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

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

Within- and between-host patterns of selection.

<p>(A) Mapping of the distributions of synonymous (S) and nonsynonymous (N) single nucleotide polymorphisms along the ORF for 13 of 28 red colobus samples, for which coverage-depth was greater than 100 reads across most (≥96%) of the ORF. (B) Distribution of average substitution rates at non-synonymous (dN) and synonymous (dS) sites, and their ratio (dN/dS), along a sliding window (100 aa window, 20 aa step) for the comparison of selection pressures among the Kibale SPgVs (accession no. KF234523, KF234526 and KF234530).</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