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Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions. Author summary: Little is known about the host response to Venezuelan Equine Encephalitis Virus (VEEV) and the cellular factors this alphavirus hijacks to support effective replication or evade cellular immune responses. Monitoring dynamics of host and viral RNA (vRNA) during viral infection at a single-cell level can provide insight into the virus-host interplay at a high resolution. Here, a single-cell RNA sequencing technology that detects host and viral RNA was used to investigate the interactions between TC-83, the vaccine strain of VEEV, with the human host during the course of infection of U-87 MG cells (human astrocytoma). Virus abundance and host transcriptome were heterogeneous across cells from the same culture. Subsets of differentially expressed genes, positively or negatively correlating with vRNA abundance, were identified and subsequently in vitro validated as candidate proviral and antiviral factors, respectively, in TC-83 and/or virulent VEEV infections. In the first replication cycle, "superproducer" cells exhibited rapid increase in vRNA abundance and unique gene expression patterns. At later time points, cells with increased structural-to-nonstructural transcript ratio demonstrated upregulation of intracellular membrane trafficking genes. Lastly, comparing the VEEV dataset with published datasets on other RNA viruses revealed unique and overlapping responses across viral clades. Overall, this study improves the understanding of VEEV-host interactions, reveals candidate targets for antiviral approaches, and establishes a comparative single-cell approach to study the evolution of virus-host interactions. [ABSTRACT FROM AUTHOR]

Summary HTSeq 2.0 provides a more extensive application programming interface including a new representation for sparse genomic data, enhancements for htseq-count to suit single-cell omics, a new script for data using cell and molecular barcodes, improved documentation, testing and deployment, bug fixes and Python 3 support. Availability and implementation HTSeq 2.0 is released as an open-source software under the GNU General Public License and is available from the Python Package Index at https://pypi.python.org/pypi/HTSeq. The source code is available on Github at https://github.com/htseq/htseq. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level. [ABSTRACT FROM AUTHOR]

HIV-1 infection cannot be cured because the virus persists as integrated proviral DNA in long-lived cells despite years of suppressive antiretroviral therapy (ART). In a previous paper (Zanini et al, 2015) we documented HIV-1 evolution in 10 untreated patients. Here we characterize establishment, turnover, and evolution of viral DNA reservoirs in the same patients after 3-18 years of suppressive ART. A median of 14% (range 0-42%) of the DNA sequences were defective due to G-to-A hypermutation. Remaining DNA sequences showed no evidence of evolution over years of suppressive ART. Most sequences from the DNA reservoirs were very similar to viruses actively replicating in plasma (RNA sequences) shortly before start of ART. The results do not support persistent HIV-1 replication as a mechanism to maintain the HIV-1 reservoir during suppressive therapy. Rather, the data indicate that DNA variants are turning over as long as patients are untreated and that suppressive ART halts this turnover. [ABSTRACT FROM AUTHOR]

Many microbial populations rapidly adapt to changing environments with multiple variants competing for survival. To quantify such complex evolutionary dynamics in vivo, time resolved and genome wide data including rare variants are essential. We performed whole-genome deep sequencing of HIV-1 populations in 9 untreated patients, with 6-12 longitudinal samples per patient spanning 5-8 years of infection. The data can be accessed and explored via an interactive web application. We show that patterns of minor diversity are reproducible between patients and mirror global HIV-1 diversity, suggesting a universal landscape of fitness costs that control diversity. Reversions towards the ancestral HIV-1 sequence are observed throughout infection and account for almost one third of all sequence changes. Reversion rates depend strongly on conservation. Frequent recombination limits linkage disequilibrium to about 100bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. [ABSTRACT FROM AUTHOR]

The dynamics of proteins in solution is a complex and hierarchical process, affected by the aqueous environment as well as temperature. We present a comprehensive study on nanosecond time and nanometer length scales below, at, and above the denaturation temperature Td. Our experimental data evidence dynamical processes in protein solutions on three distinct time scales. We suggest a consistent physical picture of hierarchical protein dynamics: (i) self-diffusion of the entire protein molecule is confirmed to agree with colloid theory for all temperatures where the protein is in its native conformational state. At higher temperatures T ＞ Td, the self-diffusion is strongly obstructed by cross-linking or entanglement. (ii) The amplitude of backbone fluctuations grows with increasing T, and a transition in its dynamics is observed above Td. (iii) The number of mobile side-chains increases sharply at Td while their average dynamics exhibits only little variations. The combination of quasi-elastic neutron scattering and the presented analytical framework provides a detailed microscopic picture of the protein molecular dynamics in solution, thereby reflecting the changes of macroscopic properties such as cluster formation and gelation. [ABSTRACT FROM AUTHOR]

