ORF but prior to the first conserved AUG ( Figure S3 A).

Three of the genome segments were monocistronic, while the other two each contained three open reading frames (ORFs) ≥ 400 nt ( Figure 1 C). The sizes and positions of the predicted ORFs were highly conserved, with the exception of viral protein (VP) 6, for which the position of the first AUG differed substantially across isolates ( Table S2 ). The two largest segments each encoded a single ORF, and both exhibited significant protein-level similarity to non-structural (NS) domains that are conserved within the genus Flavivirus. The putative NS protein (NSP) 1 on segment 1 exhibited significant similarity (e-value = 5.02e) to Pfam’s Flavi_NS5 family (PF00972), which corresponds to the RdRp, and Pfam’s FtsJ-like methyltransferase family (1.92e; PF01728). The methyltransferase domain in flaviviruses is required for the formation of the 5′ cap (). The putative NSP2 protein on segment 2 exhibited significant similarity to three Pfam families and domains, all of which correspond to the Flavivirus NS3 protein: Peptidase_S7 (5.17e; PF00949), Flavi_DEAD (2.83e; PF07652) and Helicase_C (8.01e; PF00271). These results suggest that NSP2 participates in at least two of the roles typically played by the Flavivirus NS3: serine protease activity typically used to cleave polypeptides into their mature forms and helicase activity likely involved in viral replication.

None of the segments of GCXV were polyadenylated; however, sequence conservation across segments was seen in the UTRs ( Figure S2 ). The 5′ UTRs exhibited several highly conserved sequence motifs, including the nine most terminal nt, which were strictly conserved across all segments and isolates (5′-AAAUUAAAA-3′), and a larger region just downstream, which is predicted to form a 28- to 31-base stem-loop structure ( Figure S2 A). The 3′ UTRs also exhibited regions of sequence conservation across segments, particularly at the 3′ terminus. Although some sequence variation was seen on segment 3, the last seven nt were highly conserved across segments and isolates, with a consensus sequence of 5′-CCCAUUU-3′. Notably, the four most terminal nt at the 5′ and 3′ ends were complementary. A second highly conserved motif was seen in the 3′ UTRs of segments 1–4 (5′-AAWUAC-3′). This is predicted to form the distal loop in a conserved stem-loop, although the exact size and structure of the stem-loop varies across segments ( Figure S2 ).

Segment-specific probe sets were used to visualize GCXV transcription/replication within C6/36 cells. We detected viral RNA using two combinations of probes: (1) segments 1–3 and (2) segments 3–5. At least one genome segment was detected in ≥90% of the assayed cells, and in general, when multiple segments were present, they appeared to be colocalized in the cytoplasm ( Figure 4 ). Segments 1–3 were detected together in all positive cells, whereas segments 4 and 5 were variably present within cells in which segment 3 was detected ( Figure 4 Table S1 ). The detection of different combinations of segments within individual cells is consistent with independent packaging of genome segments. Although most segments are likely required to successfully complete a full cycle of infection, only a subset of segments are likely required for viral transcription/replication. However, we cannot rule out the presence of undetected segments below our limit of detection.

A representative 63× magnification image is shown from the co-hybridization of probes for segments 3–5. Four different infected cell types were observed: closed arrowhead, missing segments 4 and 5; open arrowhead, missing segment 5; full arrow, missing segment 4 (note that segment 5 present in low abundance in the indicated cell); and dashed arrow, containing all assayed segments. The scale bar represents 10 μm. See also Figure S1 and Table S1

Multicomponent plant viruses were recognized on the basis of deviations from the expected relationship between infectious dose and the number of lesions on infected leaves (i.e., exhibiting multi-hit rather than single-hit kinetics;). We used a similar approach to assay the nature of segment packaging for GCXV using cell culture plaques instead of leaf lesions. The dose-response curve for GCXV differed significantly from expectations for a single-component virus (i.e., the number of plaques decreased more quickly than expected with dilution of the inoculant) ( Figure 3 ) (). Assuming the presence of distinct particles (each containing a subset of genome segments) present in similar amounts and with the same likelihood of invading a cell, we used our dose-response curve to estimate the presence of 3.27 ± 0.37 distinct GCXV particles required for plaque formation. Deviations from this assumption (e.g., variation in the abundance and/or probability of cell invasion for different particle types) would lead to a shallower slope and underestimation of the number of distinct particle types. Therefore, this result is consistent with three to five segments being required for plaque formation, assuming that each segment is separately packaged.

