Arteriviruses, Pegiviruses, and Lentiviruses Are Common among Wild African Monkeys.

UNLABELLED: Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks [Chlorocebus cynosuros]) and South Africa (vervet monkeys [Chlorocebus pygerythrus]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys. IMPORTANCE: Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts.

Selective Bottlenecks Shape Evolutionary Pathways Taken during Mammalian Adaptation of a 1918-like Avian Influenza Virus.

Avian influenza virus reassortants resembling the 1918 human pandemic virus can become transmissible among mammals by acquiring mutations in hemagglutinin (HA) and polymerase. Using the ferret model, we trace the evolutionary pathway by which an avian-like virus evolves the capacity for mammalian replication and airborne transmission. During initial infection, within-host HA diversity increased drastically. Then, airborne transmission fixed two polymerase mutations that do not confer a detectable replication advantage. In later transmissions, selection fixed advantageous HA1 variants. Transmission initially involved a "loose" bottleneck, which became strongly selective after additional HA mutations emerged. The stringency and evolutionary forces governing between-host bottlenecks may therefore change throughout host adaptation. Mutations occurred in multiple combinations in transmitted viruses, suggesting that mammalian transmissibility can evolve through multiple genetic pathways despite phenotypic constraints. Our data provide a glimpse into avian influenza virus adaptation in mammals, with broad implications for surveillance on potentially zoonotic viruses.

Conditional Immune Escape during Chronic Simian Immunodeficiency Virus Infection.

UNLABELLED: Anti-HIV CD8 T cells included in therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Identifying the epitopes that do not accumulate variants but retain immunogenicity depends on both host major histocompatibility complex (MHC) genetics and the likelihood for an epitope to tolerate variation. We previously found that immune escape during acute SIV infection is conditional; the accumulation of mutations in T cell epitopes is limited, and the rate of accumulation depends on the number of epitopes being targeted. We have now tested the hypothesis that conditional immune escape extends into chronic SIV infection and that epitopes with a preserved wild-type sequence have the potential to elicit epitope-specific CD8 T cells. We deep sequenced simian immunodeficiency virus (SIV) from Mauritian cynomolgus macaques (MCMs) that were homozygous and heterozygous for the M3 MHC haplotype and had been infected with SIV for about 1 year. When interrogating variation within individual epitopes restricted by M3 MHC alleles, we found three categories of epitopes, which we called categories A, B, and C. Category B epitopes readily accumulated variants in M3-homozygous MCMs, but this was less common in M3-heterozygous MCMs. We then determined that chronic CD8 T cells specific for these epitopes were more likely preserved in the M3-heterozygous MCMs than M3-homozygous MCMs. We provide evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are homozygous for a set of MHC alleles are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. IMPORTANCE: Anti-HIV CD8 T cells that are part of therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Defining these epitope sequences is a necessary precursor to designing approaches that enhance the functionality of CD8 T cells with the potential to control virus replication during chronic infection or after reactivation of latent virus. Using MHC-homozygous and -heterozygous Mauritian cynomolgus macaques, we have now obtained evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are MHC homozygous are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. Importantly, our findings support the conditional immune escape hypothesis, such that the potential to present a greater number of CD8 T cell epitopes within a single animal can delay immune escape in targeted epitopes. As a result, certain epitope sequences can retain immunogenicity into chronic infection.

SNPGenie: estimating evolutionary parameters to detect natural selection using pooled next-generation sequencing data.

UNLABELLED: New applications of next-generation sequencing technologies use pools of DNA from multiple individuals to estimate population genetic parameters. However, no publicly available tools exist to analyse single-nucleotide polymorphism (SNP) calling results directly for evolutionary parameters important in detecting natural selection, including nucleotide diversity and gene diversity. We have developed SNPGenie to fill this gap. The user submits a FASTA reference sequence(s), a Gene Transfer Format (.GTF) file with CDS information and a SNP report(s) in an increasing selection of formats. The program estimates nucleotide diversity, distance from the reference and gene diversity. Sites are flagged for multiple overlapping reading frames, and are categorized by polymorphism type: nonsynonymous, synonymous, or ambiguous. The results allow single nucleotide, single codon, sliding window, whole gene and whole genome/population analyses that aid in the detection of positive and purifying natural selection in the source population. AVAILABILITY AND IMPLEMENTATION: SNPGenie version 1.2 is a Perl program with no additional dependencies. It is free, open-source, and available for download at https://github.com/hugheslab/snpgenie. CONTACT: nelsoncw@email.sc.edu or austin@biol.sc.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Life-history evolution at the molecular level: adaptive amino acid composition of avian vitellogenins.

