Diagram of the clinical study.

<p>“unable”: patients who were reached but unable to answer to the questionnaire, “lost”: patient lost of follow up until the end of the study, “temporally lost”: not reached at a specific timepoint, “death”: dead patients.</p

Distinctive markers in serum of patients with arthralgia (ART+ in black) versus patients without arthralgia (ART−, in white) at M36.

<p>SD: standard deviation, results of Mann Whitney test are shown. * p<0.05, q = 0.056.</p

Identification of risk factors for persistence of arthralgia 14 months after CHIKV acute disease (univariate analysis).

§<p>confidence interval.</p

Evolution and characterization of arthralgia in CHIKV patients.

<p>(A) Percentage of patients with arthralgia among participating patients, (B) localization of arthralgia among patients with arthralgia, (C) number of arthralgia sites among the 76 patients answering at every time point and having arthralgia, (D) complete model presenting the probability for one joint to be painful depending on its previous state.</p

Features of patients with chronic CHIKV-associated arthralgia.

<p>Features of patients with chronic CHIKV-associated arthralgia.</p

Clinical signs and treatment in arthralgic and non-arthralgic patients 36 months after the acute phase.

§<p>ns: Non significant.</p>**<p>p<0.01.</p>***<p>p<0.001.</p

CHIKV Productively Infects Human Primary Macrophages

<div><p>(A–C) Human monocyte–derived macrophages were exposed to CHIKV for 4 h and extensively washed, and CHIKV replication was analyzed by different methods. Data are representative of at least four independent experiments, with cells from eight different donors.</p> <p>(A) CHIKV-infected macrophages. Cells were infected with CHIKV at an moi of 10. At the indicated time points, cells were stained with anti-CHIKV antibodies and analyzed by confocal microscopy. Two magnifications are depicted (objectives ×25 and ×40). NI, noninfected cells.</p> <p>(B) Release of infectious virus in supernatants<i>.</i> Macrophages were infected at various mois as stated. At the indicated time points, levels of infectious virions in supernatants were measured by limiting dilution on Vero cells. Results are expressed as TCID50/ml. Macrophages from three representative donors are depicted.</p> <p>(C) Viral RNA in supernatants<i>.</i> Levels of viral RNA in supernatants from the same experiment depicted in (B) were measured by real-time PCR.</p></div

Cell Tropism of CHIKV

<div><p>(A–C) The indicated human cell lines or primary cells were exposed to CHIKV at an moi of 10. At 24 h pi, cells were fixed, stained with anti-CHIKV antibodies, and analyzed by flow cytometry. The percentage of CHIKV-infected cells is indicated. Data are representative of at least three independent experiments. For primary cells, at least three different donors were analyzed.</p> <p>(A) Examples of sensitive and refractory cell types.</p> <p>(B) Sensitivity of adherent cells to CHIKV infection.</p> <p>(C) Sensitivity of primary blood-derived cells to CHIKV infection. The indicated cell lines, as well as nonactivated PBMCs, activated CD4+ lymphocytes, monocytes, and monocyte-derived DCs were analyzed.</p></div

CHIKV Is a Cytopathic Virus

<div><p>(A) CHIKV-infected dying cells. At 24 h after CHIKV infection, HeLa cells were fixed, stained with anti-CHIKV antibodies, and analyzed by confocal microscopy.</p> <p>(B) Viability of cell cultures. HeLa cells were infected with CHIKV at the indicated moi. After 24 h pi, cell viability was measured in a colorimetric assay (MTT cell viability test). Data are mean ±SD of triplicates and representative of five independent experiments;</p> <p>(C) Apoptosis of CHIKV-infected cells. At 24 h pi (moi 10), HeLa cells were doubly stained with anti-active caspase-3 and anti-capsid antibodies and analyzed by flow cytometry. The percentage of caspase-3 positive and negative cells among CHIKV positive cells is depicted. Similar results were obtained with another marker of apoptosis (TUNEL, not shown). Data are representative of three independent experiments.</p> <p>NI, noninfected cells.</p></div

Binding of CHIKV to Target Cells

<p>The indicated human cell lines or primary cells were exposed to CHIKV at the indicated moi for 1 h at 4 °C. Cells were fixed, stained with anti-CHIKV antibodies, and analyzed by flow cytometry. Data are representative of three independent experiments. NI, noninfected cells.</p