Plaque count is shown on the y axis, and the relative dilution of the virus stock is shown on the x axis. The analysis included five replicates per dilution (circles). The black line is the best-fitting line from a Poisson generalized linear model, with 95% confidence intervals (gray band). The gray lines represent expected slopes for multiplicities of infection of 1–5.

A reverse genetics system was established to test the ability of GCXV to replicate in the absence of segment 5. C6/36 cells were separately transfected with three combinations of in vitro transcribed RNA segments: (1) all five segments, (2) segments 1–4, and (3) segments 2–5 (i.e., no RNA-dependent RNA polymerase [RdRp]; used as a negative control). No cytopathic effect (CPE) was detected in the negative control (segments 2–5), and quantitative RT-PCR (qRT-PCR) confirmed the absence of replication. CPE was observed in the other two transfections (segments 1–5 and 1–4), and RNA extracted after post-transfection passages confirmed the establishment of successful infections both with and without segment 5 ( Figure 2 A).

Nuclease digestion assays ( Figure 1 B), along with 5′ and 3′ rapid amplification of cDNA ends (RACE), confirmed that all five genome segments were single-stranded, positive-sense RNA (ssRNA). Among the GCXV isolates, pairwise nucleotide (nt) divergences, calculated separately for each segment, ranged from 0.2% to 19.9% ( Table S4 ). The phylogenies inferred from segments 2–4 all exhibited essentially identical patterns with isolates clustered by geographic location and with TR7094 as the outlier ( Figure 2 B). However, on segment 1, TR7094 exhibited lower than expected relative levels of divergence and grouped closely with the Panamanian isolates. This is likely indicative of a segment 1 reassortment event having occurred on the lineage leading to TR7094. The phylogeny inferred from segment 5 is also inconsistent with those of the other segments. In addition to the absence of this segment in TR7094 and ACH27, this segment exhibited very low levels of nt divergence (0.4%–2.3%), with most sequence variations only present in a single isolate.

(B) Unrooted, nt-level phylogenetic trees including all six isolates of GCXV. ACH27 and TR7094 lack segment 5. Color indicates country of collection: blue for Peru, red for Panama, and green for Trinidad. Branch labels represent bootstrap support values. The scale bar indicates the number of nt changes per site.

Segment 5 of GCXV Is Not Required for Replication in C6/36 Cells and Is Variably Present in Natural Isolates

Figure 2 Segment 5 of GCXV Is Not Required for Replication in C6/36 Cells and Is Variably Present in Natural Isolates

However, only four segments were assembled for ACH27 and TR7094 (genome size ∼10.6 kb). For these two isolates, the four assembled segments corresponded to the four largest segments assembled in the other isolates ( Figure 1 , segments 1–4). The mosquito pools for both ACH27 and TR7094 contained multiple viruses capable of replicating in mosquito cells, so we were unable to obtain pure cultures for these isolates (). However, despite the complexity of these pools, very high sequencing depth was obtained for the four assembled segments (ACH27: 16,700–40,500×; TR7094: 970–11,680×). TR7094 contained very low levels of LO47 contamination (all five segments), but for ACH27, no Illumina reads aligned to the segment 5 sequences from the other isolates, and no contigs from ACH27 exhibited significant similarity to the segment 5 sequences from the other isolates. Therefore, segment 5 appears to be absent from ACH27 and TR7094.

We sequenced six isolates of GCXV from Culex spp. mosquitoes collected in three countries in Central America and South America ( Table 1 ). Complete genomes were obtained for the isolates from Peru (LO35, LO47) and Trinidad (TR7094); coding-complete (i.e., only missing pieces of non-coding, untranslated regions [UTRs] []) genomes were obtained for the isolates from Panama (GAM204, PCR18-229, ACH27). Five genome segments were assembled for four of the isolates (LO35, LO47, GAM204, and PCR18-229), resulting in a total genome size of ∼12 kb. RNA extracted from purified GCXV particles confirmed the presence of a segmented genome ( Figure 1 A).

