Full-length RNA Sequencing Research Articles

Direct Full-Length RNA Sequencing Reveals an Important Role of Epigenetics During Sexual Reversal in Chinese Soft-Shelled Turtle.

Sex dimorphism is a key feature of Chinese soft-shelled turtle (Pelodiscus sinensis). The males (M) have higher econosmic value than females (F) due to wider calipash and faster growth. Exogenous hormones like estradiol and methyltestosterone can induce sexual reversal to form new phenotypes (pseudo-female, PF; pseudo-male, PM) without changing the genotype. The possibility of inducing sexual reversal is particularly important in aquaculture breeding, but the underlying biological mechanisms remain unclear. Here we applied a direct RNA sequencing method with ultralong reads using Oxford Nanopore Technologies to study the transcriptome complexity in P. sinensis. Nanopore sequencing of the four gender types (M, F, PF, and PM) showed that the distribution of read length and gene expression was more similar between same-sex phenotypes than same-sex genotypes. Compared to turtles with an M phenotype, alternative splicing was more pronounced in F turtles, especially at alternative 3′ splice sites, alternative 5′ splice sites, and alternative first exons. Furthermore, the two RNA methylation modifications m5C and m6A were differentially distributed across gender phenotypes, with the M type having more modification sites in coding sequence regions, but fewer modification sites in 3′UTR regions. Quantitative analysis of enriched m6A RNAs revealed that the N6-methylated levels of Odf2, Pacs2, and Ak1 were significantly higher in M phenotype individuals, while the N6-methylated levels of Ube2o were reduced after sexual reversal from both M and F phenotypes. Taken together, these findings reveal an important role of epigenetics during sexual reversal in Chinese soft-shelled turtles.

Long-read isoform sequencing reveals tissue-specific isoform expression between active and hibernating brown bears (Ursus arctos).

Understanding hibernation in brown bears (Ursus arctos) can provide insight into some human diseases. During hibernation, brown bears experience periods of insulin resistance, physical inactivity, extreme bradycardia, obesity, and the absence of urine production. These states closely mimic aspects of human diseases such as type 2 diabetes, muscle atrophy, as well as renal and heart failure. The reversibility of these states from hibernation to active season enables the identification of mediators with possible therapeutic value for humans. Recent studies have identified genes and pathways that are differentially expressed between active and hibernation seasons in bears. However, little is known about the role of differential expression of gene isoforms on hibernation physiology. To identify both distinct and novel mRNA isoforms, full-length RNA-sequencing (Iso-Seq) was performed on adipose, skeletal muscle, and liver from three individual bears sampled during both active and hibernation seasons. The existing reference genome annotation was improved by combining it with the Iso-Seq data. Short-read RNA-sequencing data from six individuals were mapped to the new reference annotation to quantify differential isoform usage (DIU) between tissues and seasons. We identified differentially expressed isoforms in all three tissues, to varying degrees. Adipose had a high level of DIU with isoform switching, regardless of whether the genes were differentially expressed. Our analyses revealed that DIU, even in the absence of differential gene expression, is an important mechanism for modulating genes during hibernation. These findings demonstrate the value of isoform expression studies and will serve as the basis for deeper exploration into hibernation biology.

A Comparative Transcriptional Landscape of Two Castor Cultivars Obtained by Single-Molecule Sequencing Comparative Analysis.

Background and Objectives: Castor (Ricinus communis L.) is an important non-edible oilseed crop. Lm-type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in their inflorescence structures and leaf shapes. To better understand the mechanisms underlying these differences at the molecular level, we performed a comparative transcriptional analysis. Materials and Methods: Full-length transcriptome sequencing and short-read RNA sequencing were employed. Results: A total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm-type female strains and normal amphiprotic strains, respectively. In Lm-type female strains and normal amphiprotic strains, 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed a great variety of gene expression of common and unique transcription factors between the two cultivars. Meanwhile, a functional analysis of the isoforms was conducted. The full-length sequences were used as a reference genome, and a short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis. Conclusion: The results revealed considerable differences and expression diversity between the two cultivars, well beyond what was reported in previous studies and likely reflecting the differences in architecture between these two cultivars.

Comparative transcriptome analysis reveals regulatory network and regulators associated with proanthocyanidin accumulation in persimmon

