A Multicomponent Animal Virus Isolated from Mosquitoes

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.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

Novel, divergent simian hemorrhagic fever viruses in a wild Ugandan red colobus monkey discovered using direct pyrosequencing.

Simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but its distribution in wild primates has remained obscure. Here, we describe the discovery and genetic characterization by direct pyrosequencing of two novel, divergent SHFV variants co-infecting a single male red colobus monkey from Kibale National Park, Uganda.The viruses were detected directly from blood plasma using pyrosequencing, without prior virus isolation and with minimal PCR amplification. The two new SHFV variants, SHFV-krc1 and SHFV-krc2 are highly divergent from each other (51.9% nucleotide sequence identity) and from the SHFV type strain LVR 42-0/M6941 (52.0% and 51.8% nucleotide sequence identity, respectively) and demonstrate greater phylogenetic diversity within SHFV than has been documented within any other arterivirus. Both new variants nevertheless have the same 3' genomic architecture as the type strain, containing three open reading frames not present in the other arteriviruses.These results represent the first documentation of SHFV in a wild primate and confirm the unusual 3' genetic architecture of SHFV relative to the other arteriviruses. They also demonstrate a degree of evolutionary divergence within SHFV that is roughly equivalent to the degree of divergence between other arterivirus species. The presence of two such highly divergent SHFV variants co-infecting a single individual represents a degree of within-host viral diversity that exceeds what has previously been reported for any arterivirus. These results expand our knowledge of the natural history and diversity of the arteriviruses and underscore the importance of wild primates as reservoirs for novel pathogens

Discovery and characterization of distinct simian pegiviruses in three wild African Old World monkey species.

Within the Flaviviridae, the recently designated genus Pegivirus has expanded greatly due to new discoveries in bats, horses, and rodents. Here we report the discovery and characterization of three simian pegiviruses (SPgV) that resemble human pegivirus (HPgV) and infect red colobus monkeys (Procolobus tephrosceles), red-tailed guenons (Cercopithecus ascanius) and an olive baboon (Papio anubis). We have designated these viruses SPgVkrc, SPgVkrtg and SPgVkbab, reflecting their host species' common names, which include reference to their location of origin in Kibale National Park, Uganda. SPgVkrc and SPgVkrtg were detected in 47% (28/60) of red colobus and 42% (5/12) red-tailed guenons, respectively, while SPgVkbab infection was observed in 1 of 23 olive baboons tested. Infections were not associated with any apparent disease, despite the generally high viral loads observed for each variant. These viruses were monophyletic and equally divergent from HPgV and pegiviruses previously identified in chimpanzees (SPgVcpz). Overall, the high degree of conservation of genetic features among the novel SPgVs, HPgV and SPgVcpz suggests conservation of function among these closely related viruses. Our study describes the first primate pegiviruses detected in Old World monkeys, expanding the known genetic diversity and host range of pegiviruses and providing insight into the natural history of this genus

Pair-wise percent nucleotide identities among arteriviruses, including two new SHFV variants from Kibale red colobus (SHFV-krc1 and SHFV-krc2) and prototype <i>Arterivirus</i> strains^a.

a<p>Values are uncorrected pair-wise percent nucleotide identities of aligned, concatenated ORFs 1a, 1b, 2a, 2b, and 3–7, with reference to the EAV genome (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019056#pone-0019056-g001" target="_blank">Figure 1</a>). Other prototypical viruses included in the analysis are (GenBank accession numbers in parentheses): SHFV-LVR, the SHFV type strain LVR 42-0/M6941 (NC_003092.1); LDV-Plagemann strain (U15146.1); PRRSV-Lelystad strain (M96262.2); and EAV-Bucyrus strain (NC_002532.2).</p

Phylogenetic tree of newly discovered simian hemorrhagic fever viruses and other arteriviruses.

<p>The novel variants SHFV-krc1 and SHFV-krc2 are highlighted. Other viruses included in the analysis were chosen to represent the diversity within each viral species based on available full-genome sequences (GenBank accession numbers in parentheses): SHFV-LVR, the SHFV type strain LVR 42-0/M6941 (NC_003092.1); PRRSV-Lelystad, the European (type 1) type strain (M96262.2); PRRSV-VR2332, the North American (type 2) type strain (U87392.3); EAV-Bucyrus strain (NC_002532.2); EAV-s3685 strain (GQ903794.1); LDV-P, the Plagemann strain (U15146.1); and LDV-C, the neuro-virulent strain (L13298.1). This unrooted tree was the likeliest of all trees (−ln L 41,541) found during a maximum likelihood branch-and-bound search with the computer program PAUP* 4.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019056#pone.0019056-Swofford1" target="_blank">[17]</a>. Numbers beside internal nodes indicate statistical support for individual clades (percent), based on 1000 bootstrap replicates of the data. The scale bar indicates genetic distance (nucleotide substitutions per site).</p

Genome organization of novel simian hemorrhagic fever viruses from a Ugandan red colobus monkey.

<p>The novel variants SHFV-krc1 and SHFV-krc2 are shown in comparison to the SHFV type strain LVR 42-0/M6941 and the prototype <i>Arterivirus</i>, equine arteritis virus (EAV-Bucyrus strain). Boxes represent open reading frames and are drawn to scale. Shaded boxes indicate ORFs unique to SHFV, with dashed lines indicating the location of putative insertion relative to the EAV genome.</p

Neighbor-joining amino acid phylogeny of the NS3 helicase comprising 43 pegivirus and 25 hepacivirus sequences.

<p>This 97-aa segment (polyprotein positions 1221 to 1317 relative to HPgV, NC_001710) is highly conserved among the <i>Flaviviridae</i> and has been targeted extensively for virus discovery and phylogenetic characterization. The sequences included in this analysis encompass the full genetic diversity of identified pegiviruses within each clade, minus those for which NS3 sequence data were unavailable, namely pegiviruses infecting the common marmoset, <i>Callithrix jacchus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569-Bukh1" target="_blank">[7]</a> and several recently identified viruses infecting bats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569-Quan1" target="_blank">[12]</a>. Inclusion of the two most diverse variants of both SPgV_krc and SPgV_krtg demonstrated the relatively high within-host similarity of these viruses within the study population. GenBank accession numbers for the included taxa are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569.s005" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098569#pone.0098569.s006" target="_blank">S2</a>.</p