A recent gibbon ape leukemia virus germline integration in a rodent from New Guinea.

Germline colonization by retroviruses results in the formation of endogenous retroviruses (ERVs). Most colonization's occurred millions of years ago. However, in the Australo-Papuan region (Australia and New Guinea), several recent germline colonization events have been discovered. The Wallace Line separates much of Southeast Asia from the Australo-Papuan region restricting faunal and pathogen dispersion. West of the Wallace Line, gibbon ape leukemia viruses (GALVs) have been isolated from captive gibbons. Two microbat species from China appear to have been infected naturally. East of Wallace's Line, the woolly monkey virus (a GALV) and the closely related koala retrovirus (KoRV) have been detected in eutherians and marsupials in the Australo-Papuan region, often vertically transmitted. The detected vertically transmitted GALV-like viruses in Australo-Papuan fauna compared to sporadic horizontal transmission in Southeast Asia and China suggest the GALV-KoRV clade originates in the former region and further models of early-stage genome colonization may be found. We screened 278 samples, seven bat and one rodent family endemic to the Australo-Papuan region and bat and rodent species found on both sides of the Wallace Line. We identified two rodents (Melomys) from Australia and Papua New Guinea and no bat species harboring GALV-like retroviruses. Melomys leucogaster from New Guinea harbored a genomically complete replication-competent retrovirus with a shared integration site among individuals. The integration was only present in some individuals of the species indicating this retrovirus is at the earliest stages of germline colonization of the Melomys genome, providing a new small wild mammal model of early-stage genome colonization.

Genomic analysis of three Clostridioides difficile isolates from urban water sources.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed.

A comprehensively molecular haplotype-resolved genome of a European individual.

Independent determination of both haplotype sequences of an individual genome is essential to relate genetic variation to genome function, phenotype, and disease. To address the importance of phase, we have generated the most complete haplotype-resolved genome to date, "Max Planck One" (MP1), by fosmid pool-based next generation sequencing. Virtually all SNPs (>99%) and 80,000 indels were phased into haploid sequences of up to 6.3 Mb (N50 ~1 Mb). The completeness of phasing allowed determination of the concrete molecular haplotype pairs for the vast majority of genes (81%) including potential regulatory sequences, of which >90% were found to be constituted by two different molecular forms. A subset of 159 genes with potentially severe mutations in either cis or trans configurations exemplified in particular the role of phase for gene function, disease, and clinical interpretation of personal genomes (e.g., BRCA1). Extended genomic regions harboring manifold combinations of physically and/or functionally related genes and regulatory elements were resolved into their underlying "haploid landscapes," which may define the functional genome. Moreover, the majority of genes and functional sequences were found to contain individual or rare SNPs, which cannot be phased from population data alone, emphasizing the importance of molecular phasing for characterizing a genome in its molecular individuality. Our work provides the foundation to understand that the distinction of molecular haplotypes is essential to resolve the (inherently individual) biology of genes, genomes, and disease, establishing a reference point for "phase-sensitive" personal genomics. MP1's annotated haploid genomes are available as a public resource.

Fosmid-based whole genome haplotyping of a HapMap trio child: evaluation of Single Individual Haplotyping techniques.

Determining the underlying haplotypes of individual human genomes is an essential, but currently difficult, step toward a complete understanding of genome function. Fosmid pool-based next-generation sequencing allows genome-wide generation of 40-kb haploid DNA segments, which can be phased into contiguous molecular haplotypes computationally by Single Individual Haplotyping (SIH). Many SIH algorithms have been proposed, but the accuracy of such methods has been difficult to assess due to the lack of real benchmark data. To address this problem, we generated whole genome fosmid sequence data from a HapMap trio child, NA12878, for which reliable haplotypes have already been produced. We assembled haplotypes using eight algorithms for SIH and carried out direct comparisons of their accuracy, completeness and efficiency. Our comparisons indicate that fosmid-based haplotyping can deliver highly accurate results even at low coverage and that our SIH algorithm, ReFHap, is able to efficiently produce high-quality haplotypes. We expanded the haplotypes for NA12878 by combining the current haplotypes with our fosmid-based haplotypes, producing near-to-complete new gold-standard haplotypes containing almost 98% of heterozygous SNPs. This improvement includes notable fractions of disease-related and GWA SNPs. Integrated with other molecular biological data sets, this phase information will advance the emerging field of diploid genomics.

