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. 2019 May 30;9:8069. doi: 10.1038/s41598-019-44559-8

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Travel Distance of Endocytotic ZIKV-Laden Vesicles in Astrocytes and Neurons Increases during Infection, with the Exception of ZIKV-FP in Astrocytes. (a) Fluorescent micrographs showing internalized fluorescently labeled ZIKV-BR into astrocytes and neurons (ZIKA (DiD)) and immunolabeled with serum from a patient infected with ZIKV (infected in Brazil in 2016). Overlay panels show remarkable co-localization between vesicular structures with fluorescently labeled ZIKV and anti-ZIKV antibodies from the patient’s serum. Images were recorded at 36 hpi. (b) An image of a ZIKV-BR-infected astrocyte at 84 hpi represented in three panels: the DIC image (upper panel), the fluorescent micrograph depicting the spotted signal of DiD-labeled ZIKV-BR vesicles (middle panel), the enlarged inset of the middle panel (rectangular area) superimposed by vesicle trajectories (lower panel). Scale bars, 10 µm. (c) An image of ZIKV-BR-infected neurons at 84 hpi; the DIC image of a neuronal culture (upper panel). The fluorescent micrograph depicting puncta of DiD-labeled ZIKV-BR vesicles in neurons (middle panel). The enlarged inset of the middle panel (rectangular area) superimposed by vesicle trajectories (lower panel). Vesicle trajectories labeled as 1 and 2 are expanded in (d). Scale bars, 5 µm. (d) Vesicle trajectories of the two typical trajectories noted in all cells types. The nearly straight trajectory of vesicle 1 depicts the highly directional movement of a vesicle. Travel length (TL) is similar in distance as the maximal displacement (MD) of a vesicle. The contorted trajectory of vesicle 2 depicts non-directional movement of a vesicle. TL is longer than the MD of a vesicle. Circles depict the starting positions of vesicle trajectories. TL, travel length depicted as a white trajectory; MD, maximal displacement depicted as a gray line connecting the two most distant points in a vesicle trajectory. (e) Graphs represent the TL and MD of vesicles in astrocytes infected with strains ZIKV-BR, ZIKV-FP, and ZIKV-UG. TL and MD of endocytotic vesicles laden with ZIKV-BR and ZIKV-UG significantly increased with longer hpi, whereas a significant decrease was observed in endocytotic vesicles laden with ZIKV-FP. The number of vesicles analyzed is denoted on the graphs. (f) TL and MD of neuronal endocytotic vesicles laden with ZIKV strains increased at 84 versus 12 hpi. The number of vesicles analyzed is denoted on the graphs. 17–45 cells per time period and per strain were analyzed. Data were collected from one experiment performed in duplicates and are represented as means ± SEM (One Way ANOVA, *p < 0.05).