Discovery of Lanama Virus, a Distinct Member of Species Kunsagivirus C (Picornavirales: Picornaviridae), in Wild Vervet Monkeys (Chlorocebus pygerythrus).

We report the discovery and sequence-based molecular characterization of a novel virus, lanama virus (LNMV), in blood samples obtained from two wild vervet monkeys (Chlorocebus pygerythrus), sampled near Lake Nabugabo, Masaka District, Uganda. Sequencing of the complete viral genomes and subsequent phylogenetic analysis identified LNMV as a distinct member of species Kunsagivirus C, in the undercharacterized picornavirid genus Kunsagivirus.

Multidecade Mortality and a Homolog of Hepatitis C Virus in Bald Eagles (Haliaeetus leucocephalus), the National Bird of the USA.

The bald eagle (Haliaeetus leucocephalus) once experienced near-extinction but has since rebounded. For decades, bald eagles near the Wisconsin River, USA, have experienced a lethal syndrome with characteristic clinical and pathological features but unknown etiology. Here, we describe a novel hepacivirus-like virus (Flaviviridae: Hepacivirus) identified during an investigation of Wisconsin River eagle syndrome (WRES). Bald eagle hepacivirus (BeHV) belongs to a divergent clade of avian viruses that share features with members of the genera Hepacivirus and Pegivirus. BeHV infected 31.9% of eagles spanning 4,254 km of the coterminous USA, with negative strand viral RNA demonstrating active replication in liver tissues. Eagles from Wisconsin were approximately 10-fold more likely to be infected than eagles from elsewhere. Eagle mitochondrial DNA sequences were homogeneous and geographically unstructured, likely reflecting a recent population bottleneck, whereas BeHV envelope gene sequences showed strong population genetic substructure and isolation by distance, suggesting localized transmission. Cophylogenetic analyses showed no congruity between eagles and their viruses, supporting horizontal rather than vertical transmission. These results expand our knowledge of the Flaviviridae, reveal a striking pattern of decoupled host/virus coevolution on a continental scale, and highlight knowledge gaps about health and conservation in even the most iconic of wildlife species.

Molecular Analysis of the Complete Genome of a Simian Foamy Virus Infecting Hylobates pileatus (pileated gibbon) Reveals Ancient Co-Evolution with Lesser Apes.

Foamy viruses (FVs) are complex retroviruses present in many mammals, including nonhuman primates, where they are called simian foamy viruses (SFVs). SFVs can zoonotically infect humans, but very few complete SFV genomes are available, hampering the design of diagnostic assays. Gibbons are lesser apes widespread across Southeast Asia that can be infected with SFV, but only two partial SFV sequences are currently available. We used a metagenomics approach with next-generation sequencing of nucleic acid extracted from the cell culture of a blood specimen from a lesser ape, the pileated gibbon (Hylobates pileatus), to obtain the complete SFVhpi_SAM106 genome. We used Bayesian analysis to co-infer phylogenetic relationships and divergence dates. SFVhpi_SAM106 is ancestral to other ape SFVs with a divergence date of ~20.6 million years ago, reflecting ancient co-evolution of the host and SFVhpi_SAM106. Analysis of the complete SFVhpi_SAM106 genome shows that it has the same genetic architecture as other SFVs but has the longest recorded genome (13,885-nt) due to a longer long terminal repeat region (2,071 bp). The complete sequence of the SFVhpi_SAM106 genome fills an important knowledge gap in SFV genetics and will facilitate future studies of FV infection, transmission, and evolutionary history.

Selective constraint and adaptive potential of West Nile virus within and among naturally infected avian hosts and mosquito vectors.

