Kinetic analysis of mouse brain proteome alterations following chikungunya virus infection before and after appearance of clinical symptoms

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV in

Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

Background:The recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical sympt

Assessment of <it>Anopheles</it> salivary antigens as individual exposure biomarkers to species-specific malaria vector bites

Abstract Background Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites. Methods For this purpose, salivary gland proteins 6 (SG6) and 5′nucleotidases (5′nuc) from An. gambiae (gSG6 and g-5′nuc) and An. funestus (fSG6 and f-5′nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages (NDiop, n = 50; Dielmo, n = 38; and Diama, n = 46) that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls (Marseille, n = 45). Results The IgG responses against SG6 recombinant proteins from these two Anopheles species and against g-5′nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5′nucleotidase protein. Conclusion This study revealed an Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed.</p

Western blot validations of differentially regulated proteins identified by 2D-DIGE and/or iTRAQ analyses.

<p>(A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock (M), early (E), late paralytic (LP) and late tetanus-like (LT), separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye; green) and the specific immunoreactive proteins (FITC or Cy5 dye; red). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean ± S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons. The significance of the differential protein expression are indicated *, p<0.05; **, p<0.01; ***, p<0.001. A.U., arbitrary units. ANXA2: annexin A2; ARRB1: β-arrestin; GABRA1: γ-aminobutyric acid receptor subunit alpha-1; GRASP1: GRIP-associated protein; ITGAV: integrin αV; MYPT1: myosin phosphatase target subunit 1; N-Ras: N-Ras; RABEP1: rabaptin-5; SYNGR3: synaptogyrin-3.</p

Proteins identified from the 2D-DIGE (pH 3–10) analysis of mouse brain lysates collected at early (E), late paralytic (LP) or late tetanus-like (LT) time-points after CHIKV infection.

<p>The proteins were identified by mass spectrometry following in-gel trypsin digestion. The spot numbers correspond to the same numbers as indicated on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091397#pone-0091397-g002" target="_blank">Figure 2</a>. The identities of the spots, their SwissProt accession numbers, and the theoretical molecular masses and <i>pI</i> values as well as the number of peptide sequences, the corresponding percent sequence coverage, and the Mascot score are listed for MS/MS analysis. Protein scores greater than 35 were considered as significant (<i>p< 0.05</i>). Paired average volume ratio and <i>p</i>-values (ANOVA) between each paired groups compared, were defined using Progenesis Samespot software. n.i., no identification.</p><p>M; mock-infected samples.</p

Top-2 most significant protein networks of differentially regulated proteins following CHIKV infection.

<p>Ingenuity Pathway Analysis of the total 177 proteins identified as differentially expressed generated 2 emerging networks with high score and including more than 20 molecules with direct relationships. Network 1 (A,B,C) was associated with Cell Morphology, Tissue Morphology, Infectious disease and Network 2 (D,E,F) with Cell-To-Cell Signaling and Interaction, Cellular Assembly and Organization, Cellular Compromise. Each network was overlaid with the protein expression fold-change determined in each separate comparison (early (E) <i>vs</i> mock (M) (A, D), late paralytic (LP) <i>vs</i> E (B, E) or late tetanus-like (LT) <i>vs</i> E (C, F)), to highlight the proteins modified during the time-course of infection. Individual proteins are represented as nodes colored in red and green corresponding to up- and down-regulated proteins, respectively, while the nodes (proteins) in white have been added by IPA to maximize the network connectivity. The different shapes of the nodes represent functional classification of the proteins as indicated in the legend.</p

2D-DIGE analysis (pH 3–10) of mock (M)-, early (E), late paralytic (LP) and late tetanus-like (LT) CHIKV-infected brain samples.

<p>(A) Representative data from a 2D-DIGE experiment using a 10% SDS-polyacrylamide gel with the pH 3–10 range is shown. Proteins from M-, E- and LP- and LT- CHIKV-infected brain samples were labeled with Cy5 or Cy3 cyanine dyes, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091397#pone.0091397.s002" target="_blank">Table S1</a>. As determined by Progenesis SameSpot software, protein spots that were differentially regulated between the four experimental groups (|FC| ≥1.3 and <i>p</i> ≤0.05), were submitted to mass spectrometry for identification. The numbers annotated on the gel corresponded to master gel numbers of deregulated protein spots. Spots were all identified as <i>Mus musculus</i> proteins and were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091397#pone-0091397-t001" target="_blank">Table 1</a>. Spots differentially modified between E and mock- (B), LP- and E- (C), LT and E- (D), LP and M- (E) and LT and M- (F) infected samples are represented by red (up-regulated) or blue (down-regulated) dots.</p