Early evolution of conserved regulatory sequences associated with development in vertebrates.

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.

Serological Evidence That SARS-CoV-2 Has Not Emerged in Deer in Germany or Austria during the COVID-19 Pandemic.

Spillover of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) to North American white-tailed deer (Odocoileus virginianus) has been documented. However, it is unclear if this is a phenomenon specific to North American deer or is a broader problem. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white-tailed deer, we found no evidence of SARS-CoV-2 exposure.

Seasonal host and ecological drivers may promote restricted water as a viral vector.

In climates with seasonally limited precipitation, terrestrial animals congregate at high densities at scarce water sources. We hypothesize that viruses can exploit the recurrence of these diverse animal congregations to spread. In this study, we test the central prediction of this hypothesis - that viruses employing this transmission strategy remain stable and infectious in water. Equid herpesviruses (EHVs) were chosen as a model as they have been shown to remain stable and infectious in water for weeks under laboratory conditions. Using fecal data from wild equids from a previous study, we establish that EHVs are shed more frequently by their hosts during the dry season, increasing the probability of water source contamination with EHV. We document the presence of several strains of EHVs present in high genome copy number from the surface water and sediments of waterholes sampled across a variety of mammalian assemblages, locations, temperatures and pH. Phylogenetic analysis reveals that the different EHV strains found exhibit little divergence despite representing ancient lineages. We employed molecular approaches to show that EHVs shed remain stable in waterholes with detection decreasing with increasing temperature in sediments. Infectivity experiments using cell culture reveals that EHVs remain infectious in water derived from waterholes. The results are supportive of water as an abiotic viral vector for EHV.

Retroviral integrations contribute to elevated host cancer rates during germline invasion.

Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by high cancer prevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages.

Defining a genomic radius for long-range enhancer action: duplicated conserved non-coding elements hold the key.

Many conserved non-coding elements (CNEs) in vertebrate genomes have been shown to function as tissue-specific enhancers. However, the target genes of most CNEs are unknown. Here we show that the target genes of duplicated CNEs can be predicted by considering their neighbouring paralogous genes. This enables us to provide the first systematic estimate of the genomic range for distal cis-regulatory interactions in the human genome: half of CNEs are >250 kb away from their associated gene.

Terrestrial mammal surveillance using hybridization capture of environmental DNA from African waterholes.

Determining species distributions can be extremely challenging but is crucial to ecological and conservation research. Environmental DNA (eDNA) approaches have shown particular promise in aquatic systems for several vertebrate and invertebrate species. For terrestrial animals, however, eDNA-based surveys are considerably more difficult due to the lack of or difficulty in obtaining appropriate sampling substrate. In water-limited ecosystem where terrestrial mammals are often forced to congregate at waterholes, water and sediment from shared water sources may be a suitable substrate for noninvasive eDNA approaches. We characterized mitochondrial DNA sequences from a broad range of terrestrial mammal species in two different African ecosystems (in Namibia and Tanzania) using eDNA isolated from native water, sediment and water filtered through glass fibre filters. A hybridization capture enrichment with RNA probes targeting the mitochondrial genomes of 38 mammal species representing the genera/families expected at the respective ecosystems was employed, and 16 species were identified, with a maximum mitogenome coverage of 99.8%. Conventional genus-specific PCRs were tested on environmental samples for two genera producing fewer positive results than hybridization capture enrichment. An experiment with mock samples using DNA from non-African mammals showed that baits covering 30% of nontarget mitogenomes produced 91% mitogenome coverage after capture. In the mock samples, over-representation of DNA of one species still allowed for the detection of DNA of other species that was at a 100-fold lower concentration. Hybridization capture enrichment of eDNA is therefore an effective method for monitoring terrestrial mammal species from shared water sources.

Highly conserved non-coding sequences are associated with vertebrate development.

In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH), in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.

Metabolite changes in blood predict the onset of tuberculosis.