(D) Treatment with NP40 resulted in highly diminished RNA copy numbers, lack of growth, and absence of CPE in C6/36 cells, indicating that GCXV is enveloped.

(C) Genome schematic with all ORFs ≥ 400 nt. Dotted lines indicate regions putatively translated through −1 ribosomal frameshifting (arrows indicate slippery heptanucleotides); solid lines indicate the predicted ORFs based on the first conserved AUG.

Discussion

+) have been described with both four and five genome segments; the fifth segment is not required for successful infection or transmission but, when present, is thought to play a role in modulating pathogenesis ( Tamada et al., 1989 Tamada T.

Shirako Y.

Abe H.

Saito M.

Kiguchi T.

Harada T. Production and pathogenicity of isolates of beet necrotic yellow vein virus with different numbers of RNA components. Here we describe a segmented virus that is distantly related to the flaviviruses, which we have tentatively designated GCXV. In vitro and in vivo replication experiments suggest GCXV is mosquito specific, but we cannot definitively rule out replication in other organisms. In contrast to the prototypical flavivirus (i.e., unsegmented, single polyprotein), the genome of GCXV is composed of four or five distinct segments, depending on the isolate. The four largest segments were found in all isolates and are therefore assumed to be necessary for infection and transmission, whereas the fifth segment seems to be optional. VP7 peptides were not detected in purified GCXV virions, suggesting that segment 5 likely encodes an NSP. There was no correlation between the presence and absence of this fifth segment and phylogenetic relationships, as inferred from the four “core” segments ( Figure 2 B), and in vitro experiments confirmed that GCXV can replicate without segment 5. Similarly, natural isolates of Beet Necrotic Yellow Vein Virus (BNYVV; ssRNA) have been described with both four and five genome segments; the fifth segment is not required for successful infection or transmission but, when present, is thought to play a role in modulating pathogenesis (). Also similar to BNYVV, GCXV appears to individually package its genome segments (i.e., is multicomponent). Although a multicomponent organization is not required for segment loss, it provides a straightforward mechanism for the establishment of infections with a subset of genome segments.

Fulton, 1980 Fulton R.W. Biological significance of multicomponent viruses. King et al., 2011 King A.M.

Adams M.J.

Lefkowitz E.J. Flint et al., 2009 Flint S.J.

Enquist L.W.

Racaniello V.R.

Skalka A.M.

Barnum D.R.

de Evaluación E. Principles of Virology Volume I: Molecular Biology. Cui et al. (2014) Cui J.

Schlub T.E.

Holmes E.C. An allometric relationship between the genome length and virion volume of viruses. Multicomponent genome organizations are relatively common among viruses that infect plants and fungi, but no multicomponent animal viruses have been described (). The dose-response kinetics of GCXV indicate that at least three different particles are required for plaque formation. Given that the vast majority of animal viruses require only a single particle to form a plaque (), this result provides strong evidence for a multicomponent virus. Our results from RNA fluorescence in situ hybridization (FISH) and electron microscopy are also consistent with GCXV’s being multicomponent. Although segments 1–3 were detected in all infected cells, segments 4 and 5 were variably present, and the measured particle size for GCXV, ∼30–35 nm, is considerably smaller than that of other flaviviruses. In fact, the spherical volume calculated from this size lies below the 95% confidence limit calculated byfor viruses with 12 kb genomes. Similar to the flaviviruses, however, GCXV particles are enveloped, whereas particles from the multicomponent viruses known to infect plants and fungi do not contain envelopes.

Fulton, 1980 Fulton R.W. Biological significance of multicomponent viruses. Pressing and Reanney, 1984 Pressing J.

Reanney D.C. Divided genomes and intrinsic noise. Reijnders, 1978 Reijnders L. The origin of multicomponent small ribonucleoprotein viruses. García-Arriaza et al., 2004 García-Arriaza J.

Manrubia S.C.

Toja M.

Domingo E.

