Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.

BACKGROUND: Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies. RESULTS: The MinION run of the HCMV (strain TB40/E) library resulted in ~ 47,000 reads from a single R9 flowcell and in ~ 100× average read depth across the virus genome. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage of the bioinformatics algorithm, long contigs (N50 = 21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50 = 5686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50 = 41,056). We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions. CONCLUSIONS: Third generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis

Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

Background & AimsMucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored.MethodsThe phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1.ResultsIntrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17.ConclusionsOur findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future

Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.

BACKGROUND: Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies. RESULTS: The MinION run of the HCMV (strain TB40/E) library resulted in ~ 47,000 reads from a single R9 flowcell and in ~ 100× average read depth across the virus genome. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage of the bioinformatics algorithm, long contigs (N50 = 21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50 = 5686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50 = 41,056). We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions. CONCLUSIONS: Third generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis

Investigation into sub-cellular CD4 distribution in human embryonic stem cell derived macrophages and its role in HIV-1 infection

Human macrophages are one of the main targets for HIV-1 infection, despite their moderately low surface expression levels of the main HIV-1 receptor, CD4. The site of HIV-1 fusion can occur at the surface or following uptake through an endosomal pathway and it might be anticipated that the site would affect the progress of HIV-1 through the cell to the nucleus. Previous pharmacological studies provide one line of evidence for an endosomal entry route which is dependent on Detergent Resistant Membranes (DRMs). However, these findings need confirmation using a genetic approach, as small molecules may have multiple non-specific effects. For this study, a novel genetic approach was developed to manipulate sub-cellular CD4 distribution and investigate whether it determines the HIV-1 entry pathway in macrophages. This was achieved by transducing human embryonic stem cells (hESC) with lentiviral vectors and differentiating these cells into homogeneous genetically modified macrophages. This cellular system by-passes the challenges posed by the refractoriness to direct genetic manipulation of heterogeneous primary macrophages. Firstly, as proof of principle, a short hairpin RNA targeting CD4 was expressed in hESC-macrophages, resulting in knockdown of CD4 and, as anticipated, strong inhibition of HIV-1 infection. Secondly, expression of LCK in hESC-macrophages effectively tethered CD4 at the cell surface, and sequestered HIV-1 into an unproductive pathway, presumably through surface fusion, rather than progressing successfully to the nucleus. Thirdly, endogenous CD4 was substituted with CD4 mutants designed to be excluded from DRMs, which resulted in reduced successful HIV-1 entry versus substituted control CD4. The results support the model in which the productive entry pathway of HIV-1 in macrophages occurs via fusion after a raft-dependent endocytic uptake pathway, and requires CD4 localization to lipid rafts.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions

<div><p>Human macrophages are specialised hosts for HIV-1, dengue virus, <i>Leishmania</i> and <i>Mycobacterium tuberculosis</i>. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×10⁷ monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14⁺, CD16^low, CD163⁺). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.</p></div

Monocytopoiesis characterization in differentiation cultures.

<p>A) Time-course analysis of cell surfaces marker expressed during monocytopoiesis. Adherent EBs were harvested at 12, 21 and 33 days after EB formation and stained for various cell surface markers. Immuno-fluorescence dot plot analysis of CD38 and CD34; Thy-1 and CD34; CD45 and CD14. Gates were determined by using the relevant isotype control antibodies. B) Forward Scatter (FSC) and Side Scatter (SSC) dot plot of harvested PSC-MC showing a gate around the homogenous cell population. C) Non-adherent PSC-MC were harvested from the supernatant of differentiation cultures and counted using a cell counter (Chemometec). The media were replaced for repeated PSC-MC harvests over a period of 52 week. Data represent the cumulative number of viable PSC-MC from 6 wells.</p

PSC-MDM characterization compared to b-MC.

<p>A) Morphology of MDM. From left to right: Representative brightfield image (scale bare 200 µM), Scanning Electron Microscopy and Transmission Electron Microscopy (scale bar, 10 µM) D) Diameter of PSC-MDM (n = 3) and b-MDM (n = 6) were determined by an AO-DAPI stain (Chemometec). Bars represent the mean diameter ± SEM. C) Phenotype of MC. Surface expression of CD14, CD16, CD163, CD86 and MHC II were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). D) The symbols reflect the relative ratio of the geometric mean fluorescence intensity (MFI) over the isotype control of independent experiments.</p