Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Untersuchungen zum intrazellulären Transportmechanismus von Marburgvirus Nukleokapsiden

Das Marburgvirus bildet zusammen mit dem Ebolavirus die Familie der Filoviridae. Beide Viren lösen beim Menschen und bei nicht-menschlichen Primaten ein starkes hämorrhagisches Fie-ber mit hoher Sterblichkeitsrate aus. Die Viren besitzen ein einzelsträngiges, nicht-segmentiertes RNA-Genom in negativer Orientierung und gehören somit in die Ordnung der Mononegavirales. Die RNA codiert für sieben virale Strukturproteine, von denen fünf das Ge-nom enkapsidieren: das Nukleoprotein NP, die viralen Proteine VP24, VP30 und VP35 sowie die Polymerase L. VP40 umgibt als Matrixprotein den als Nukleokapsid bezeichneten Komplex. In die Lipidhülle, die das längliche Virus umgibt, ist das Glykoprotein GP inseriert. Diese Arbeit beschäftigt sich mit dem intrazellulären Transport von Nukleokapsiden in der Marburgvirus-infizierten Zelle. In erster Linie wurde hierfür die Methode der Lebendzellmikroskopie ver-wendet, die von konfokaler Mikroskopie und Scanning Elektronenmikroskopie ergänzt wurde. Durch ein reverses Marburgvirus-spezifisches Genetiksystem wurde ein zusätzlicher Leserah-men für ein rot markiertes VP40 in das virale Genom integriert und ein rekombinantes Virus hergestellt (rMARVRFP-VP40). In Kombination mit der Plasmid-gestützten Expression von grün fluoreszierenden VP30 (VP30GFP) konnte in rMARVRFP-VP40-infizierten Zellen, neben dem Mat-rixprotein auch das Nukleokapsid sichtbar gemacht werden. Die Zweifarbmarkierung erlaubte es, sogenannte Inclusions als den Ursprungsort von neu gebildeten Nukleokapsiden zu identi-fizieren. Obwohl VP40 in Inclusions vorliegt, tragen neugebildete Nukleokapside nach Freiset-zung aus den Einschlusskörpern keine detektierbare Menge des Matrixproteins. Nukleokapsi-de werden in einem scheinbar zufällig ablaufenden, aktinbasierten Langstreckentransport mit Geschwindigkeiten zwischen 200 und 500 nm/s entlang von Aktinfilamenten transportiert. Nach einem mehrere Mikrometer langen Transportweg verlangsamen Nukleokapside ihre Bewegung auf ca. 100 nm/s und an der Plasmamembran treffen Matrixprotein und Nukleoka-pside aufeinander. Es kommt zur dynamischen Assoziation mit dem Matrixprotein, die von der Phosphorylierung des Matrixproteins an Tyrosinresten abhängig ist. Die Assoziation der Nuk-leokapside mit dem Matrixprotein ist erforderlich für den Austrittsprozess der Viren. Mar-burgviren nutzen längliche Zellausläufer (Filopodien), um die Zelle zu verlassen. Die Nukleo-kapside werden in den Filopodien gemeinsam mit Myosin 10 entlang von Aktinbündeln transportiert

Untersuchungen zum intrazellulären Transportmechanismus von Marburgvirus Nukleokapsiden