BackgroundProanthocyanidins (PAs) are important plant secondary metabolites that confer flavor, nutritional value, and resistance to pathogens. Persimmon is one of the PA richest crops. Mature fruits can be inedible because of the astringency caused by high PA levels and need to go through a de-astringency treatment before consumption. The molecular basis for PA accumulation is poorly known, particularly transcriptional regulators. We characterised three genotypes (‘Luotiantianshi’ (LT), ‘Mopanshi’ (MP), and ‘Youhou’ (YH)) with different PA accumulation patterns using an approach that combined PacBio full-length sequencing and Illumina-based RNA sequencing to build high-quality full-length transcriptomes. Additionally, we analysed transcriptome dynamics of the three genotypes (LT, MP, and YH) at four key fruit developmental stages.ResultsA total of 96,463 transcripts were obtained. We identified 80,075 protein-coding sequences (CDSs), 71,137 simple sequence repeats (SSRs), and 27,845 long noncoding RNAs (lncRNAs). Pearson correlation coefficient (PCC), principal component analysis (PCA), and differentially expressed transcripts (DETs) analyses indicated that the four different developmental stages within a genotype exhibited similar transcriptome activities. A total of 2,164 transcripts specific to each fruit developmental stage were detected. The transcripts specific to early stages were attributed to phenylpropanoid and flavonoid biosynthesis. Co-expression network analyses revealed MEbrown and MEblue modules were strongly associated to PA accumulation. From these two modules, 20 hub TFs are potential regulators for PA accumulation. Among them, Cluster_78388 (SBP protein), Cluster_63454 (bZIP protein), and Cluster_66595 (MYB protein) appear to involve in the PA biosynthesis in Chinese genotypes.ConclusionsThis is the first high-quality reference transcriptome for commercial persimmon. Our work provides insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout fruit development.

Dissecting spatial heterogeneity and the immune-evasion mechanism of CTCs by single-cell RNA-seq in hepatocellular carcinoma

Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

Single cell transcriptome sequencing on the Nanopore platform with ScNapBar.

The current ecosystem of single-cell RNA-seq platforms is rapidly expanding, but robust solutions for single-cell and single-molecule full-length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single-cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single-cell sequencing method on the 10× Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g., for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing saturation is low. ScNapBar uses unique molecular identifier (UMI) or Naïve Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore data sets. We further applied ScNapBar to pools of cells with an active or a silenced nonsense-mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.

Characterization of local gut microbiome and intestinal transcriptome responses to rosiglitazone treatment in diabetic db/db mice

The gut microbiota has been implicated in the therapeutic effects of antidiabetics. It is unclear if antidiabetics directly influences gut microbiome-host interaction. Oral peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists, such as rosiglitazone, are potent insulin sensitizers used in the treatment of type 2 diabetes (T2D). PPAR-γ is abundantly expressed in the intestine, making it possible that PPAR-γ agonists directly influences gut microbiome-host homeostasis. The presented study therefore aimed to characterize local gut microbiome and intestinal transcriptome responses in diabetic db/db mice following rosiglitazone treatment. Diabetic B6.BKS(D)-Leprdb/J (db/db) mice (8 weeks of age) received oral dosing once daily with vehicle (n = 12) or rosiglitazone (3 mg/kg, n = 12) for 8 weeks. Gut segments (duodenum, jejunum, ileum, caecum, and colon) were sampled for paired analysis of gut microbiota and host transcriptome signatures using full-length bacterial 16S rRNA sequencing and RNA sequencing (n = 5–6 per group). Treatment with rosiglitazone improved glucose homeostasis without influencing local gut microbiome composition in db/db mice. In contrast, rosiglitazone promoted marked changes in ileal and colonic gene expression signatures associated with peroxisomal and mitochondrial lipid metabolism, carbohydrate utilization and immune regulation. In conclusion, rosiglitazone treatment markedly affected transcriptional markers of intestinal lipid metabolism and immune regulation but had no effect on the gut microbiome in diabetic db/db mice.

Recent advances and future perspectives in vector-omics.

We have reviewed recent progress and the remaining challenges in vector-omics. We have highlighted several technologies and applications that facilitate novel biological insights beyond achieving a reference-quality genome assembly. Among other topics, we have discussed the applications of chromatin conformation capture, chromatin accessibility assays, optical mapping, full-length RNA sequencing, single cell RNA analysis, proteomics, and population genomics. We anticipate that we will witness a great expansion in vector-omics research not only in its application in a broad range of species, but also its ability to uncover novel genetic elements and tackle previously inaccessible regions of the genome. It is our hope that the continued innovation in device portability, cost reduction, and informatics support will in the foreseeable future bring vector-omics to every vector laboratory and field station in the world, which will have an unparalleled impact on basic research and the control of vector-borne infectious diseases.

Abstract A92: Assessing the T-cell exhaustion status of TCR discovery outputs

Abstract Identification of antigen-specific T cell receptors (TCRs) is an essential component of the ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) platform at Immunocore. ImmTAC molecules are formed of a soluble TCR fused to an anti-CD3 effector function that engages, redirects and activates T cells to kill cancerous cells. TCRs recognize peptide antigens presented on the surface of the cancer cell by human leukocyte antigen (HLA). Thus, a fundamental step in our pipeline is to isolate T-cell receptors specific for validated cancer antigens for downstream development. T cells are isolated from whole blood of healthy donors to identify antigen-specific T cell clones using an in vitro model that consists of several stimulation steps. Activated CD8+ T cells are sorted into single cells for subsequent analysis and characterization. Despite the relative success of this approach, often analysis of T-cell clones is limited by low or absent mRNA expression for either or both TCR alpha (TRAV) and beta (TRBV) chains. We predict that TCR downregulation may result from overstimulation of T cells, leading to exhaustion. Here, we aimed to establish an in vitro model of T-cell exhaustion that would enable us to better understand the molecular mechanisms behind this process and thus facilitate method development. We induced extreme conditions of stimulation using CD3/CD28 beads and recombinant human IL-2 (rhIL-2) for 2-3 weeks. Whole-transcriptome analysis of the resulting activated T cells was compared to rested cells using a 5′ single-cell RNA-seq method from 10X Genomics (California, USA). Our preliminary analysis indicates that the activated T cells express several checkpoint inhibitors at higher levels. Our findings will be further investigated by performing full-length RNA sequencing of previously isolated single T-cell clones with low TRAV and/or TRBV detection rates. By identifying a molecular signature that represents the phenotype of exhausted T cells, we can potentially assess the state of T cells in TCR discovery outputs. Most importantly, determination of the expression levels of inhibitory receptors that are responsible for the decrease or loss of TCR expression is expected to enhance our understanding of the potential impact of T-cell exhaustion. Citation Format: Paraskevi Mallini, Filipa Bravo-Lopes, Karolina Lech, Sunir Malla, Maria Busz, Nathaniel Davies, Jacob Hurst, Luke Williams, Sterenn Davis, Michelle Teng. Assessing the T-cell exhaustion status of TCR discovery outputs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A92.