Arthropod-borne viruses are among the most genetically constrained RNA viruses, yet they have a remarkable propensity to adapt and emerge. We studied wild birds and mosquitoes naturally infected with West Nile virus (WNV) in a 'hot spot' of virus transmission in Chicago, IL, USA. We generated full coding WNV genome sequences from spatiotemporally matched bird and mosquito samples using high-throughput sequencing, allowing a molecular evolutionary assessment with deep coverage. Mean FST among samples was 0.66 (±0.02 SE) and was bimodal, with mean nucleotide diversity being higher between samples (interhost πN = 0.001; πS = 0.024) than within them (intrahost πN < 0.0001; πS < 0.001). Eight genomic sites with FST > 1.01 (in the PrM, NS2a, NS3, NS4b, and 5'-noncoding genomic regions) showed bird versus mosquito variant frequency differences of >30 per cent and/or polymorphisms fixed in ≥5 host or vector individuals, suggesting host tropism for these variants. However, phylogenetic analyses demonstrated a lack of grouping by bird or mosquito, most inter-sample differences were synonymous (mean interhost πN/πS = 0.04), and there was no significant difference between hosts and vectors in either their nucleotide diversities or levels of purifying selection (mean intrahost πN/πS = 0.28 in birds and πN/πS = 0.21 in mosquitoes). This finding contrasts with the 'trade-off' and 'selective sieve' hypotheses that have been proposed and tested in the laboratory, which predict strong host versus vector effects on WNV genetic variation, with heightened selective constraint in birds alternating with heightened viral diversity in mosquitoes. Overall, our data show WNV to be highly selectively constrained within and between both hosts and vectors but still able to vary at a limited number of sites across the genome. Such site-specific plasticity in the face of overall selective constraint may offer a mechanism whereby highly constrained viruses such as WNV and its relatives can still adapt and emerge.

Severe neurologic disease and chick mortality in crested screamers (Chauna torquata) infected with a novel Gyrovirus.

Gyroviruses are small, single stranded DNA viruses in the family Anelloviridae. In chickens, the type virus (chicken anemia virus; CAV) causes epidemic disease in poultry flocks worldwide. In 2007 and 2008, young crested screamers (Chauna torquata) at a zoo in Wisconsin, USA, died of neurologic disease with clinical and pathological features resembling CAV infection. Conventional diagnostics were negative, but molecular analyses revealed coinfection of an affected bird with three variants of a novel Gyrovirus lineage, GyV10. Analysis of ten additional screamers from this and another zoo revealed infection in all but one bird, with co-infections and persistent infections common. The association between GyV10 ("screamer anemia virus," provisionally) and the disease remains unproven, but certain immunological and neurologic features of the syndrome would expand the known pathologic consequences of Gyrovirus infection. To control the virus, autogenous vaccines, environmental decontamination, and management strategies to limit vertical and horizontal transmission might prove effective.

Metagenomic assessment of adventitious viruses in commercial bovine sera.

Animal serum is an essential supplement for cell culture media. Contamination of animal serum with adventitious viruses has led to major regulatory action and product recalls. We used metagenomic methods to detect and characterize viral contaminants in 26 bovine serum samples from 12 manufacturers. Across samples, we detected sequences with homology to 20 viruses at depths of up to 50,000 viral reads per million. The viruses detected represented nine viral families plus four taxonomically unassigned viruses and had both RNA genomes and DNA genomes. Sequences ranged from 28% to 96% similar at the amino acid level to viruses in the GenBank database. The number of viruses varied from zero to 11 among samples and from one to 11 among suppliers, with only one product from one supplier being entirely "clean." For one common adventitious virus, bovine viral diarrhea virus (BVDV), abundance estimates calculated from metagenomic data (viral reads per million) closely corresponded to Ct values from quantitative real-time reverse transcription polymerase chain reaction (rtq-PCR), with metagenomics being approximately as sensitive as rtq-PCR. Metagenomics is useful for detecting taxonomically and genetically diverse adventitious viruses in commercial serum products, and it provides sensitive and quantitative information.

Identification by next-generation sequencing of Aichivirus B in a calf with enterocolitis and neurologic signs.

