Comparative analysis of infection and apoptosis in response to CHIKV infection.

<p>Summary of percentage infection (A) and apoptosis induction (B) in cell lines infected with CHIKV ECSA E1:226 V (226 V), CHIKV ECSA E1: A226 (A226), Ross strain (ROSS) or mock infected (Mock).</p

Chikungunya Virus Infection of Cell Lines: Analysis of the East, Central and South African Lineage

<div><p>Chikungunya virus (CHIKV) is a re-emerging mosquito borne alphavirus that has caused large scale epidemics in the countries around the Indian Ocean, as well as leading to autochthonous transmission in some European countries. The transmission of the disease has been driven by the emergence of an African lineage of CHIKV with enhanced transmission and dissemination in <em>Aedes</em> mosquito hosts. Two main genotypes of this lineage have been circulating, characterized by the presence of a substitution of a valine for an alanine at position 226 of the E1 protein. The outbreak, numbering in millions of cases in the infected areas, has been associated with increasing numbers of cases with non-classical presentation including encephalitis and meningitis. This study sought to compare the original Ross strain with two isolates from the recent outbreak of chikungunya fever in respect of infectivity and the induction of apoptosis in eight mammalian cell lines and two insect cell lines, in addition to generating a comprehensive virus production profile for one of the newer isolates. Results showed that in mammalian cells there were few differences in either tropism or pathogenicity as assessed by induction of apoptosis with the exception of Hela cells were the recent valine isolate showed less infectivity. The Aedes albopictus C6/36 cell line was however significantly more permissive for both of the more recent isolates than the Ross strain. The results suggest that the increased infectivity seen in insect cells derives from an evolution of the CHIKV genome not solely associated with the E1:226 substitution.</p> </div

Summary of susceptibility to infection and induction of apoptosis for three CHIKV isolates.

<p>0%≤⁃≤20%; 21%≤▴≤40%; 41%≤▴▴≤60%; 61%≤▴▴▴≤80%; 81%≤▴▴▴▴≤100%; <b>ND</b>: Not determined.</p

CHIKV infection of CHME-5 human microglial cells.

<p>A. CHME-5 cells were mock infected (M) or infected with CHIKV ECSA E1: 226 V (EV) at m.o.i. of 1. Infected cells and culture medium were collected daily and the medium assayed for levels of infectious CHIKV at the times indicated by standard plaque assay on Vero cells, while cells were assayed for infectivity and induction of apoptosis by flow cytometry after staining with an anti-alphavirus monoclonal antibody and FITC-conjugated Annexin V/propidium iodide respectively. B. CHME-5 cells were mock infected (M) or infected with ECSA CHIKV E1: A226 (EA), ECSA CHIKV E1: 226 V (EV) or Ross strain (RO) at m.o.i. of 1 and assayed for infectivity and induction of apoptosis by flow cytometry on the days indicated. All experiments were undertaken independently in triplicate with duplicate analysis of virus titers. Error bars show S.D. C to F. CHME-5 cells were mock infected (C) or infected with ECSA CHIKV E1: 226 V (D), ECSA CHIKV E1: A226 (E) or Ross strain (F) and on day 2 p.i. stained with a mouse anti-alphavirus monoclonal antibody followed by a FITC conjugated goat anti-mouse IgG polyclonal antibody (green). Nuclei of cells were stained with TO-PRO-3 iodide (red). Non-contrast adjusted merged images are shown.</p

Infection and apoptosis in response to CHIKV E1:226 V infection.

<p>Summary of percentage infection (A) and apoptosis induction (B) in cell lines infected with CHIKV ECSA E1: 226 V (226 V) or mock infected (Mock).</p

Values of fold change in response to CHIKV infection, significance and half life of selected proteins.

<p>Values of fold change in response to CHIKV infection, significance and half life of selected proteins.</p

Infection and apoptosis in CHIKV infected CHME-5 cells.