Avian genomes typically encode three distinct vitellogenin (VTG) egg yolk proteins (VTG1, VTG2 and VTG3), which arose by gene duplication prior to the most recent common ancestor of birds. Analysis of VTG sequences from 34 avian species in a phylogenetic framework supported the hypothesis that VTG amino acid composition has co-evolved with embryo incubation time. Embryo incubation time was positively correlated with the proportions of dietary essential amino acids (EAAs) in VTG1 and VTG2, and with the proportion of sulfur-containing amino acids in VTG3. These patterns were seen even when only semi-altricial and/or altricial species were considered, suggesting that the duration of embryo incubation is a major selective factor on the amino acid composition of VTGs, rather than developmental mode alone. The results are consistent with the hypothesis that the level of EAAs provided to the egg represents an adaptation to the loss of amino acids through breakdown over the course of incubation and imply that life-history phenotypes and VTG amino acid composition have co-evolved throughout the evolutionary history of birds.

Selection Maintains Low Genomic GC Content in Marine SAR11 Lineages.

The genomic G+C content of ocean bacteria varies from below 30% to over 60%. This broad range of base composition is likely shaped by distinct mutational processes, recombination, effective population size, and selection driven by environmental factors. A number of studies have hypothesized that depletion of G/C in genomes of marine bacterioplankton cells is an adaptation to the nitrogen-poor pelagic oceans, but they failed to disentangle environmental factors from mutational biases and population history. Here, we reconstructed the evolutionary changes of bases at synonymous sites in genomes of two marine SAR11 populations and a freshwater counterpart with its evolutionary origin rooted in the marine lineage. Although they all have similar genome sizes, DNA repair gene repertoire, and base compositions, there is a stronger bias toward A/T changes, a reduced frequency of nitrogenous amino acids, and an exclusive occurrence of polyamine, opine, and taurine transport systems in the ocean populations, consistent with a greater nitrogen stress in surface oceans compared with freshwater lakes. Furthermore, the ratio of nonsynoymous to synonymous nucleotide diversity is not statistically distinguishable among these populations, suggesting that population history has a limited effect. Taken together, the ecological transition of SAR11 from ocean to freshwater habitats makes nitrogen more available to these organisms, and thus relaxation of purifying selection drove a genome-wide reduction in the frequency of G/C to A/T changes in the freshwater population.

Stability of social behavior in captive American Flamingos (Phoenicopterus ruber): A quantitative case study.

Over a three-year period a captive American flamingo (Phoenicopterus ruber) group showed recurring seasonal changes in the pattern of agonistic behavior. In spite of seasonal changes in the rates of agonistic behavior, dominance relations were generally stable across seasons. Males had significantly higher dominance status than females, and birds in long-term male-female pair bonds had significantly higher dominance than others. Unresolved agonistic encounters (URI), those with no clear winner or loser, were frequently observed; and their pattern of occurrence changed over the course of the year. URI were most frequent on the breeding "island" during the breeding season, and at the feeder in the post-breeding season. Thus, the frequency of URI reliably tracked seasonal changes regarding the resources most in contention; and, the extent of an individual's involvement in URI was indicative of privileged access to resources over which competition occurred.

Adaptive amino acid composition in collagens of parasitic nematodes.

Amino acid composition was analyzed in the glycine-rich repeat region of 306 collagens belonging to three major families of collagens from both parasitic and free-living nematodes. The collagens of parasitic species showed a tendency toward decreased usage of the hydrophilic residues A, D, and Q and increased usage of the hydrophobic resides I, L, and M; and this trend was seen in parasitic species of both the order Rhabdita and the order Spirurida. The amino acid composition of collagens of parasitic Rhabdita thus tended to resemble those of Spirurida more than that of free-living Rhabdita, suggesting an association between amino acid composition and a parasitic lifestyle. Computer predictions suggested that the more hydrophobic amino acid composition was associated with a reduction of the propensity towards B-cell epitope formation, suggesting that evasion of host immune responses may be a major selective factor responsible for the parasite-specific trend in collagen amino acid composition.

Within-host nucleotide diversity of virus populations: insights from next-generation sequencing.

Next-generation sequencing (NGS) technology offers new opportunities for understanding the evolution and dynamics of viral populations within individual hosts over the course of infection. We review simple methods for estimating synonymous and nonsynonymous nucleotide diversity in viral genes from NGS data without the need for inferring linkage. We discuss the potential usefulness of these data for addressing questions of both practical and theoretical interest, including fundamental questions regarding the effective population sizes of within-host viral populations and the modes of natural selection acting on them.

Cross-clade simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase inhibitor mutations by Illumina MiSeq.

