Joe Parker

Genome-wide signatures of convergent evolution in echolocating mammals

Evolution is typically thought to proceed through divergence of genes, proteins and ultimately phenotypes. However, similar traits might also evolve convergently in unrelated taxa owing to similar selection pressures. Adaptive phenotypic convergence is widespread in nature, and recent results from several genes have suggested that this phenomenon is powerful enough to also drive recurrent evolution at the sequence level. Where homoplasious substitutions do occur these have long been considered the result of neutral processes. However, recent studies have demonstrated that adaptive convergent sequence evolution can be detected in vertebrates using statistical methods that model parallel evolution, although the extent to which sequence convergence between genera occurs across genomes is unknown. Here we analyse genomic sequence data in mammals that have independently evolved echolocation and show that convergence is not a rare process restricted to several loci but is instead widespread, continuously distributed and commonly driven by natural selection acting on a small number of sites per locus. Systematic analyses of convergent sequence evolution in 805,053 amino acids within 2,326 orthologous coding gene sequences compared across 22 mammals (including four newly sequenced bat genomes) revealed signatures consistent with convergence in nearly 200 loci. Strong and significant support for convergence among bats and the bottlenose dolphin was seen in numerous genes linked to hearing or deafness, consistent with an involvement in echolocation. Unexpectedly, we also found convergence in many genes linked to vision: the convergent signal of many sensory genes was robustly correlated with the strength of natural selection. This first attempt to detect genome-wide convergent sequence evolution across divergent taxa reveals the phenomenon to be much more pervasive than previously recognized.

Phylogenomic Analyses Elucidate the Evolutionary Relationships of Bats

Molecular phylogenetics has rapidly established the evolutionary positions of most major mammal groups, yet analyses have repeatedly failed to agree on that of bats (order Chiroptera). Moreover, the relationship among the major bat lineages has proven equally contentious, with ongoing disagreements about whether echolocating bats are paraphyletic or a true group having profound implications for whether echolocation evolved once or possibly multiple times. By generating new bat genome data and applying model-based phylogenomic analyses designed to accommodate heterogeneous evolutionary processes, we show that-contrary to recent suggestions-bats are not closely related to odd-toed ungulates but instead have a more ancient origin as sister group to a large clade of carnivores, ungulates, and cetaceans. Additionally, we provide the first genome-scale support showing that laryngeal echolocating bats are not a true group and that this paraphyly is robust to their position within mammals. We suggest that earlier disagreements in the literature may reflect model misspecification, long-branch artifacts, poor taxonomic coverage, and differences in the phylogenetic markers used. These findings are a timely reminder of the relevance of experimental design and careful statistical analysis as we move into the phylogenomic era.

Safety and immunogenicity of novel recombinant BCG and modified vaccinia virus Ankara vaccines in neonate rhesus macaques.

ABSTRACT Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA401 or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA401 induced high frequencies of BCG-specific IFN-γ-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-γ responses. MVA.HIVA elicited HIV-1-specific IFN-γ responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.

Molecular Epidemiology and Phylogeny Reveal Complex Spatial Dynamics in Areas Where Canine Parvovirus Is Endemic

ABSTRACT Canine parvovirus type 2 (CPV-2) is a severe enteric pathogen of dogs, causing high mortality in unvaccinated dogs. After emerging, CPV-2 spread rapidly worldwide. However, there is now some evidence to suggest that international transmission appears to be more restricted. In order to investigate the transmission and evolution of CPV-2 both nationally and in relation to the global situation, we have used a long-range PCR to amplify and sequence the full VP2 gene of 150 canine parvoviruses obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the United Kingdom, over a 2-year period. Among these 150 strains, 50 different DNA sequence types (S) were identified, and apart from one case, all appeared unique to the United Kingdom. Phylogenetic analysis provided clear evidence for spatial clustering at the international level and for the first time also at the national level, with the geographical range of some sequence types appearing to be highly restricted within the United Kingdom. Evolution of the VP2 gene in this data set was associated with a lack of positive selection. In addition, the majority of predicted amino acid sequences were identical to those found elsewhere in the world, suggesting that CPV VP2 has evolved a highly fit conformation. Based on typing systems using key amino acid mutations, 43% of viruses were CPV-2a, and 57% CPV-2b, with no type 2 or 2c found. However, phylogenetic analysis suggested complex antigenic evolution of this virus, with both type 2a and 2b viruses appearing polyphyletic. As such, typing based on specific amino acid mutations may not reflect the true epidemiology of this virus. The geographical restriction that we observed both within the United Kingdom and between the United Kingdom and other countries, together with the lack of CPV-2c in this population, strongly suggests the spread of CPV within its population may be heterogeneously subject to limiting factors. This cross-sectional study of national and global CPV phylogeographic segregation reveals a substantially more complex epidemic structure than previously described.

