BackgroundSimian 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.Methodology/Principal FindingsThe 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.Conclusions/SignificanceThese 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.

h GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.

Primate gastrointestinal microbial communities are becoming increasingly appreciated for their relevance to comparative medicine and conservation, but the factors that structure primate “microbiomes” remain controversial. This study examined a community of primates in Kibale National Park, Uganda, to assess the relative importance of host species and location in structuring gastrointestinal microbiomes. Fecal samples were collected from primates in intact forest and from primates in highly disturbed forest fragments. People and livestock living nearby were also included, as was a geographically distant population of related red colobus in Kenya. A culture-free microbial community fingerprinting technique was used to analyze fecal microbiomes from 124 individual red colobus (Procolobus rufomitratus), 100 individual black-and-white colobus (Colobus guereza), 111 individual red-tailed guenons (Cercopithecus ascanius), 578 human volunteers, and 364 domestic animals, including cattle (Bos indicus and B. indicus × B. taurus crosses), goats (Caprus hircus), sheep (Ovis aries), and pigs (Sus scrofa). Microbiomes sorted strongly by host species, and forest fragmentation did not alter this pattern. Microbiomes of Kenyan red colobus sorted distinctly from microbiomes of Ugandan red colobus, but microbiomes from these two red colobus populations clustered more closely with each other than with any other species. Microbiomes from red colobus and black-and-white colobus were more differentiated than would be predicted by the phylogenetic relatedness of these two species, perhaps reflecting heretofore underappreciated differences in digestive physiology between the species. Within Kibale, social group membership influenced intra-specific variation among microbiomes. However, intra-specific variation was higher among primates in forest fragments than among primates in intact forest, perhaps reflecting the physical separation of fragments. These results suggest that, in this system, species-specific processes such as gastrointestinal physiology strongly structure microbial communities, and that primate microbiomes are relatively resistant to perturbation, even across large geographic distances or in the face of habitat disturbance.

h Simian hemorrhagic fever virus (SHFV) is an arterivirus that causes severe disease in captive macaques. We describe two new SHFV variants subclinically infecting wild African red-tailed guenons (Cercopithecus ascanius). Both variants are highly divergent from the prototype virus and variants infecting sympatric red colobus (Procolobus rufomitratus). All known SHFV variants are monophyletic and share three open reading frames not present in other arteriviruses. Our data suggest a need to modify the current arterivirus classification. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, together with equine arteritis virus (EAV), lactate dehydrogenase elevating virus (LDV), and porcine reproductive and respiratory syndrome virus (PRRSV) (1). SHFV was first isolated from captive macaques (Macaca sp.) in 1964 after nearly simultaneous outbreaks in Soviet and American primate centers (2-4), possibly having originated from subclinically infected wild-caught patas monkeys (Erythrocebus patas), green monkeys (Chlorocebus aethiops), or guinea baboons (Papio papio) (5,6). Much of what is known about SHFV comes from prototype variants LVR 42-0/M6941 and Sukhumi, isolated during the original two outbreaks, and their derivatives (4, 7).We recently discovered two novel SHFV variants infecting a male red colobus monkey (Procolobus rufomitratus) from Kibale National Park, Uganda (8). These viruses are highly divergent from each other and the prototype variants but, like the prototype variants, contain three unique open reading frames (ORFs) (2a, 2b, and 3) downstream from the replicase-encoding ORFs (8,9). This genomic architecture may be characteristic of the SHFV taxon.Here, we report the discovery of two novel SHFV variants in red-tailed guenons (Cercopithecus ascanius) from the same location where we previously reported SHFV in a red colobus (8). In 2010, we sampled 13 Kibale red-tailed guenons as part of a larger study of primate ecology, conservation, and health (10). Animals were anesthetized and samples were collected as previously described (8). Viral RNA was prepared from blood plasma for direct sequencing as previously described (8), with minor modifications for sequencing on an Illumina MiSeq instrument. De novo assembly of sequence reads yielded complete SHFV coding genomes from three individuals (two females, RT05 and RT11, and one male, RT10). A fourth nearly complete SHFV coding genome was recovered from another male individual, RT09, and small gaps were filled by PCR and 3= rapid amplification of cDNA ends (RACE) followed by Sanger sequencing (8).Viral genomes were annotated with CLC Genomics Workbench version 5.5 (CLC Bio, Aarhus, Denmark), and putative ORFs were confirmed by querying the NCBI GenBank database (11). Open reading frames were individually aligned with prototype variant LVR 42-0/M6941 and red colobus variants SHFV-krc1 and SHFV-krc2 using a codon-based version of the MAFFT algorithm (12) implemented in Translator X (13). Individual ORF alignments were then concatenated...