An 11-d-old Holstein bull calf was presented to the Veterinary Medical Teaching Hospital at the University of Wisconsin-Madison because of a 4-d history of diarrhea and persistent low-grade fever. Initial diagnosis was enteritis caused by Cryptosporidium and rotavirus. During hospitalization, the calf became stuporous and was only responsive to noxious stimuli, with hypotonia of all 4 limbs, tail, head, and neck. A cerebrospinal fluid analysis revealed xanthochromia, with marked lymphocytic pleocytosis, which was suggestive of viral meningitis and/or encephalitis. Aichivirus B, which belongs to the Kobuvirus genus, was tentatively identified in spinal fluid by next-generation DNA sequencing. This virus can affect a multitude of species, including humans and cattle, and has been isolated from both healthy and diarrheic individuals. However, to date, a possible connection with neurologic disease has not been described, to our knowledge.

Novel reovirus associated with epidemic mortality in wild largemouth bass (Micropterus salmoides).

Reoviruses (family Reoviridae) infect vertebrate and invertebrate hosts with clinical effects ranging from inapparent to lethal. Here, we describe the discovery and characterization of Largemouth bass reovirus (LMBRV), found during investigation of a mortality event in wild largemouth bass (Micropterus salmoides) in 2015 in WI, USA. LMBRV has spherical virions of approximately 80 nm diameter containing 10 segments of linear dsRNA, aligning it with members of the genus Orthoreovirus, which infect mammals and birds, rather than members of the genus Aquareovirus, which contain 11 segments and infect teleost fishes. LMBRV is only between 24 % and 68 % similar at the amino acid level to its closest relative, Piscine reovirus (PRV), the putative cause of heart and skeletal muscle inflammation of farmed salmon. LMBRV expands the known diversity and host range of its lineage, which suggests that an undiscovered diversity of related pathogenic reoviruses may exist in wild fishes.

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.

Definitive Hosts of Versteria Tapeworms (Cestoda: Taeniidae) Causing Fatal Infection in North America.

We previously reported fatal infection of a captive Bornean orangutan with metacestodes of a novel taeniid tapeworm, Versteria sp. New data implicate mustelids as definitive hosts of these tapeworms in North America. At least 2 parasite genetic lineages circulate in North America, representing separate introductions from Eurasia.

Zoonotic Potential of Simian Arteriviruses.

Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be "preemergent" zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.

Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection.

Human pegivirus (HPgV)-formerly known as GB virus C and hepatitis G virus-is a poorly characterized RNA virus that infects about one-sixth of the global human population and is transmitted frequently in the blood supply. We create an animal model of HPgV infection by infecting macaque monkeys with a new simian pegivirus (SPgV) discovered in wild baboons. Using this model, we provide a high-resolution, longitudinal picture of SPgV viremia where the dose, route, and timing of infection are known. We detail the highly variable acute phase of SPgV infection, showing that the viral load trajectory early in infection is dependent on the infecting dose, whereas the chronic-phase viremic set point is not. We also show that SPgV has an extremely low propensity for accumulating sequence variation, with no consensus-level variants detected during the acute phase of infection and an average of only 1.5 variants generated per 100 infection-days. Finally, we show that SPgV RNA is highly concentrated in only two tissues: spleen and bone marrow, with bone marrow likely producing most of the virus detected in plasma. Together, these results reconcile several paradoxical observations from cross-sectional analyses of HPgV in humans and provide an animal model for studying pegivirus biology.

Two novel simian arteriviruses in captive and wild baboons (Papio spp.).