Escarmís C. Evolutionary transition toward defective RNAs that are infectious by complementation. Goldbach, 1986 Goldbach R. Molecular evolution of plant RNA viruses. Bolling et al., 2012 Bolling B.G.

Olea-Popelka F.J.

Eisen L.

Moore C.G.

Blair C.D. Transmission dynamics of an insect-specific flavivirus in a naturally infected Culex pipiens laboratory colony and effects of co-infection on vector competence for West Nile virus. Multiple theories have been proposed regarding potential benefits of segmented genomes; however, it is unclear whether independent packaging could provide additional benefits (over packaging within a single virion;) or whether this arrangement may simply be a byproduct of the mechanism of segmentation (most likely the formation of complementary defective viral particles;). It has been proposed that multicomponent viruses may represent specialized forms of unsegmented genomes, which are facilitated by the existence of efficient mechanisms of transmission. In fact, transmission inefficiency is thought to be the primary reason for the general lack of multicomponent animal viruses (). Vertical transmission has been shown to be the primary mechanism for the insect-specific Culex flavivirus (); however, vertical transmission was not detected in our experiments with GCXV. Further investigation into the transmission mechanism for GCXV will provide insight into the transmission requirements for multicomponent viruses and the unequal distribution of these viruses among eukaryotic lineages.

Maruyama et al., 2014 Maruyama S.R.

Castro-Jorge L.A.

Ribeiro J.M.C.

Gardinassi L.G.

Garcia G.R.

Brandão L.G.

Rodrigues A.R.

Okada M.I.

Abrão E.P.

Ferreira B.R.

et al. Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil. Webster et al. (2015) Webster C.L.

Waldron F.M.

Robertson S.

Crowson D.

Ferrari G.

Quintana J.F.

Brouqui J.M.

Bayne E.H.

Longdon B.

Buck A.H.

et al. The discovery, distribution, and evolution of viruses associated with Drosophila melanogaster. Shi et al., 2015 Shi M.

Lin X.D.

Vasilakis N.

Tian J.H.

Li C.X.

Chen L.J.

Eastwood G.

Diao X.N.

Chen M.H.

Chen X.

et al. Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the Flaviviridae and related viruses. Callister et al., 2008 Callister D.M.

Winter A.D.

Page A.P.

Maizels R.M. Four abundant novel transcript genes from Toxocara canis with unrelated coding sequences share untranslated region tracts implicated in the control of gene expression. Qin et al., 2014 Qin X.-C.

Shi M.

Tian J.-H.

Lin X.-D.

Gao D.-Y.

He J.-R.

Wang J.-B.

Li C.-X.

Kang Y.-J.

Yu B.

et al. A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors. All of the sequenced Jingmenviruses appear to have at least four genome segments, and homology can be inferred for at least three of these segments across all viruses. Although MGTV was not initially recognized as being segmented, we used the published Illumina data set (SRR525284;) to assemble nearly full-length contigs with sequence homology to all four segments of JMTV (see Supplemental Experimental Procedures ). Similarly, althoughonly reported one segment (encoding the putative RdRp) for the three Jingmenviruses they sequenced, we identified several additional contigs in their data set with significant sequence homology to segments 1–4 from GCXV (see Supplemental Experimental Procedures ). All of these viruses contain separate monocistronic segments with sequence homology to the Flavivirus NS3 and NS5 proteins. Additionally, they all have one multicistronic segment with two partially overlapping ORFs, consistent with −1 ribosomal frameshifting; the first ORF is predicted to have a signal peptide, and the second contains many predicted transmembrane helices. Protein sequence homology is detectable for this segment between TCLA (ANT-3) and JMTV/MGTV (segment 4) and also among the nine insect-associated viruses, including GCXV (segment 4 in, segment 3 for GCXV). However, no sequence-level similarity is detectable for this segment between these two groups. GCXV, JMTV, and TCLA all exhibit some degree of sequence conservation (across segments) in the non-coding UTRs (), and these regions may play a role in the initiation of translation and/or replication.

