Interactions of different-length NS1 protein with PABII.

<p>293T cells were co-transfected with the MYC-tagged vector pMYC-PABII and the FLAG-tagged vector pCMV-tag2B (empty vector), pCMV-Mex NS1/219 or pCMV-Mex NS1/230. At 48 h post-transfection, cells were lysed, supplemented with a protease inhibitor cocktail and subjected to anti-FLAG affinity chromatography. After extensive washing, the precipitated proteins were dissociated from the resin using elution buffer and analyzed by SDS-PAGE followed by Western bloting with anti-FLAG and anti-MYC antibodies. The proteins were visualized using a SuperSignal West Pico and detected with a MF-ChemiBIS system (DNR). PD: Pull Down.</p

A validation of microarray data by qRT-PCR.

<p>A validation of microarray data by qRT-PCR.</p

Inhibition of host gene expression by different-length NS1 protein.

<p>(A) A549 cells were co-transfected with Renilla luciferase plasmid pRL-TK and expression plasmid pcDNA-GST, pcDNA-Mex NS1/219 or pcDNA-Mex NS1/230. Renilla luciferase expression was measured at 24 h post-transfection. The results showed the means and standard deviations of triplicate values (normalized to GST). The statistical analysis was performed using Student's t test (★★, <i>p</i><0.01; ★, <i>p</i><0.05). (B) The 219-aa NS1 and 230-aa NS1 protein expression at 24 h post-transfection in transfected A549 cells was detected by Western bloting using rabbit anti-serum against a fusion protein of GST and Mex NS1/219, tubulin was used as control. (C) A549 cells were infected at an MOI of 3 TCID₅₀ per cell. At 6, 12 and 24 h p.i., host antiviral gene IFN-β mRNA and viral M gene mRNA production was deteced by qRT-PCR. The statistical analysis was performed using Student's t test (★★, <i>p</i><0.01; ★, <i>p</i><0.05).</p

In vitro characterization of the recombinant viruses encoding different-length NS1 protein.

<p>(A) A549 or PK-15 cells were infected at an MOI of 0.001 TCID₅₀ per cell. After washed with PBS, fresh medium containing 0.1 µg/ml TPCK-trypsin was added. Supernatants were sampled at 12, 24, 36 and 48 h p.i. and virus titers were determined as log₁₀TCID₅₀/ml in MDCK cells. The statistical analysis was performed using Student's t test. (B) Virus plaque assay was performed by inoculating confluent MDCK cells in 6-well plates with appropriate dilutions of viruses. After washed with PBS, cells were overlaid with DMEM (without phenol red)-0.8% agarose mixture containing 1% bovine serum albumin and 1 µg/ml TPCK-trypsin. After incubated at 37°C for 4 days, a second agar overlay containing 1∶10,000 neutral red was added.</p

Pathogenicity and growth property of the recombinant viruses encoding different-length NS1 protein in mice.

<p>Groups of mice were inoculated i.n. with 50 µl of 10² TCID₅₀ of the recombinant virus rPR8-Mex NS1/219 or rPR8-Mex NS1/230. (A) Body weight of five mice in each group was measured for either 14 days p.i. or until the loss of 30% of their body weight. The statistical analysis was performed using Student's t test (★★, <i>p</i><0.01; ★, <i>p</i><0.05). (B) The survival percentage of four mice in each group was monitored for 14 days p.i.. (C) At days 3 and 6 p.i., three mice in each group were sacrificed, lungs were removed and homogenized in 1 ml PBS to determine virus titers by log₁₀TCID₅₀/ml in MDCK cells. (D) At days 3 and 6 p.i., three mice in each group were sacrificed and lungs were fixed with formalin, embedded in paraffin and stained with hematoxylin and eosin. Images were captured at 40X magnification.</p

Intracellular localization of different-length NS1 protein in transfected or infected cells.

<p>(A) A549 cells were transfected with the plasmid pEGFP-Mex NS1/219 or pEGFP-Mex NS1/230. At 24 h post-transfection, cells were imaged using an inverted fluorescence microscope. (B) A549 cells were infected with recombinant virus rPR8-Mex NS1/219 or rPR8-Mex NS1/230 at an MOI of 3 TCID₅₀ per cell. At 8 h p.i., cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and incubated for one hour with 2% bovine serum albumin. An NS1-specific rabbit anti-serum incubation was performed overnight at 4°C followed by FITC-labelled goat anti-rabbit IgG and DAPI. Cells were imaged on an LSM510 Meta confocal laser scanning microscope.</p

Rapid deployment of a mobile biosafety level-3 laboratory in Sierra Leone during the 2014 Ebola virus epidemic

<div><p>Background</p><p>Ebola virus emerged in West Africa in December 2013. The high population mobility and poor public health infrastructure in this region led to the development of the largest Ebola virus disease (EVD) outbreak to date.</p><p>Methodology/Principal findings</p><p>On September 26, 2014, China dispatched a Mobile Biosafety Level-3 Laboratory (MBSL-3 Lab) and a well-trained diagnostic team to Sierra Leone to assist in EVD diagnosis using quantitative real-time PCR, which allowed the diagnosis of suspected EVD cases in less than 4 hours from the time of sample receiving. This laboratory was composed of three container vehicles equipped with advanced ventilation system, communication system, electricity and gas supply system. We strictly applied multiple safety precautions to reduce exposure risks. Personnel, materials, water and air flow management were the key elements of the biosafety measures in the MBSL-3 Lab. Air samples were regularly collected from the MBSL-3 Lab, but no evidence of Ebola virus infectious aerosols was detected. Potentially contaminated objects were also tested by collecting swabs. On one occasion, a pipette tested positive for EVD. A total of 1,635 suspected EVD cases (824 positive [50.4%]) were tested from September 28 to November 11, 2014, and no member of the diagnostic team was infected with Ebola virus or other pathogens, including Lassa fever. The specimens tested included blood (69.2%) and oral swabs (30.8%) with positivity rates of 54.2% and 41.9%, respectively. The China mobile laboratory was thus instrumental in the EVD outbreak response by providing timely and reliable diagnostics.</p><p>Conclusions/Significance</p><p>The MBSL-3 Lab significantly contributed to establishing a suitable laboratory response capacity during the emergence of EVD in Sierra Leone.</p></div

Schematic diagram of the “Four Flows”.

<p>Personnel, materials, water and air flow management were the key elements of biosafety measures in the MBSL-3 Lab.</p

Diagnostic algorithm for laboratory testing.

<p>Diagnostic algorithm for laboratory testing.</p

Layout of the mobile biosafety level-3 laboratory.

<p>The main and auxiliary containers were connected by an airtight soft connection and together formed a complete biosafety level-3 (BSL-3) lab. The instruments represented by letters were listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005622#pntd.0005622.s003" target="_blank">S1 Table</a>.</p