UNLABELLED: Since the 1960s, simian hemorrhagic fever virus (SHFV; Nidovirales, Arteriviridae) has caused highly fatal outbreaks of viral hemorrhagic fever in captive Asian macaque colonies. However, the source(s) of these outbreaks and the natural reservoir(s) of this virus remain obscure. Here we report the identification of two novel, highly divergent simian arteriviruses related to SHFV, Mikumi yellow baboon virus 1 (MYBV-1) and Southwest baboon virus 1 (SWBV-1), in wild and captive baboons, respectively, and demonstrate the recent transmission of SWBV-1 among captive baboons. These findings extend our knowledge of the genetic and geographic diversity of the simian arteriviruses, identify baboons as a natural host of these viruses, and provide further evidence that baboons may have played a role in previous outbreaks of simian hemorrhagic fever in macaques, as has long been suspected. This knowledge should aid in the prevention of disease outbreaks in captive macaques and supports the growing body of evidence that suggests that simian arterivirus infections are common in Old World monkeys of many different species throughout Africa. IMPORTANCE: Historically, the emergence of primate viruses both in humans and in other primate species has caused devastating outbreaks of disease. One strategy for preventing the emergence of novel primate pathogens is to identify microbes with the potential for cross-species transmission in their natural state within reservoir species from which they might emerge. Here, we detail the discovery and characterization of two related simian members of the Arteriviridae family that have a history of disease emergence and host switching. Our results expand the phylogenetic and geographic range of the simian arteriviruses and define baboons as a natural host for these viruses. Our findings also identify a potential threat to captive macaque colonies by showing that simian arteriviruses are actively circulating in captive baboons.

Discovery and full genome characterization of a new SIV lineage infecting red-tailed guenons (Cercopithecus ascanius schmidti) in Kibale National Park, Uganda.

BACKGROUND: Human immunodeficiency virus (HIV) type 1 and 2, the causative agents of acquired immunodeficiency syndrome (AIDS), emerged from African non-human primates (NHPs) through zoonotic transmission of simian immunodeficiency viruses (SIV). Among African NHPs, the Cercopithecus genus contains the largest number of species known to harbor SIV. However, our understanding of the diversity and evolution of SIVs infecting this genus is limited by incomplete taxonomic and geographic sampling, particularly in East Africa. In this study, we screened blood specimens from red-tailed guenons (Cercopithecus ascanius schmidti) from Kibale National Park, Uganda, for the presence of novel SIVs using unbiased deep-sequencing. FINDINGS: We describe and characterize the first full-length SIV genomes from wild red-tailed guenons in Kibale National Park, Uganda. This new virus, tentatively named SIVrtg_Kib, was detected in five out of twelve animals and is highly divergent from other Cercopithecus SIVs as well as from previously identified SIVs infecting red-tailed guenons, thus forming a new SIV lineage. CONCLUSIONS: Our results show that the genetic diversity of SIVs infecting red-tailed guenons is greater than previously appreciated. This diversity could be the result of cross-species transmission between different guenon species or limited gene flow due to geographic separation among guenon populations.

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.

High genetic diversity and adaptive potential of two simian hemorrhagic fever viruses in a wild primate population.

Key biological properties such as high genetic diversity and high evolutionary rate enhance the potential of certain RNA viruses to adapt and emerge. Identifying viruses with these properties in their natural hosts could dramatically improve disease forecasting and surveillance. Recently, we discovered two novel members of the viral family Arteriviridae: simian hemorrhagic fever virus (SHFV)-krc1 and SHFV-krc2, infecting a single wild red colobus (Procolobus rufomitratus tephrosceles) in Kibale National Park, Uganda. Nearly nothing is known about the biological properties of SHFVs in nature, although the SHFV type strain, SHFV-LVR, has caused devastating outbreaks of viral hemorrhagic fever in captive macaques. Here we detected SHFV-krc1 and SHFV-krc2 in 40% and 47% of 60 wild red colobus tested, respectively. We found viral loads in excess of 10(6)-10(7) RNA copies per milliliter of blood plasma for each of these viruses. SHFV-krc1 and SHFV-krc2 also showed high genetic diversity at both the inter- and intra-host levels. Analyses of synonymous and non-synonymous nucleotide diversity across viral genomes revealed patterns suggestive of positive selection in SHFV open reading frames (ORF) 5 (SHFV-krc2 only) and 7 (SHFV-krc1 and SHFV-krc2). Thus, these viruses share several important properties with some of the most rapidly evolving, emergent RNA viruses.