Das Marburgvirus bildet zusammen mit dem Ebolavirus die Familie der Filoviridae. Beide Viren lösen beim Menschen und bei nicht-menschlichen Primaten ein starkes hämorrhagisches Fie-ber mit hoher Sterblichkeitsrate aus. Die Viren besitzen ein einzelsträngiges, nicht-segmentiertes RNA-Genom in negativer Orientierung und gehören somit in die Ordnung der Mononegavirales. Die RNA codiert für sieben virale Strukturproteine, von denen fünf das Ge-nom enkapsidieren: das Nukleoprotein NP, die viralen Proteine VP24, VP30 und VP35 sowie die Polymerase L. VP40 umgibt als Matrixprotein den als Nukleokapsid bezeichneten Komplex. In die Lipidhülle, die das längliche Virus umgibt, ist das Glykoprotein GP inseriert. Diese Arbeit beschäftigt sich mit dem intrazellulären Transport von Nukleokapsiden in der Marburgvirus-infizierten Zelle. In erster Linie wurde hierfür die Methode der Lebendzellmikroskopie ver-wendet, die von konfokaler Mikroskopie und Scanning Elektronenmikroskopie ergänzt wurde. Durch ein reverses Marburgvirus-spezifisches Genetiksystem wurde ein zusätzlicher Leserah-men für ein rot markiertes VP40 in das virale Genom integriert und ein rekombinantes Virus hergestellt (rMARVRFP-VP40). In Kombination mit der Plasmid-gestützten Expression von grün fluoreszierenden VP30 (VP30GFP) konnte in rMARVRFP-VP40-infizierten Zellen, neben dem Mat-rixprotein auch das Nukleokapsid sichtbar gemacht werden. Die Zweifarbmarkierung erlaubte es, sogenannte Inclusions als den Ursprungsort von neu gebildeten Nukleokapsiden zu identi-fizieren. Obwohl VP40 in Inclusions vorliegt, tragen neugebildete Nukleokapside nach Freiset-zung aus den Einschlusskörpern keine detektierbare Menge des Matrixproteins. Nukleokapsi-de werden in einem scheinbar zufällig ablaufenden, aktinbasierten Langstreckentransport mit Geschwindigkeiten zwischen 200 und 500 nm/s entlang von Aktinfilamenten transportiert. Nach einem mehrere Mikrometer langen Transportweg verlangsamen Nukleokapside ihre Bewegung auf ca. 100 nm/s und an der Plasmamembran treffen Matrixprotein und Nukleoka-pside aufeinander. Es kommt zur dynamischen Assoziation mit dem Matrixprotein, die von der Phosphorylierung des Matrixproteins an Tyrosinresten abhängig ist. Die Assoziation der Nuk-leokapside mit dem Matrixprotein ist erforderlich für den Austrittsprozess der Viren. Mar-burgviren nutzen längliche Zellausläufer (Filopodien), um die Zelle zu verlassen. Die Nukleo-kapside werden in den Filopodien gemeinsam mit Myosin 10 entlang von Aktinbündeln transportiert

Interaction with Tsg101 Is Necessary for the Efficient Transport and Release of Nucleocapsids in Marburg Virus-Infected Cells

<div><p>Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV_PSAPmut). rMARV_PSAPmut was attenuated by up to one log compared with recombinant wild-type MARV (rMARV_wt), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV_PSAPmut-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV_wt-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV_wt-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV_PSAPmut nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.</p></div

rMARV_PSAPmut exhibits delayed growth kinetics.

<p>Vero E6 cells were inoculated with either rMARV_PSAPmut or rMARV_wt. Supernatants and cell lysates were collected at indicated time points p.i. and viral titres determined by TCID₅₀ assay or subjected to Western blot analysis. (<b>A</b>) Growth kinetics of rMARV_PSAPmut (grey circle) or rMARV_wt (black square) at MOI of 0.01. (<b>B</b>) Western Blot analysis of viral protein levels during an infection at MOI of 0.01. Cell lysates and culture supernatants were collected at indicated time points and were analyzed by SDS-PAGE and Western Blotting using NP- and VP40-specific antibodies. (<b>C</b>) Growth kinetics of rMARV_PSAPmut (grey circle) or rMARV_wt (black square) at MOI of 0.1. (<b>D</b>) rMARV_PSAPmut- or rMARV_wt– or mock-infected cells were analyzed for CPE formation during infection at MOI of 0.1 at 3 days p.i. (<b>E</b>) Western Blot analysis of viral protein levels during an infection at MOI of 0.1. Cell lysates and culture supernatants were collected at indicated time points and were analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004463#ppat-1004463-g001" target="_blank">Fig. 1B</a>. P-values are indicated (_*, P≤0.05; _**, P≤0.001; _***, P≤0.0001).</p

rMARV_PSAPmut particles incorporate less Tsg101 and display similar infectivity.

