Correction: Viral genetic variation accounts for a third of variability in HIV-1 set-point viral load in Europe.

[This corrects the article DOI: 10.1371/journal.pbio.2001855.].

Viral genetic variation accounts for a third of variability in HIV-1 set-point viral load in Europe.

HIV-1 set-point viral load-the approximately stable value of viraemia in the first years of chronic infection-is a strong predictor of clinical outcome and is highly variable across infected individuals. To better understand HIV-1 pathogenesis and the evolution of the viral population, we must quantify the heritability of set-point viral load, which is the fraction of variation in this phenotype attributable to viral genetic variation. However, current estimates of heritability vary widely, from 6% to 59%. Here we used a dataset of 2,028 seroconverters infected between 1985 and 2013 from 5 European countries (Belgium, Switzerland, France, the Netherlands and the United Kingdom) and estimated the heritability of set-point viral load at 31% (CI 15%-43%). Specifically, heritability was measured using models of character evolution describing how viral load evolves on the phylogeny of whole-genome viral sequences. In contrast to previous studies, (i) we measured viral loads using standardized assays on a sample collected in a strict time window of 6 to 24 months after infection, from which the viral genome was also sequenced; (ii) we compared 2 models of character evolution, the classical "Brownian motion" model and another model ("Ornstein-Uhlenbeck") that includes stabilising selection on viral load; (iii) we controlled for covariates, including age and sex, which may inflate estimates of heritability; and (iv) we developed a goodness of fit test based on the correlation of viral loads in cherries of the phylogenetic tree, showing that both models of character evolution fit the data well. An overall heritability of 31% (CI 15%-43%) is consistent with other studies based on regression of viral load in donor-recipient pairs. Thus, about a third of variation in HIV-1 virulence is attributable to viral genetic variation.

Comparative analysis of the chicken IFITM locus by targeted genome sequencing reveals evolution of the locus and positive selection in IFITM1 and IFITM3.

BACKGROUND: The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species. RESULTS: Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection. CONCLUSIONS: Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.

Accurate characterization of the IFITM locus using MiSeq and PacBio sequencing shows genetic variation in Galliformes.

BACKGROUND: Interferon inducible transmembrane (IFITM) proteins are effectors of the immune system widely characterized for their role in restricting infection by diverse enveloped and non-enveloped viruses. The chicken IFITM (chIFITM) genes are clustered on chromosome 5 and to date four genes have been annotated, namely chIFITM1, chIFITM3, chIFITM5 and chIFITM10. However, due to poor assembly of this locus in the Gallus Gallus v4 genome, accurate characterization has so far proven problematic. Recently, a new chicken reference genome assembly Gallus Gallus v5 was generated using Sanger, 454, Illumina and PacBio sequencing technologies identifying considerable differences in the chIFITM locus over the previous genome releases. METHODS: We re-sequenced the locus using both Illumina MiSeq and PacBio RS II sequencing technologies and we mapped RNA-seq data from the European Nucleotide Archive (ENA) to this finalized chIFITM locus. Using SureSelect probes capture probes designed to the finalized chIFITM locus, we sequenced the locus of a different chicken breed, namely a White Leghorn, and a turkey. RESULTS: We confirmed the Gallus Gallus v5 consensus except for two insertions of 5 and 1 base pair within the chIFITM3 and B4GALNT4 genes, respectively, and a single base pair deletion within the B4GALNT4 gene. The pull down revealed a single amino acid substitution of A63V in the CIL domain of IFITM2 compared to Red Jungle fowl and 13, 13 and 11 differences between IFITM1, 2 and 3 of chickens and turkeys, respectively. RNA-seq shows chIFITM2 and chIFITM3 expression in numerous tissue types of different chicken breeds and avian cell lines, while the expression of the putative chIFITM1 is limited to the testis, caecum and ileum tissues. CONCLUSIONS: Locus resequencing using these capture probes and RNA-seq based expression analysis will allow the further characterization of genetic diversity within Galliformes.

Systems consequences of amplicon formation in human breast cancer.

Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer.

IVA: accurate de novo assembly of RNA virus genomes.

MOTIVATION: An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods. RESULTS: We developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers. AVAILABILITY AND IMPLEMENTATION: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva

Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting.

