Physical and psychological consequences of serious road traffic injuries, deliverable 7.2 of the H2020 project SafetyCube

SafetyCube aims to develop an innovative road safety Decision Support System (DSS) that will enable policy-makers and stakeholders to select the most appropriate strategies, measures and cost-effective approaches to reduce casualties of all road user types and all severities. Work Package 7 of SafetyCube is dedicated to serious road traffic injuries, their health impacts and their costs. This Deliverable discusses health impacts of (serious) road traffic injuries

Comparative analysis at days 3, 5 and 7 of cells replicating the wt-JFH-1, FL-J6/JFH and C10M-JFH-1 RNAs.

<p><b>(A)</b> Intracellular replication and virus production. Left panel: Replication of HCV RNAs in transfected Huh7.5 cells, as determined by RT-qPCR. Values are expressed relative to wt-JFH-1 at day 3 (D3), for which the value is fixed at 1. Right panel: Virus production in transfected Huh7.5 cells, as determined by intracellular core protein determinations with the Abbot-Architect HCV Ag. Values are normalized for intracellular core levels and RNA detection with the number of infected cells, to exclude variations caused by reinfection. <b>(B)</b> Virus release. Left panel: HCV RNA in the supernatants of transfected cells, as determined with the Abbott Realtime HCV assay. Right panel: Core protein quantification in the supernatant of transfected cells, with the Abbot-Architect HCV Ag. <b>(C)</b> Determination of the extracellular (left panel) and intracellular (right panel) infectivity of the viruses produced by transfected cells. For extracellular infectivity assays, naïve Huh7.5 cells were inoculated with supernatants obtained from cells harvested at 3, 5 and 7 days post-transfection. Intracellular infectivity was measured by assessing the infection levels of cleared cell lysates obtained following the hypotonic shock of transfected cells, on days 3, 5, and 7. The numbers of FFUs were determined after 3 days of cell culture. The mean values ± SD of three independent experiments are shown. <b>(D)</b> Analysis of the intrinsic infectivity of infectious particles produced by cells replicating the wt-JFH-1, FL-J6/JFH and C10M-JFH-1 RNAs. Left panel: Calculated intrinsic infectivity deduced from the [Core released from transfected cells] / [Extracellular infectivity] ratio from panels <b>B</b> and <b>C</b>. Right panel: After standardized infections of Huh7.5 cells with supernatants normalized on the basis of HCV RNA titration (Abbott Realtime HCV assay), intracellular HCV RNAs were quantified with the Abbott Realtime HCV assay. Mean values for one experiment performed in triplicate and means ± SD are shown. Calculated p values (Student’s t test) in <b>B</b> and <b>C</b> were presented as follows: **, p value 0.01; ***, p value 0.001.</p

Subcellular localization of HCV viral proteins and lipid droplets by confocal microscopy.

<p>Huh7.5 cells were transfected with <i>in vitro</i>-transcribed HCV RNAs and were grown on coverslips. Cells were fixed 5 days post-transfection and analyzed for the immunofluorescence of <b>(A)</b> core protein and LDs; <b>(B)</b> core and NS5A proteins; <b>(C)</b> NS5A and LDs and <b>(D)</b> core and envelope E2 proteins. Immunofluorescence analyses were performed with the C7.50 anti-core monoclonal antibody, the AR3A anti-E2 monoclonal antibody, and anti-NS5A polyclonal antibodies. LDs were stained with Nile red, as described in the materials and methods. The value ± SD right to the image series indicates the extent of colocalization from confocal images stacks of 5 independent cells, measured as the Pearson coefficient in the cell volume dataset.</p

Analysis of the 1066 HCV core protein sequences extracted from the HCV Los Alamos database.

<p>Each line represents the core protein sequence of a strain (highlighted in gray) or a core protein consensus sequence (no highlighting), with a representation of amino-acid polymorphism at the 10 positions identified. Core protein domains and amino-acids positions are shown at the top. The real residue frequency (<b>Freq.</b>) or the frequency calculated as a percentage (<b>Freq (%)</b>) of all the sequences analyzed (first right column) is shown below each amino-acid. *This line corresponds to a consensus sequence from 130 core protein sequences, including 13 genotype 3 sequences, 41 genotype 4 sequences, 5 genotypes 5 sequences, 70 genotype 6 sequences and 1 genotype 7 sequence.</p

Electron micrographs of HCV core mutants expressed using SFV vectors and analysis of their ability to self-assemble into HCV-like particles.