Estimating the date of origin of an HIV-1 circulating recombinant form.

HIV is capable of frequent genetic exchange through recombination. Despite the pandemic spread of HIV-1 recombinants, their times of origin are not well understood. We investigate the epidemic history of a HIV-1 circulating recombinant form (CRF) by estimating the time of the recombination event that lead to the emergence of CRF33_01B, a recently described recombinant descended from CRF01_AE and subtype B. The gag, pol and env genes were analyzed using a combined coalescent and relaxed molecular clock model, implemented in a Bayesian Markov chain Monte Carlo framework. Using linked genealogical trees we calculated the time interval between the common ancestor of CRF33_01B and the ancestors it shares with closely related parental lineages. The recombination event that generated CRF33_01B (t(rec)) occurred sometime between 1991 and 1993, suggesting that recombination is common in the early evolutionary history of HIV-1. The proof-of-concept approach provides a new tool for the investigation of HIV molecular epidemiology and evolution.

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

ABSTRACT Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.

Evolutionary and Phylogenetic Analysis of the Hepaciviruses and Pegiviruses

Abstract The known genetic diversity of the hepaciviruses and pegiviruses has increased greatly in recent years through the discovery of viruses related to hepatitis C virus and human pegivirus in bats, bovines, equines, primates, and rodents. Analysis of these new species is important for research into animal models of hepatitis C virus infection and into the zoonotic origins of human viruses. Here, we provide the first systematic phylogenetic and evolutionary analysis of these two genera at the whole-genome level. Phylogenies confirmed that hepatitis C virus is most closely related to viruses from horses whereas human pegiviruses clustered with viruses from African primates. Within each genus, several well-supported lineages were identified and viral diversity was structured by both host species and location of sampling. Recombination analyses provided evidence of interspecific recombination in hepaciviruses, but none in the pegiviruses. Putative mosaic genome structures were identified in NS5B gene region and were supported by multiple tests. The identification of interspecific recombination in the hepaciviruses represents an important evolutionary event that could be clarified by future sampling of novel viruses. We also identified parallel amino acid changes shared by distantly related lineages that infect similar types of host. Notable parallel changes were clustered in the NS3 and NS4B genes and provide a useful starting point for experimental studies of the evolution of Hepacivirus host–virus interactions.

Selected HIV-1 Env Trimeric Formulations Act as Potent Immunogens in a Rabbit Vaccination Model

Background Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant. Methods Based on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments. Results It was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region. Conclusions Our results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.

Field-based species identification of closely-related plants using real-time nanopore sequencing

Advances in DNA sequencing and informatics have revolutionised biology over the past four decades, but technological limitations have left many applications unexplored. Recently, portable, real-time, nanopore sequencing (RTnS) has become available. This offers opportunities to rapidly collect and analyse genomic data anywhere. However, generation of datasets from large, complex genomes has been constrained to laboratories. The portability and long DNA sequences of RTnS offer great potential for field-based species identification, but the feasibility and accuracy of these technologies for this purpose have not been assessed. Here, we show that a field-based RTnS analysis of closely-related plant species (Arabidopsis spp.) has many advantages over laboratory-based high-throughput sequencing (HTS) methods for species level identification and phylogenomics. Samples were collected and sequenced in a single day by RTnS using a portable, “al fresco” laboratory. Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification. Individually annotated RTnS reads can be used to infer the evolutionary relationships of A. thaliana. Furthermore, hybrid genome assembly with RTnS and HTS reads substantially improved upon a genome assembled from HTS reads alone. Field-based RTnS makes real-time, rapid specimen identification and genome wide analyses possible.

Generation of neutralizing antibodies and divergence of SIVmac239 in cynomolgus macaques following short-term early antiretroviral therapy.

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4+ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 104 copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4+ T-cell numbers, low viral load and limited viral divergence.