Highlights: [•] The Beijing family of M. tuberculosis is an emerging lineage in the area of Milan. [•] Beijing strains affect mainly young foreign-born patients. [•] Beijing strains are associated with MDR, but not with HIV infection. [•] Constant prevalence over time of the founder Beijing sublineages. [Copyright &y& Elsevier]

Intrapatient evolution of human immunodeficiency virus type 1 (HIV-1) is driven by the adaptive immune system resulting in rapid change of HIV-1 proteins. When cytotoxic CD8+ T cells or neutralizing antibodies target a new epitope, the virus often escapes via nonsynonymous mutations that impair recognition. Synonymous mutations do not affect this interplay and are often assumed to be neutral. We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. We find that most synonymous variants are lost even though they often reach high frequencies in the viral population, suggesting a cost to the virus. Using published data from SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) assays, we find that synonymous mutations that disrupt base pairs in RNA stems flanking the variable loops of gp120 are more likely to be lost than other synonymous changes: these RNA hairpins might be important for HIV-1. Computational modeling indicates that, to be consistent with the data, a large fraction of synonymous mutations in this genomic region need to be deleterious with a cost on the order of 0.002 per day. This weak selection against synonymous substitutions does not result in a strong pattern of conservation in cross-sectional data but slows down the rate of evolution considerably. Our findings are consistent with the notion that large-scale patterns of RNA structure are functionally relevant, whereas the precise base pairing pattern is not. [ABSTRACT FROM AUTHOR]

Mechanisms by which HIV affects the thymus are multiple and only partially known, and the role of thymic dysfunction in HIV/AIDS immunopathogenesis remains poorly understood. To evaluate the effects of HIV infection on intra-thymic precursors of T cells in HIV-infected adults, we conducted a detailed immunophenotypic study of thymic tissue isolated from 7 HIV-infected and 10 HIV-negative adults who were to undergo heart surgery. We found that thymuses of HIVinfected individuals were characterized by a relative depletion of CD4+ single positive T cells and a corresponding enrichment of CD8+ single positive T cells. In addition, thymocytes derived from HIV-infected subjects showed increased levels of activated and proliferating cells. Our analysis also revealed a decreased expression of interleukin-7 receptor in early thymocytes from HIV-infected individuals, along with an increase in this same expression in mature double- and singlepositive cells. Frequency of regulatory T cells (CD25+FoxP3+) was significantly increased in HIV-infected thymuses, particularly in priorly-committed CD4 single positive cells. Our data suggest that HIV infection is associated with a complex set of changes in the immunophenotype of thymocytes, including a reduction of intrathymic CD4+ T cell precursors, increased expression of activation markers, changes in the expression pattern of IL-7R and enrichment of T regulatory cells generation. [ABSTRACT FROM AUTHOR]

Single cell transcriptomics is revolutionising our understanding of tissue and disease heterogeneity, yet cell type identification remains a partially manual task. Published algorithms for automatic cell annotation are limited to known cell types and fail to capture novel populations, especially cancer cells. We developed northstar, a computational approach to classify thousands of cells based on published data within seconds while simultaneously identifying and highlighting new cell states such as malignancies. We tested northstar on data from glioblastoma, melanoma, and seven different healthy tissues and obtained high accuracy and robustness. We collected eleven pancreatic tumors and identified three shared and five private neoplastic cell populations, offering insight into the origins of neuroendocrine and exocrine tumors. Northstar is a useful tool to assign known and novel cell type and states in the age of cell atlases. [ABSTRACT FROM AUTHOR]

Introduction: The present study consists in a bibliographical' review concerning the articles related to the different manifestations and forms of the septum nasal perforation, with a main emphasis in the description of its techniques of surgical correction. As the etiology, that is fundamentally iatrogenic, accordingly surgical trauma. Other causes include exhibition to chemical industrial reagents, cocaine use, intranasal steroidal therapeutic, neoplasia and, infectious conditions. There is a few data concerning the prevalence of septum perforation in the general population. The decision making about surgical correction or medical treatment depend on the anatomic characteristics and of the pathogenesis of the septal perforation.