BACKGROUND: Viral resistance to antiretroviral therapy threatens our best methods to control and prevent HIV infection. Current drug resistance genotyping methods are costly, optimized for subtype B virus, and primarily detect resistance mutations to protease and reverse transcriptase inhibitors. With the increasing use of integrase inhibitors in first-line therapies, monitoring for integrase inhibitor drug resistance mutations is a priority. We designed a universal primer pair to PCR amplify all major group M HIV-1 viruses for genotyping using Illumina MiSeq to simultaneously detect drug resistance mutations associated with protease, nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, and integrase inhibitors. RESULTS: A universal primer pair targeting the HIV pol gene was used to successfully PCR amplify HIV isolates representing subtypes A, B, C, D, CRF01_AE and CRF02_AG. The universal primers were then tested on 62 samples from a US cohort of injection drug users failing treatment after release from prison. 94% of the samples were successfully genotyped for known drug resistance mutations in the protease, reverse transcriptase and integrase gene products. Control experiments demonstrate that mutations present at ≥ 2% frequency are reliably detected and above the threshold of error for this method. New drug resistance mutations not found in the baseline sample were identified in 54% of the patient samples after treatment failure. 86% of patients with major drug resistance mutations had 1 or more mutations associated with drug resistance to the treatment regimen at the time point of treatment failure. 59% of the emerging mutations were found at frequencies between 2% and 20% of the total sequences generated, below the estimated limit of detection of current FDA-approved genotyping techniques. Primary plasma samples with viral loads as low as 799 copies/ml were successfully genotyped using this method. CONCLUSIONS: Here we present an Illumina MiSeq-based HIV drug resistance genotyping assay. Our data suggests that this universal assay works across all major group M HIV-1 subtypes and identifies all drug resistance mutations in the pol gene known to confer resistance to protease, reverse transcriptase and integrase inhibitors. This high-throughput and sensitive assay could significantly improve access to drug resistance genotyping worldwide.

Evolutionary diversification of insect innexins.

Phylogenetic analysis of insect innexins supported the hypothesis that six major clades of insect innexins arose by gene duplication prior to the origin of the endopterygote insects. Within one of the six clades (the Zpg Clade), two independent gene duplication events were inferred to have occurred in the lineage of Drosophila, after the most recent common ancestor of the dipteran families Culicidae and Drosophilidae. The relationships among this clades were poorly resolved, except for a sister relationship between ShakB and Ogre. Gene expression data from FlyAtlas supported the hypothesis that the latter gene duplication events gave rise to functional differentiation, with Zpg showing a high level of expression in ovary, and Inx5 and Inx6 showing a high level of expression in testis. Because unduplicated members of this clade in Bombyx mori and Anopheles gambiae showed high levels of expression in both ovary and tests, the expression patterns of the Drosophila members of this clade provide evidence of subdivision of an ancestral gene function after gene duplication.

Evolutionary diversification of the vertebrate transferrin multi-gene family.

In a phylogenetic analysis of vertebrate transferrins (TFs), six major clades (subfamilies) were identified: (a) S, the mammalian serotransferrins; (b) ICA, the mammalian inhibitor of carbonic anhydrase (ICA) homologs; (c) L, the mammalian lactoferrins; (d) O, the ovotransferrins of birds and reptiles; (e) M, the melanotransferrins of bony fishes, amphibians, reptiles, birds, and mammals; and (f) M-like, a newly identified TF subfamily found in bony fishes, amphibians, reptiles, and birds. A phylogenetic tree based on the joint alignment of N-lobes and C-lobes supported the hypothesis that three separate events of internal duplication occurred in vertebrate TFs: (a) in the common ancestor of the M subfamily, (b) in the common ancestor of the M-like subfamily, and (c) in the common ancestor of other vertebrate TFs. The S, ICA, and L subfamilies were found only in placental mammals, and the phylogenetic analysis supported the hypothesis that these three subfamilies arose by gene duplication after the divergence of placental mammals from marsupials. The M-like subfamily was unusual in several respects, including the presence of a uniquely high proportion of clade-specific conserved residues, including distinctive but conserved residues in the sites homologous to those functioning in carbonate binding of human serotransferrin. The M-like family also showed an unusually high proportion of cationic residues in the positively charged region corresponding to human lactoferrampin, suggesting a distinctive role of this region in the M-like subfamily, perhaps in antimicrobial defense.

Comparing effective population sizes of dominant marine alphaproteobacteria lineages.

A fundamental question in marine microbial ecology is how microbes adapt to ocean environments. Although numerically dominant populations are typically considered more successful, higher census population sizes (Nc) do not equate directly to a greater capability for adaptation. Instead, effective population size (Ne) determines the fate of deleterious and favourable mutations, and thus is a key parameter for determining the adaptive potential of a population. In the case of the SAR11 and Roseobacter lineages, two abundant heterotrophic bacteria in ocean surface waters with contrasting life history strategies, culture-independent population surveys suggest that SAR11s have greater Nc than Roseobacters. To determine relative Ne, we compared the ratio of nonsynonymous to synonymous substitution rates (ω) of recently diverged lineages of these taxa. Values of ω associated with several of the Roseobacter subclades were lower than for SAR11 subclades, suggesting greater Ne in these cases. Most Roseobacter lineages also had smaller ω values compared with an atypical basal Roseobacter lineage with a large Nc. This finding provides insight into variability in Ne across two important marine bacterial lineages, and provides an evolutionary context for considering how heterotrophic marine bacteria may differ in their ability to adapt to changing ocean habitats.