New biomarkers of tuberculosis (TB) risk and disease are critical for the urgently needed control of the ongoing TB pandemic. In a prospective multisite study across Subsaharan Africa, we analyzed metabolic profiles in serum and plasma from HIV-negative, TB-exposed individuals who either progressed to TB 3-24 months post-exposure (progressors) or remained healthy (controls). We generated a trans-African metabolic biosignature for TB, which identifies future progressors both on blinded test samples and in external data sets and shows a performance of 69% sensitivity at 75% specificity in samples within 5 months of diagnosis. These prognostic metabolic signatures are consistent with development of subclinical disease prior to manifestation of active TB. Metabolic changes associated with pre-symptomatic disease are observed as early as 12 months prior to TB diagnosis, thus enabling timely interventions to prevent disease progression and transmission.

CONDOR: a database resource of developmentally associated conserved non-coding elements.

BACKGROUND: Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal cis-regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them. DESCRIPTION: Here we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: http://condor.fugu.biology.qmul.ac.uk. In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing in vivo zebrafish enhancer assays of the CNEs carried out in-house, which currently number approximately 100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental cis-regulation. CONCLUSION: By providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development.

Integration of Metabolomics and Transcriptomics Reveals a Complex Diet of Mycobacterium tuberculosis during Early Macrophage Infection.

Nutrient acquisition from the host environment is crucial for the survival of intracellular pathogens, but conceptual and technical challenges limit our knowledge of pathogen diets. To overcome some of these technical roadblocks, we exploited an experimentally accessible model for early infection of human macrophages by Mycobacterium tuberculosis, the etiological agent of tuberculosis, to study host-pathogen interactions with a multi-omics approach. We collected metabolomics and complete transcriptome RNA sequencing (dual RNA-seq) data of the infected macrophages, integrated them in a genome-wide reaction pair network, and identified metabolic subnetworks in host cells and M. tuberculosis that are modularly regulated during infection. Up- and downregulation of these metabolic subnetworks suggested that the pathogen utilizes a wide range of host-derived compounds, concomitant with the measured metabolic and transcriptional changes in both bacteria and host. To quantify metabolic interactions between the host and intracellular pathogen, we used a combined genome-scale model of macrophage and M. tuberculosis metabolism constrained by the dual RNA-seq data. Metabolic flux balance analysis predicted coutilization of a total of 33 different carbon sources and enabled us to distinguish between the pathogen's substrates directly used as biomass precursors and the ones further metabolized to gain energy or to synthesize building blocks. This multiple-substrate fueling confers high robustness to interventions with the pathogen's metabolism. The presented approach combining multi-omics data as a starting point to simulate system-wide host-pathogen metabolic interactions is a useful tool to better understand the intracellular lifestyle of pathogens and their metabolic robustness and resistance to metabolic interventions. IMPORTANCE The nutrients consumed by intracellular pathogens are mostly unknown. This is mainly due to the challenge of disentangling host and pathogen metabolism sharing the majority of metabolic pathways and hence metabolites. Here, we investigated the metabolic changes of Mycobacterium tuberculosis, the causative agent of tuberculosis, and its human host cell during early infection. To this aim, we combined gene expression data of both organisms and metabolite changes during the course of infection through integration into a genome-wide metabolic network. This led to the identification of infection-specific metabolic alterations, which we further exploited to model host-pathogen interactions quantitatively by flux balance analysis. These in silico data suggested that tubercle bacilli consume up to 33 different nutrients during early macrophage infection, which the bacteria utilize to generate energy and biomass to establish intracellular growth. Such multisubstrate fueling strategy renders the pathogen's metabolism robust toward perturbations, such as innate immune responses or antibiotic treatments.

Concise gene signature for point-of-care classification of tuberculosis.

There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expression profiles based on blood signatures have proved useful in identifying genes that enable classification of TB patients, but have thus far been complex. Using real-time PCR analysis, we evaluated the expression profiles from a large panel of genes in TB patients and healthy individuals in an Indian cohort. Classification models were built and validated for their capacity to discriminate samples from TB patients and controls within this cohort and on external independent gene expression datasets. A combination of only four genes distinguished TB patients from healthy individuals in both cross-validations and on separate validation datasets with very high accuracy. An external validation on two distinct cohorts using a real-time PCR setting confirmed the predictive power of this 4-gene tool reaching sensitivity scores of 88% with a specificity of around 75%. Moreover, this gene signature demonstrated good classification power in HIV(+) populations and also between TB and several other pulmonary diseases. Here we present proof of concept that our 4-gene signature and the top classifier genes from our models provide excellent candidates for the development of molecular point-of-care TB diagnosis in endemic areas.