On the basis of the apparent homology of at least three segments, it is likely that the Jingmenviruses shared a segmented common ancestor. The existence of a common, segmented ancestor suggests that the multicomponent organization seen in GCXV is likely also shared by the other Jingmenviruses. However, the method of packaging has not been investigated in any of these other viruses.

Despite a common origin, levels of sequence divergence among the different Jingmenviruses were high. In fact, average levels of amino acid divergence among the Jingmenviruses (NS5: 59.8%; NS3: 70.2%) were higher than the maximum divergence seen among viruses within the Flavivirus genus (NS5: 57%; NS3: 69%). Structural differences are also evident among the Jingmenviruses. These differences include genome segments with distinct coding strategies and no measurable homology (e.g., GCXV multicistronic segment 4 versus JMTV monocistronic segment 2) and the presence of polyadenylated 3′ termini only in JMTV/MGTV and TCLA ( Table 2 ). On the basis of the extensive sequence-level and structural genomic diversity, this clade of segmented flavi-like viruses appears to be quite old, and it likely includes substantial viral diversity that has yet to be described.

Simon-Loriere and Holmes, 2011 Simon-Loriere E.

Holmes E.C. Why do RNA viruses recombine?. Zeddam et al., 2010 Zeddam J.L.

Gordon K.H.

Lauber C.

Alves C.A.

Luke B.T.

Hanzlik T.N.

Ward V.K.

Gorbalenya A.E. Euprosterna elaeasa virus genome sequence and evolution of the Tetraviridae family: emergence of bipartite genomes and conservation of the VPg signal with the dsRNA Birnaviridae family. Because of the high levels of divergence and the lack of an appropriate outgroup, it is not possible to use sequence information to accurately determine the polarity of the transition between segmented and unsegmented genomes. One possibility is that the segmented genome is ancestral and that these pieces were later joined together to form the prototypical, unsegmented flavivirus genome. Such a transition could be mediated by repeated episodes of non-homologous copy-choice recombination (); however, to our knowledge no such transitions have been described. The alternative is that these segmented viruses evolved from an unsegmented ancestor. Phylogenetic analysis of the Tetraviridae supports a similar transition between the betatetraviruses (monopartite) and the omegatetraviruses (bipartite), which is hypothesized to have been mediated through the formation of subgenomic RNA molecules ().

Qin et al. (2014) Qin X.-C.

Shi M.

Tian J.-H.

Lin X.-D.

Gao D.-Y.

He J.-R.

Wang J.-B.

Li C.-X.

Kang Y.-J.

Yu B.

et al. A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors. Chambers et al., 1990 Chambers T.J.

Hahn C.S.

Galler R.

Rice C.M. Flavivirus genome organization, expression, and replication. On the basis of the lack of sequence similarity to flaviviruses at two of four segments (those thought to encode the structural proteins),argued that JMTV is likely of hybrid origin, resulting from the coinfection of a flavivirus and a second, as of yet, uncharacterized virus. Although we cannot rule out this scenario, segmentation of a single flavivirus-like ancestor presents a more parsimonious explanation. The patterns of sequence divergence and similarity seen between members of the genus Flavivirus and both JMTV and GCXV are as expected between very divergent flaviviruses, with weak but significant similarity at the highly conserved NS genes, but a lack of detectable homology at the structural genes, which typically exhibit higher rates of evolution (). In fact, this is exactly the same pattern seen between GCXV and JMTV. Therefore, in the absence of an identified “donor” group for the putative structural genes of JMTV and GCXV, the most parsimonious scenario involves the segmentation of a single, flavivirus-like ancestor.

In summary, we have described a multicomponent virus, which belongs to a diverse clade of segmented viruses related to the family Flaviviridae. We have also described a variant of JMTV, the prototype species for this clade, from a non-human primate, thus substantially expanding the host range of the group and indicating potential implications for animal and human health. This clade establishes a strong link between two distinct genome organizations, which may help uncover some of the mysteries associated with the evolution of genome architecture in RNA viruses; however, methods of inference beyond sequence-level homology will be needed to reconstruct the evolutionary history that connects these genome types. The existence of an enveloped, multicomponent animal virus will require us to rethink historical perspectives regarding the advantages and requirements of this type of genome organization.