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.

Evolutionary diversification of aminopeptidase N in Lepidoptera by conserved clade-specific amino acid residues.

Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family.

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Evolutionary analysis of a streamlined lineage of surface ocean Roseobacters.

The vast majority of surface ocean bacteria are uncultivated. Compared with their cultured relatives, they frequently exhibit a streamlined genome, reduced G+C content and distinct gene repertoire. These genomic traits are relevant to environmental adaptation, and have generally been thought to become fixed in marine bacterial populations through selection. Using single-cell genomics, we sequenced four uncultivated cells affiliated with the ecologically relevant Roseobacter clade and used a composition-heterogeneous Bayesian phylogenomic model to resolve these single-cell genomes into a new clade. This lineage has no representatives in culture, yet accounts for ∼35% of Roseobacters in some surface ocean waters. Analyses of multiple genomic traits, including genome size, G+C content and percentage of noncoding DNA, suggest that these single cells are representative of oceanic Roseobacters but divergent from isolates. Population genetic analyses showed that substitution of physicochemically dissimilar amino acids and replacement of G+C-rich to G+C-poor codons are accelerated in the uncultivated clade, processes that are explained equally well by genetic drift as by the more frequently invoked explanation of natural selection. The relative importance of drift vs selection in this clade, and perhaps in other marine bacterial clades with streamlined G+C-poor genomes, remains unresolved until more evidence is accumulated.

Selection on haemagglutinin imposes a bottleneck during mammalian transmission of reassortant H5N1 influenza viruses.

The emergence of human-transmissible H5N1 avian influenza viruses poses a major pandemic threat. H5N1 viruses are thought to be highly genetically diverse both among and within hosts; however, the effects of this diversity on viral replication and transmission are poorly understood. Here we use deep sequencing to investigate the impact of within-host viral variation on adaptation and transmission of H5N1 viruses in ferrets. We show that, although within-host genetic diversity in haemagglutinin (HA) increases during replication in inoculated ferrets, HA diversity is dramatically reduced upon respiratory droplet transmission, in which infection is established by only 1-2 distinct HA segments from a diverse source virus population in transmitting animals. Moreover, minor HA variants present in as little as 5.9% of viruses within the source animal become dominant in ferrets infected via respiratory droplets. These findings demonstrate that selective pressures acting during influenza virus transmission among mammals impose a significant bottleneck.

Evolution of divergent life history strategies in marine alphaproteobacteria.

UNLABELLED: Marine bacteria in the Roseobacter and SAR11 lineages successfully exploit the ocean habitat, together accounting for ~40% of bacteria in surface waters, yet have divergent life histories that exemplify patch-adapted versus free-living ecological roles. Here, we use a phylogenetic birth-and-death model to understand how genome content supporting different life history strategies evolved in these related alphaproteobacterial taxa, showing that the streamlined genomes of free-living SAR11 were gradually downsized from a common ancestral genome only slightly larger than the extant members (~2,000 genes), while the larger and variably sized genomes of roseobacters evolved along dynamic pathways from a sizeable common ancestor (~8,000 genes). Genome changes in the SAR11 lineage occurred gradually over ~800 million years, whereas Roseobacter genomes underwent more substantial modifications, including major periods of expansion, over ~260 million years. The timing of the first Roseobacter genome expansion was coincident with the predicted radiation of modern marine eukaryotic phytoplankton of sufficient size to create nutrient-enriched microzones and is consistent with present-day ecological associations between these microbial groups. We suggest that diversification of red-lineage phytoplankton is an important driver of divergent life history strategies among the heterotrophic bacterioplankton taxa that dominate the present-day ocean. IMPORTANCE: One-half of global primary production occurs in the oceans, and more than half of this is processed by heterotrophic bacterioplankton through the marine microbial food web. The diversity of life history strategies that characterize different bacterioplankton taxa is an important subject, since the locations and mechanisms whereby bacteria interact with seawater organic matter has effects on microbial growth rates, metabolic pathways, and growth efficiencies, and these in turn affect rates of carbon mineralization to the atmosphere and sequestration into the deep sea. Understanding the evolutionary origins of the ecological strategies that underlie biochemical interactions of bacteria with the ocean system, and which scale up to affect globally important biogeochemical processes, will improve understanding of how microbial diversity is maintained and enable useful predictions about microbial response in the future ocean.