Correction: The CARD9 Polymorphisms rs4077515, rs10870077 and rs10781499 Are Uncoupled from Susceptibility to and Severity of Pulmonary Tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0163662.].

The CARD9 Polymorphisms rs4077515, rs10870077 and rs10781499 Are Uncoupled from Susceptibility to and Severity of Pulmonary Tuberculosis.

Genetic variants in the CARD9 gene predispose to inflammatory disorders and chronic infectious diseases. Tuberculosis (TB), a chronic infectious disease affecting the lung, is lethal in Card9-deficient mice. We hypothesized that polymorphisms in the CARD9 gene influence TB progression and disease-associated lung damage in humans. We tested genotype distributions of the CARD9 polymorphisms rs4077515, rs10781499 and rs10870077 in TB patients and healthy subjects in a Caucasian cohort. SNPs were in linkage disequilibrium and none of the haplotypes was significantly enriched in the TB group. We determined total and differential leukocyte count, erythrocyte sedimentation rate and plasma abundance of cytokines and chemokines as markers for systemic inflammation and scored chest X-rays to assess lung involvement in TB subjects. Most disease parameters segregated independently of the CARD9 haplotypes. In contrast to multifactorial chronic inflammation, selected genetic variants in the CARD9 gene leave host responses apparently unaffected in TB, at least in the population analyzed here.

CXCL5-secreting pulmonary epithelial cells drive destructive neutrophilic inflammation in tuberculosis.

Successful host defense against numerous pulmonary infections depends on bacterial clearance by polymorphonuclear leukocytes (PMNs); however, excessive PMN accumulation can result in life-threatening lung injury. Local expression of CXC chemokines is critical for PMN recruitment. The impact of chemokine-dependent PMN recruitment during pulmonary Mycobacterium tuberculosis infection is not fully understood. Here, we analyzed expression of genes encoding CXC chemokines in M. tuberculosis-infected murine lung tissue and found that M. tuberculosis infection promotes upregulation of Cxcr2 and its ligand Cxcl5. To determine the contribution of CXCL5 in pulmonary PMN recruitment, we generated Cxcl5(-/-) mice and analyzed their immune response against M. tuberculosis. Both Cxcr2(-/-) mice and Cxcl5(-/-) mice, which are deficient for only one of numerous CXCR2 ligands, exhibited enhanced survival compared with that of WT mice following high-dose M. tuberculosis infection. The resistance of Cxcl5(-/-) mice to M. tuberculosis infection was not due to heightened M. tuberculosis clearance but was the result of impaired PMN recruitment, which reduced pulmonary inflammation. Lung epithelial cells were the main source of CXCL5 upon M. tuberculosis infection, and secretion of CXCL5 was reduced by blocking TLR2 signaling. Together, our data indicate that TLR2-induced epithelial-derived CXCL5 is critical for PMN-driven destructive inflammation in pulmonary tuberculosis.

Functional analysis of conserved non-coding regions around the short stature hox gene (shox) in whole zebrafish embryos.

BACKGROUND: Mutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene. METHODOLOGY/PRINCIPAL FINDINGS: Expression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al., resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.'s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs. CONCLUSION/SIGNIFICANCE: Our results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs.

Pebble and rock band: heuristic resolution of repeats and scaffolding in the velvet short-read de novo assembler.

BACKGROUND: Despite the short length of their reads, micro-read sequencing technologies have shown their usefulness for de novo sequencing. However, especially in eukaryotic genomes, complex repeat patterns are an obstacle to large assemblies. PRINCIPAL FINDINGS: We present a novel heuristic algorithm, Pebble, which uses paired-end read information to resolve repeats and scaffold contigs to produce large-scale assemblies. In simulations, we can achieve weighted median scaffold lengths (N50) of above 1 Mbp in Bacteria and above 100 kbp in more complex organisms. Using real datasets we obtained a 96 kbp N50 in Pseudomonas syringae and a unique 147 kbp scaffold of a ferret BAC clone. We also present an efficient algorithm called Rock Band for the resolution of repeats in the case of mixed length assemblies, where different sequencing platforms are combined to obtain a cost-effective assembly. CONCLUSIONS: These algorithms extend the utility of short read only assemblies into large complex genomes. They have been implemented and made available within the open-source Velvet short-read de novo assembler.