<p>(<b>A</b>) Tsg101 incorporation into MARV particles. Vero E6 cells were infected with rMARV_wt or rMARV_PSAPmut and virus particles released into the supernatant were pelleted through a 20% sucrose cushion at 48 h p.i. Virus pellets and cell lysates were subjected to SDS-PAGE and Western Blot analysis using Tsg101-specific antibody. (<b>B</b>) Detection of ubiquitinated form of Tsg101 in viral particles. Vero E6 cells were infected with rMARV_wt or rMARV_PSAPmut and subsequently transfected with HA-Ub expression plasmid. Virus particles were pelleted from the supernatants and analyzed by SDS-PAGE and Western Blot analysis using anti-Tsg101 and anti-HA specific primary antibodies and secondary antibodies for detection with the Odyssey imaging system (see merge image). (<b>C, D</b>) Comparison of virus infectivity. (<b>C</b>) Equal amounts of TCID₅₀ units of rMARV_PSAPmut and rMARV_wt stock viruses were pelleted through 20% sucrose cushion, separated by SDS-PAGE and analyzed by Western Blot using NP- and VP40-specific antibodies. (<b>D</b>) Huh-7 cells grown on glass cover slips were inoculated with rMARV_PSAPmut and rMARV_wt normalized to nucleoprotein amount, fixed at 17 h p.i. and stained with DAPI and NP-specific antibody for detection of infected cells by immunofluorescence assay.</p

Budding from filopodia is reduced in rMARV_PSAPmut-infected cells.

<p>(<b>A</b>) Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut at an MOI of 1, and fixed at 19–23 h p.i. The whole mounted cells were analyzed by electron microscopy on grids after negative staining. Graphics shows the percentage of nucleocapsids in the process of budding from the filopodia (20 micrographs were analyzed for each virus), p-value (*** P≤0.0001). (<b>B</b>) Western Blot analysis of viral proteins in cell lysates. Huh-7 cells were infected as indicated in (A), harvested at 19 h p.i., and analyzed by using NP- and VP40-specific antibodies. (<b>C</b>) Live cell imaging of Huh-7 cells were infected with rMARV_wt- or rMARV_PSAPmut. At 28 h (rMARV_PSAPmut) and 43 h p.i. (rMARV_wt), a series of 600 pictures was taken every second for a period of 10 min. Maximal projection of the picture series is displayed. Boxes in the left panels indicate areas that are shown at higher magnification in the middle panels. Trajectories of individual nucleocapsids are highlighted with white dashed lines. White asterisks indicate regions with several static rMARV_PSAPmut nucleocapsids. Right panels show the trajectories of nucleocapsids in rMARV_wt- or rMARV_PSAPmut-infected cells. Bars, 10 µm. (<b>D</b>) Length of nucleocapsid trajectories. The length of nucleocapsid trajectories was measured in rMARV_wt- or rMARV_PSAPmut-infected cells using the Leica LAS AF software, p-value (***, p≤0.0001). (<b>E</b>) Co-localization of Tsg101-Venus1/2 with MARV inclusions and nucleocapsids. Huh-7 cells were infected either with rMARV_wt or rMARV_PSAPmut and transfected with Venus1-Tsg101 and Venus2-Tsg101 plasmids. Cells were fixed at 22 h p.i. and subjected to immunofluorescence analysis with a NP-specific antibody. The Tsg101-Venus1/2 signal is displayed in green and the NP signal in red. Merged pictures show the overlay. The grey boxes indicate marginal region of cells, which are shown at higher magnification in the right panels. Arrows indicate nucleocapsids (approximately 1 µm in length).</p

rMARV_PSAPmut-infected cells contain more nucleocapsids in the cytoplasm and at early steps of budding than rMARV_wt-infected cells.