<p>(A and B) CHME-5 cells either mock infected or infected with CHIKV at MOI 2.5 or 5 were collected at day 2 p.i. and (A) cells were stained with an anti-alphavirus antibody and the percentage of infected cells was determined by flow cytometry or (B) cells were stained with Annexin V-FITC and PI and the percentage of induced apoptosis determined by flow cytometery. Bar graphs represent the means ± SD of 3 replications per group. (C and D) CHME-5 cells either mock infected or infected with CHIKV at MOI 2.5 or 5 were collected on days 2 and 4 p.i. and analyzed by flow cytometry after double staining with antibodies directed against active caspase 3 and alphavirus. Experiment was undertaken in three independent replicates. Representative flow cytometry dot plot is shown in (C) and data is shown graphically in (D). Bar graphs represent the means ± SD of 3 replications per group.</p

Validation of proteins differentially expressed in response to CHIKV infection in CHME-5 cells.

<p>(A) CHME-5 cells were either mock infected or infected with CHIKV at MOI 0.1 before extraction of proteins and analysis by Western blot analysis on 1 and 2 d.p.i. hnRNP: heterogeneous nuclear ribonucleoprotein; NCL: nucleolin; JAK2: tyrosine-protein kinase JAK2; Hsp70: heat shock protein 70; Hsp90: heat shock protein 90. (B). CHME-5 cells were either mock infected or infected with CHIKV at MOI 0.1 before extraction of total RNA and analysis by RT-PCR on 1, 2 and 3 d.p.i. BRE1B: E3 ubiquitin-protein ligase; CUL9: Cullin-9; CHD2: chromodomain-helicase-DNA binding protein 2; MTERF: mitochondrial precursor transcription termination factor; ROD1: regulator of differentiation 1 isoform; PIK3CD: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta; GCDH: mitochondrial glutaryl-CoA dehydrogenase isoform precursor; HSDL2: hydroxysteroid dehydrogenase-like protein 2; PLCH2: 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase eta-2; ALOX12: 12-lipoxygenase; DRPLA: Dentatorubral pallidoluysian atrophy protein; DENND3: DENN domain-containing protein 3; HIS1H2B: Histone 2B.</p

Immunogenic Properties of a BCG Adjuvanted Chitosan Nanoparticle-Based Dengue Vaccine in Human Dendritic Cells

<div><p>Dengue viruses (DENVs) are among the most rapidly and efficiently spreading arboviruses. WHO recently estimated that about half of the world’s population is now at risk for DENV infection. There is no specific treatment or vaccine available to treat or prevent DENV infections. Here, we report the development of a novel dengue nanovaccine (DNV) composed of UV-inactivated DENV-2 (UVI-DENV) and <i>Mycobacterium bovis</i> Bacillus Calmette-Guerin cell wall components (BCG-CWCs) loaded into chitosan nanoparticles (CS-NPs). CS-NPs were prepared by an emulsion polymerization method prior to loading of the BCG-CWCs and UVI-DENV components. Using a scanning electron microscope and a zetasizer, DNV was determined to be of spherical shape with a diameter of 372.0 ± 11.2 nm in average and cationic surface properties. The loading efficacies of BCG-CWCs and UVI-DENV into the CS-NPs and BCG-CS-NPs were up to 97.2 and 98.4%, respectively. THP-1 cellular uptake of UVI-DENV present in the DNV was higher than soluble UVI-DENV alone. DNV stimulation of immature dendritic cells (iDCs) resulted in a significantly higher expression of DCs maturation markers (CD80, CD86 and HLA-DR) and induction of various cytokine and chemokine productions than in UVI-DENV-treated iDCs, suggesting a potential use of BCG- CS-NPs as adjuvant and delivery system for dengue vaccines.</p></div

Sub-cellular distribution and functional annotation of proteins significantly differentially expressed in response to CHIKV infection of CHME-5 cells.

<p>The sub-cellular (A) and functional (B) categorization of the proteins was performed using the GoCat software.</p