UNLABELLED: Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure. IMPORTANCE: The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance.

Plasmablasts generated during repeated dengue infection are virus glycoprotein-specific and bind to multiple virus serotypes.

Dengue virus immune protection is specific to the serotype encountered and is thought to persist throughout one's lifetime. Many serotype cross-reactive memory B cells isolated from humans with previous dengue infection are specific for the nonstructural and the prM structural viral proteins, and they can enhance infection in vitro. However, plasmablasts circulating in enormous numbers during acute secondary infection have not been studied. In this study, we analyzed single plasmablasts from two patients by sorting the cells for Ig sequence analysis and for recombinant expression of Abs. In contrast to memory B cells, most plasmablast-derived Abs bound to the structural E protein of dengue, and protection experiments in mice revealed that virus serotypes encountered during past infections were neutralized more efficiently than were the serotypes of the current infection. Together with genetic analyses, we show evidence that plasmablasts in dengue patients are a polyclonal pool of activated E protein-specific memory B cells and that their specificity is not representative of the serum Abs secreted by long-lived plasma cells in the memory phase. These results contribute to the understanding of the phenomenon of original antigenic sin in dengue.

LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets.

The study of cell-population heterogeneity in a range of biological systems, from viruses to bacterial isolates to tumor samples, has been transformed by recent advances in sequencing throughput. While the high-coverage afforded can be used, in principle, to identify very rare variants in a population, existing ad hoc approaches frequently fail to distinguish true variants from sequencing errors. We report a method (LoFreq) that models sequencing run-specific error rates to accurately call variants occurring in <0.05% of a population. Using simulated and real datasets (viral, bacterial and human), we show that LoFreq has near-perfect specificity, with significantly improved sensitivity compared with existing methods and can efficiently analyze deep Illumina sequencing datasets without resorting to approximations or heuristics. We also present experimental validation for LoFreq on two different platforms (Fluidigm and Sequenom) and its application to call rare somatic variants from exome sequencing datasets for gastric cancer. Source code and executables for LoFreq are freely available at http://sourceforge.net/projects/lofreq/.

Notch1 mutations drive clonal expansion in normal esophageal epithelium but impair tumor growth.

NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. Sequencing NOTCH1 mutant clones in aging human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. Widespread Notch1 loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. Notch1 null tumors showed reduced proliferation. We conclude that Notch1 mutations in normal epithelium are beneficial as wild-type Notch1 favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.

p53 mutation in normal esophagus promotes multiple stages of carcinogenesis but is constrained by clonal competition.

Aging normal human oesophagus accumulates TP53 mutant clones. These are the origin of most oesophageal squamous carcinomas, in which biallelic TP53 disruption is almost universal. However, how p53 mutant clones expand and contribute to cancer development is unclear. Here we show that inducing the p53R245W mutant in single oesophageal progenitor cells in transgenic mice confers a proliferative advantage and clonal expansion but does not disrupt normal epithelial structure. Loss of the remaining p53 allele in mutant cells results in genomically unstable p53R245W/null epithelium with giant polyaneuploid cells and copy number altered clones. In carcinogenesis, p53 mutation does not initiate tumour formation, but tumours developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.

AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kß/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors.

The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kß inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kßi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kßi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kßi sensitivity while modulation of RRAGC, LAMTOR1, LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kßi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kß/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kßi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kßi/AKTi sensitive and resistant breast tumours.

Phylogenetic estimation of the viral fitness landscape of HIV-1 set-point viral load.

Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 104 and 105 copies of viral RNA per ml. Here we aimed to detect transmission fitness differences between viruses from individuals with different SPVLs directly from phylogenetic trees inferred from whole-genome sequences. We used the local branching index (LBI) as a proxy for transmission fitness. We found that LBI is more sensitive to differences in infectiousness than to differences in the duration of the infectious state. By analysing subtype-B samples from the Bridging the Evolution and Epidemiology of HIV in Europe project, we inferred a significant positive relationship between SPVL and LBI up to approximately 105 copies/ml, with some evidence for a peak around this value of SPVL. This is evidence of selection against low values of SPVL in HIV-1 subtype-B strains, likely related to lower infectiousness, and perhaps a peak in the transmission fitness in the expected range of SPVL. The less prominent signatures of selection against higher SPVL could be explained by an inherent limit of the method or the deployment of antiretroviral therapy.