Deep sequencing identifies two genotypes and high viral genetic diversity of human pegivirus (GB virus C) in rural Ugandan patients.

Human pegivirus (HPgV), formerly 'GB virus C' or 'hepatitis G virus', is a member of the genus Flavivirus (Flaviviridae) that has garnered significant attention due to its inhibition of HIV, including slowing disease progression and prolonging survival in HIV-infected patients. Currently, there are six proposed HPgV genotypes that have roughly distinct geographical distributions. Genotypes 2 and 3 are the most comprehensively characterized, whereas those genotypes occurring on the African continent, where HPgV prevalence is highest, are less well studied. Using deep sequencing methods, we identified complete coding HPgV sequences in four of 28 patients (14.3%) in rural Uganda, east Africa. One of these sequences corresponds to genotype 1 and is the first complete genome of this genotype from east Africa. The remaining three sequences correspond to genotype 5, a genotype that was previously considered exclusively South African. All four positive samples were collected within a geographical area of less than 25 km(2), showing that multiple HPgV genotypes co-circulate in this area. Analysis of intra-host viral genetic diversity revealed that total single-nucleotide polymorphism frequency was approximately tenfold lower in HPgV than in hepatitis C virus. Finally, one patient was co-infected with HPgV and HIV, which, in combination with the high prevalence of HIV, suggests that this region would be a useful locale to study the interactions and co-evolution of these viruses.

Discovery and full genome characterization of two highly divergent simian immunodeficiency viruses infecting black-and-white colobus monkeys (Colobus guereza) in Kibale National Park, Uganda.

BACKGROUND: African non-human primates (NHPs) are natural hosts for simian immunodeficiency viruses (SIV), the zoonotic transmission of which led to the emergence of HIV-1 and HIV-2. However, our understanding of SIV diversity and evolution is limited by incomplete taxonomic and geographic sampling of NHPs, particularly in East Africa. In this study, we screened blood specimens from nine black-and-white colobus monkeys (Colobus guereza occidentalis) from Kibale National Park, Uganda, for novel SIVs using a combination of serology and "unbiased" deep-sequencing, a method that does not rely on genetic similarity to previously characterized viruses. RESULTS: We identified two novel and divergent SIVs, tentatively named SIVkcol-1 and SIVkcol-2, and assembled genomes covering the entire coding region for each virus. SIVkcol-1 and SIVkcol-2 were detected in three and four animals, respectively, but with no animals co-infected. Phylogenetic analyses showed that SIVkcol-1 and SIVkcol-2 form a lineage with SIVcol, previously discovered in black-and-white colobus from Cameroon. Although SIVkcol-1 and SIVkcol-2 were isolated from the same host population in Uganda, SIVkcol-1 is more closely related to SIVcol than to SIVkcol-2. Analysis of functional motifs in the extracellular envelope glycoprotein (gp120) revealed that SIVkcol-2 is unique among primate lentiviruses in containing only 16 conserved cysteine residues instead of the usual 18 or more. CONCLUSIONS: Our results demonstrate that the genetic diversity of SIVs infecting black-and-white colobus across equatorial Africa is greater than previously appreciated and that divergent SIVs can co-circulate in the same colobine population. We also show that the use of "unbiased" deep sequencing for the detection of SIV has great advantages over traditional serological approaches, especially for studies of unknown or poorly characterized viruses. Finally, the detection of the first SIV containing only 16 conserved cysteines in the extracellular envelope protein gp120 further expands the range of functional motifs observed among SIVs and highlights the complex evolutionary history of simian retroviruses.

A novel hepacivirus with an unusually long and intrinsically disordered NS5A protein in a wild Old World primate.

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.