<p>Huh-7 and Vero E6 cells were infected with rMARV_PSAPmut- or rMARV_wt, fixed at 26 h p.i and embedded in epoxy resin. Cells were analyzed by thin section transmission electron microscopy (<b>A–D</b>) or electron tomography (<b>E–J</b>). (<b>A–B</b>) rMARV_wt-infected Huh-7 cell displaying free virions (black arrows) and a nucleocapsid in the cytoplasm near the plasma membrane (white arrow in A). (<b>C–D</b>) rMARV_PSAPmut-infected Huh-7 cells displaying fully protruded (grey arrows) or partially protruded (blue arrow in D) virus buds, and nucleocapsids bound to the plasma membrane (light blue arrows in C) or in the cytoplasm near the plasma membrane (white arrow in D). (<b>E</b>) 10 nm digital z-slice of an electron tomogram showing several nucleocapsids in the process of budding or in fully protruded virus buds in the periphery of rMARV_PSAPmut-infected Huh-7 cell. (<b>G</b>) 9 nm digital z-slice of an electron tomogram showing accumulated nucleocapsids in the cytoplasm of rMARV_PSAPmut-infected Huh-7 cell. (<b>F, H</b>) 3D surface representations of nucleocapsids (blue) and cytoplasm (yellow, semi-transparent) in the full tomograms for which z-slices are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004463#ppat-1004463-g007" target="_blank">Fig. 7E and 7G</a>, respectively. Bars, 500 nm. (<b>I–J</b>) Quantification of the nucleocapsid distribution in tomograms from 300 nm thick sections of rMARV_wt- or rMARV_PSAPmut-infected Huh-7 or Vero E6 cells. Intracellular nucleocapsids (including cytoplasmic and those bound to plasma membrane, or partially extruded nucleocapsids) and fully extruded nucleocapsids were counted in a set (5 or more) of representative tomograms (p-value, *P≤0.05).</p

Infection with MARV_PSAPmut results in compact inclusion bodies and accumulation of nucleocapsids in the periphery of cells.

<p>Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut, fixed at 24 h p.i. and subjected to immunofluorescence staining using NP-specific antibodies. Samples were then analyzed by confocal laser scanning microscopy. Left panels: rMARV_wt infection. Right panels: rMARV_PSAPmut infection. Grey boxes in the upper pictures indicate different regions of the same cell that are shown in higher magnification below. (<b>A</b>) and (<b>B</b>) periphery of cells. (<b>C</b>) and (<b>D</b>) inclusion bodies. Arrows indicate nucleocapsids.</p

IQGAP1 is co-localized with inclusions and nucleocapsids and supports MARV release.

<p>(<b>A</b>) Co-localization of Tsg101 and IQGAP1 in NP inclusions. Huh-7 cells were co-transfected with plasmids encoding NP_wt or NP_PSAPmut and VP40, mCherry-Tsg101 and IQGAP1-YFP. Cells were fixed 24 h p.tr., stained with NP-specific antibody and subjected to immunofluorescence analysis. White arrow shows co-localization of mCherry-Tsg101 (red), IQGAP1-YFP (green) with NP inclusions (blue) in the merge picture. In NP_PSAPmut transfected cells only mCherry-Tsg101 and IQGAP1-YFP are co-localized (arrowhead). (<b>B</b>) Co-localization of Tsg101 and IQGAP1 in MARV infected cell. Huh-7 cells were infected with rMARV_wt or rMARV_PSAPmut and co-transfected with mCherry-Tsg101 and IQGAP1-YFP encoding plasmids. Cells were fixed 24 h p.i. stained with NP-specific antibody and analyzed by CLSM. Lower panels show higher magnification of boxed area and white arrow indicates co-localization of nucleocapsid with mCherry-Tsg101 and IQGAP1-YFP in wild type infected cells whereas mutant nucleocapsids did not show any co-localization. (<b>C</b>) IQGAP1 depletion of infected cells. MARV-infected Huh-7 cells (MOI of 1) were transfected with IQGAP1-specific siRNA or control siRNA at 1 h p.i. Cells and culture supernatants were harvested at 48 h and 72 h p.i. Lysates and supernatants collected at 72 h p.i. were subjected to Western Blot analysis. (<b>D</b>) Virus titers in the supernatants of MARV infected cells transfected with IQGAP1-specific or control siRNA were determined by TCID₅₀ titration, p-value (_*, P≤0.05). (<b>E</b>) Phenotype of IQGAP1-knockdown in MARV-infected cells at 48 h p.i. Huh-7 cells grown on cover slips were infected with MARV_wt and treated with IQGAP1 specific or control siRNA and subjected to immunofluorescence analysis using NP-specific antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrow indicates accumulation of nucleocapsids upon IQGAP1 knockdown at cell periphery marked with dashed line. Bars, 10 µm.</p