A highly virulent variant of HIV-1 circulating in the Netherlands.

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence.

KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.

Selection of Oncogenic Mutant Clones in Normal Human Skin Varies with Body Site.

Skin cancer risk varies substantially across the body, yet how this relates to the mutations found in normal skin is unknown. Here we mapped mutant clones in skin from high- and low-risk sites. The density of mutations varied by location. The prevalence of NOTCH1 and FAT1 mutations in forearm, trunk, and leg skin was similar to that in keratinocyte cancers. Most mutations were caused by ultraviolet light, but mutational signature analysis suggested differences in DNA-repair processes between sites. Eleven mutant genes were under positive selection, with TP53 preferentially selected in the head and FAT1 in the leg. Fine-scale mapping revealed 10% of clones had copy-number alterations. Analysis of hair follicles showed mutations in the upper follicle resembled adjacent skin, but the lower follicle was sparsely mutated. Normal skin is a dense patchwork of mutant clones arising from competitive selection that varies by location. SIGNIFICANCE: Mapping mutant clones across the body reveals normal skin is a dense patchwork of mutant cells. The variation in cancer risk between sites substantially exceeds that in mutant clone density. More generally, mutant genes cannot be assigned as cancer drivers until their prevalence in normal tissue is known.See related commentary by De Dominici and DeGregori, p. 227.This article is highlighted in the In This Issue feature, p. 211.

Genomic epidemiology of a dengue virus epidemic in urban Singapore.

Dengue is one of the most important emerging diseases of humans, with no preventative vaccines or antiviral cures available at present. Although one-third of the world's population live at risk of infection, little is known about the pattern and dynamics of dengue virus (DENV) within outbreak situations. By exploiting genomic data from an intensively studied major outbreak, we are able to describe the molecular epidemiology of DENV at a uniquely fine-scaled temporal and spatial resolution. Two DENV serotypes (DENV-1 and DENV-3), and multiple component genotypes, spread concurrently and with similar epidemiological and evolutionary profiles during the initial outbreak phase of a major dengue epidemic that took place in Singapore during 2005. Although DENV-1 and DENV-3 differed in viremia and clinical outcome, there was no evidence for adaptive evolution before, during, or after the outbreak, indicating that ecological or immunological rather than virological factors were the key determinants of epidemic dynamics.

IDBD: infectious disease biomarker database.

Biomarkers enable early diagnosis, guide molecularly targeted therapy and monitor the activity and therapeutic responses across a variety of diseases. Despite intensified interest and research, however, the overall rate of development of novel biomarkers has been falling. Moreover, no solution is yet available that efficiently retrieves and processes biomarker information pertaining to infectious diseases. Infectious Disease Biomarker Database (IDBD) is one of the first efforts to build an easily accessible and comprehensive literature-derived database covering known infectious disease biomarkers. IDBD is a community annotation database, utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It allows users to link infectious diseases or pathogens to protein, gene or carbohydrate biomarkers through the use of search tools. It supports various types of data searches and application tools to analyze sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 611 biomarkers for 66 infectious diseases and 70 pathogens. It is publicly accessible at http://biomarker.cdc.go.kr and http://biomarker.korea.ac.kr.

Spatial competition shapes the dynamic mutational landscape of normal esophageal epithelium.

During aging, progenitor cells acquire mutations, which may generate clones that colonize the surrounding tissue. By middle age, normal human tissues, including the esophageal epithelium (EE), become a patchwork of mutant clones. Despite their relevance for understanding aging and cancer, the processes that underpin mutational selection in normal tissues remain poorly understood. Here, we investigated this issue in the esophageal epithelium of mutagen-treated mice. Deep sequencing identified numerous mutant clones with multiple genes under positive selection, including Notch1, Notch2 and Trp53, which are also selected in human esophageal epithelium. Transgenic lineage tracing revealed strong clonal competition that evolved over time. Clone dynamics were consistent with a simple model in which the proliferative advantage conferred by positively selected mutations depends on the nature of the neighboring cells. When clones with similar competitive fitness collide, mutant cell fate reverts towards homeostasis, a constraint that explains how selection operates in normal-appearing epithelium.