<p>The production of all core proteins induced circonvoluted ER membranes, while no ultra-structural change of ER was identified in cells expressing the β-galactosidase, as previously described. The budding of HCV-LPs at these convoluted ER membrane was observed with the Dj core protein, while the oligomerization of the J6 core protein led to the formation of tubular structures. HCV-LPs or tubular structures were never observed with the JFH-1 core protein, as for all the core single, dual or triple mutants. Bars: 0,5 μm. LD: lipid droplet.</p

Alignment of amino acids 119–191 of the core protein consensus sequences from 5 patients with little or no steatosis (in green) and 6 patients with severe steatosis (in red).

<p>The 5 patients in with little or no steatosis had a LD area <1000 µm² per 50,000 µm² of liver tissue. The 6 patients with severe steatosis had a LD area >10,000 µm² per 50,000 µm² of tissue. Patients infected with genotype 3 strains are indicated with an asterisk (*) and such patients are present in both groups. The residues at each position are indicated with the single-letter amino-acid code. Amino acids identical to the first sequence are indicated by a dot. The positions of the residues shown in previous studies to increase LD accumulation <i>in vitro</i>, in cultured cells, are indicated in red. No association was found between a particular amino-acid residue or motif and the presence of severe steatosis.</p

TEM observation of representative liver biopsy specimens from three patients chronically infected with HCV.

<p>The liver in A (patient P16) shows a small number of small LD (arrows) and was found to have a lipid droplet area <1000 µm² per 50,000 µm² of tissue. The liver in B (patient P20) has extremely large LD and was found to have a LD area >10,000 µm² per 50,000 µm² of tissue. The TEM photograph in C shows a high magnification of an individual hepatocyte with several large LD, taken from a liver biopsy specimen (patient P03) considered intermediate to the other two in terms of steatosis severity. Scale bars: 20 µm in A and B; 2 µm in C.</p

Demographic, biochemical and histological characteristics of the patients at the time of liver biopsy.

a<p>Determined with the Versant HCV Genotype Assay.</p>b<p>Data missing for 4 patients.</p>c<p>Data missing for 2 patients.</p>d<p>Data missing for 1 patient.</p

Conservation of amino acids 161–181 of the NS5A protein sequences of all variants identified in the 27 patients included in the study.

<p>The sequences were classified into three different groups : (i) patients with little or no steatosis, with a LD area <1000 µm² per 50,000 µm² of liver tissue (n = 5) ; (ii) patients with severe steatosis, with a LD area >10,000 µm² per 50,000 µm² of tissue (n = 6) ; (iii) patients with a steatosis of intermediate severity (n = 16). Using CLC Main Workbench 6 software, we determined, for each group, the most frequent residue in each position and the percentage conservation of these residues. The D in position 362 in the core-NS5A fused sequence (arrow, residue 171 in the NS5A protein) was highly conserved in all genotype 3 viruses from all groups. For the other genotypes, the D on position 362/171 was characteristic of the variants found in patients with little or no steatosis, whereas an E in the same position was characteristic of the variants found in patients with severe steatosis.</p

Comparative analysis of the cumulative LD areas determined by TEM on liver biopsy specimens from patients infected with genotype 3 viruses and viruses of other genotypes, in µm².

<p>Cumulative LD areas were summed for all patients with genotype 3 infection (left boxplot) and for patients infected with viruses of other genotypes (right boxplot). Boxplots show the median LD area (horizontal black line), the 25^th and the 75^th percentile. Dot plots indicate the extreme values of cumulative LD area. There was no significant difference between the two groups (5461.1[2165.2; 10027.9] for the genotype 3 group versus 3538.7[2184.6; 4984.4] for the non-3 group, p = 0.581).</p