Analysis of Alternative Splicing and Alternative Polyadenylation in Populus alba var. pyramidalis by Single-Molecular Long-Read Sequencing.

Poplars are worldwidely cultivated with ecologically and economically important value. Populus alba var. pyramidalis (= P. bolleana) is a main tree of the farmland shelter-belt system in the arid region of Northwest China due to its rapid growth, erect stems, and high biomass production. However, the full-length messenger RNA (mRNA) sequences and complete structure of P. alba var. pyramidalis remain unclear. In this study, using single-molecular real-time (SMRT) and next-generation high-throughput sequencing (NGS) platform, we sequenced transcripts from leaf, root, xylem, and phloem of P. alba var. pyramidalis, to obtain the full-length mRNA transcripts and annotate the complete structure. In total, 86,327 mapped full-length non-chimeric (FLNC) reads were identified, with 705 previously unannotated loci and 3,410 long noncoding RNAs (lncRNAs) and 174 fusion genes found. Alternative spicing (AS) events were detected in 7,536 genes, of which 4,652 genes had multiple AS events. A total of 10,213 alternative polyadenylation (APA) sites were identified, with two or more APA sites observed in 2,212 genes. Our transcriptome data provided the full-length sequences and gene isoforms of transcripts for P. alba var. pyramidalis, which will be helpful in improving our understanding for the genome annotation and gene structures of P. alba var. pyramidalis.

Direct full-length RNA sequencing reveals unexpected transcriptome complexity during Caenorhabditis elegans development.

Massively parallel sequencing of the polyadenylated RNAs has played a key role in delineating transcriptome complexity, including alternative use of an exon, promoter, 5′ or 3′ splice site or polyadenylation site, and RNA modification. However, reads derived from the current RNA-seq technologies are usually short and deprived of information on modification, compromising their potential in defining transcriptome complexity. Here, we applied a direct RNA sequencing method with ultralong reads using Oxford Nanopore Technologies to study the transcriptome complexity in Caenorhabditis elegans. We generated approximately six million reads using native poly(A)-tailed mRNAs from three developmental stages, with average read lengths ranging from 900 to 1100 nt. Around half of the reads represent full-length transcripts. To utilize the full-length transcripts in defining transcriptome complexity, we devised a method to classify the long reads as the same as existing transcripts or as a novel transcript using sequence mapping tracks rather than existing intron/exon structures, which allowed us to identify roughly 57,000 novel isoforms and recover at least 26,000 out of the 33,500 existing isoforms. The sets of genes with differential expression versus differential isoform usage over development are largely different, implying a fine-tuned regulation at isoform level. We also observed an unexpected increase in putative RNA modification in all bases in the coding region relative to the UTR, suggesting their possible roles in translation. The RNA reads and the method for read classification are expected to deliver new insights into RNA processing and modification and their underlying biology in the future.

CircSLNN: Identifying RBP-Binding Sites on circRNAs via Sequence Labeling Neural Networks.

The interactions between RNAs and RNA binding proteins (RBPs) are crucial for understanding post-transcriptional regulation mechanisms. A lot of computational tools have been developed to automatically predict the binding relationship between RNAs and RBPs. However, most of the methods can only predict the presence or absence of binding sites for a sequence fragment, without providing specific information on the position or length of the binding sites. Besides, the existing tools focus on the interaction between RBPs and linear RNAs, while the binding sites on circular RNAs (circRNAs) have been rarely studied. In this study, we model the prediction of binding sites on RNAs as a sequence labeling problem, and propose a new model called circSLNN to identify the specific location of RBP-binding sites on circRNAs. CircSLNN is driven by pretrained RNA embedding vectors and a composite labeling model. On our constructed circRNA datasets, our model has an average F 1 score of 0.790. We assess the performance on full-length RNA sequences, the proposed model outperforms previous classification-based models by a large margin.

Tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing.

Polyadenylation at the 3′-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3′-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr’s performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far. Although single splicing events have been described for ≤200 single cells with statistical confidence, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3' sequencing enables the identification of cellular subtypes, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms.

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir.

The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection.

A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene

Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766NIID, the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.