Phylogenesys and homology modeling in Zika virus epidemic: food for thought

<p>Zika virus (ZIKV) is an emerging Flavivirus that have recently caused an outbreak in Brazil and rapid spread in several countries. In this study, the consequences of ZIKV evolution on protein recognition by the host immune system have been analyzed. Evolutionary analysis was combined with homology modeling and T-B cells epitope predictions. Two separate clades, the African one with the Uganda sequence, as the most probable ancestor, and the second one containing all the most recent sequences from the equatorial belt were identified. Brazilian strains clustered all together and closely related to the French Polynesia isolates. A strong presence of a negatively selected site in the envelope gene (<i>Env</i>) protein was evidenced, suggesting a probable purging of deleterious polymorphisms in functionally important genes. Our results show relative conservancy of ZIKV sequences when envelope and other non-structural proteins (NS3 and NS5) are analyzed by homology modeling. However, some regions within the consensus sequence of NS5 protein and to a lesser extent in the envelope protein, show localized high mutation frequency corresponding to a considerable alteration in protein stability. In terms of viral immune escape, envelope protein is under a higher selective pressure than NS5 and NS3 proteins for HLA class I and II molecules. Moreover, envelope mutations that are not strictly related to T-cell immune responses are mostly located on the surface of the protein in putative B-cell epitopes, suggesting an important contribution of B cells in the immune response as well.</p

Principal Component Analysis of healthy controls and IPF patients.

<p>The PCA plot was computed using the differentially expressed gene list (FDR<0.05, log2FC>0.6 filter).</p

Bronchoalveolar lavage (BAL) cells in idiopathic pulmonary fibrosis express a complex pro-inflammatory, pro-repair, angiogenic activation pattern, likely associated with macrophage iron accumulation

<div><p>Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown cause characterized by alveolar epithelial damage, patchy interstitial fibrosis and diffuse microvascular abnormalities. In IPF, alveolar clustering of iron-laden alveolar macrophages—a common sign of microhemorrhage, has been associated with vascular abnormalities and worsening of pulmonary hypertension. As iron-dependent ROS generation has been shown to induce unrestrained macrophage activation in disease models of vascular damage, we explored alveolar macrophage activation phenotype in IPF patients (n = 16) and healthy controls (CTR, n = 7) by RNA sequencing of bronchoalveolar lavage (BAL) cells. The frequencies of macrophages in BAL cells were 86+4% and 83.4+8% in IPF and CTR groups, respectively (p-value = 0.41). In IPF patients, BAL cells showed increased iron-dependent ROS generation (p-value<0.05 vs CTR). Gene expression analysis showed overrepresentation of Gene Ontology processes/functions and KEGG pathways enriched in upregulated M1-type inflammatory (p-value<0.01), M2-type anti-inflammatory/tissue remodeling (p-value<0.0001), and MTPP-type chronic inflammatory/angiogenic (p-value<0.0001) chemokine and cytokine genes. The <i>ex vivo</i> finding was confirmed by the induction of iron-dependent ROS generation and chemokine/cytokine overexpression of Ccl4, Cxcl10 (M1), Il1rn (M2), Cxcl2, and Cxcl7 (MTPP) in MH-S murine immortalized alveolar macrophages exposed to ferric ammonium citrate in culture (p-value<0.05 vs CTR). The data show alveolar macrophage expression of a pro-inflammatory, tissue remodeling and angiogenic complex activation pattern, suggesting that iron accumulation may play a role in macrophage activation.</p></div

RNA-seq differential gene expression validation by qRT-PCR.

<p>Relative expression of selected chemokines in IPF (N = 7, closed circles) vs. Healthy control (N = 4, open circles) BAL cells represented by normalized ΔCt value. ΔCt value was normalized to the GNB2L1 housekeeping gene expression by subtracting GNB2L1 raw Ct from the selected gene raw Ct and by further adding the absolute GNB2L1 minimum value (the same for all samples) to the gene ΔCt. Lower cycle values correspond to higher expression level. Mann-Whitney test (*) and (**) denote statistical significance (P < 0.05, P < 0.01, respectively) vs. control.</p

Intracellular iron and ROS levels in BAL cells.

<p>Alveolar macrophage intracellular iron was assessed using the Prussian Blue Iron Staining and the Golde score was calculated based on the staining intensity 14 IPF patients and 7 controls. Reactive oxygen species (ROS) were measured in 11 IPF patients and 7 controls, using CM-H2DCFDA fluorimetry. Shown are Golde scores and chelatable iron-dependent ROS levels per 10⁵ BAL cells in healthy controls and IPF affected individuals.</p

Quantification of BAL fluid chemokine levels by protein immunochemistry.

<p>Chemokines were measured in un-concentrated BAL fluid by protein immunochemistry on IPF affected individuals (closed circles) and Healthy Controls (open circles). CXCL-5 was quantified using the Chemokine Human 5-Plex Panel II for the Luminex 100/200 platform. PPBP/CXCL-7, CXCL-10, CCL13 and CCL18 were measured using enzyme-linked immunosorbent assay (ELISA) kits. Mann-Whitney test (*) and (**) denote statistical significance (P < 0.05, P < 0.01, respectively) vs. control.</p

Gene Ontology (GO) term and KEGG pathway analysis.

<p>Gene Ontology (GO) term and KEGG pathway analysis.</p

Study population characteristics of IPF patients and controls undergoing fiberoptic bronchoscopy with BAL.

<p>Study population characteristics of IPF patients and controls undergoing fiberoptic bronchoscopy with BAL.</p

Study flow diagram.

<p>Abbreviations: TB: tuberculosis; RD: Region of Difference; Indeterm: indeterminate; TST: tuberculin skin test; QF: QuantiFERON-TB GOLD In-Tube.</p

Estimates of sensitivities for the combination of the diagnostic tests studied.

<p><b>Abbreviations:</b></p><p>RD: region of difference; TST: tuberculin skin test; CI: confidence interval.</p