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Zika Virus Cell Tropism in the Developing Human Brain and Inhibition by Azithromycin

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Zika Virus Cell Tropism in the Developing Human Brain and Inhibition by Azithromycin Zika virus cell tropism in the developing human brain and inhibition by azithromycin Hanna Retallacka,1, Elizabeth Di Lullob,c,1, Carolina Ariasa,d,KristeeneA.Knoppa, Matthew T. Lauriea, Carmen Sandoval-Espinosab,c, Walter R. Mancia Leonb,c, Robert Krencike,f, Erik M. Ulliane, Julien Spatazzab,g, Alex A. Pollenb,c, Caleigh Mandel-Brehma, Tomasz J. Nowakowskib,c, Arnold R. Kriegsteinb,c,2, and Joseph L. DeRisia,2 aDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; bEli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA 94143; cDepartment of Neurology, University of California, San Francisco, CA 94158; dDepartment of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106; eDepartment of Ophthalmology, University of California, San Francisco, CA 94122; fCenter for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX 77030; and gDepartment of Neurological Surgery, University of California, San Francisco, CA 94143 Contributed by Joseph L. DeRisi, November 1, 2016 (sent for review October 7, 2016; reviewed by Nenad Sestan and Pei-Yong Shi) The rapid spread of Zika virus (ZIKV) and its association with abnormal cell requirements. Building on studies that suggested that enriched brain development constitute a global health emergency. Congenital expression of the candidate entry factor AXL could confer vul- ZIKV infection produces a range of mild to severe pathologies, nerability to ZIKV entry (6–8), we used AXL expression levels to including microcephaly. To understand the pathophysiology of ZIKV predict that radial glia, astrocytes, microglia, and endothelial cells infection, we used models of the developing brain that faithfully would be particularly vulnerable to infection (9). A recent study recapitulate the tissue architecture in early to midgestation. We highlighted the utility of ex vivo models using primary human tis- identify the brain cell populations that are most susceptible to ZIKV sue samples to analyze the consequences of ZIKV infection in the infection in primary human tissue, provide evidence for a mechanism human prenatal brain (7). Here we further use primary tissue of viral entry, and show that a commonly used antibiotic protects samples from distinct stages of brain development corresponding cultured brain cells by reducing viral proliferation. In the brain, ZIKV to periods of peak neurogenesis and early gliogenesis. preferentially infected neural stem cells, astrocytes, oligodendrocyte Determining the tropism of ZIKV for specific cell types will precursor cells, and microglia, whereas neurons were less susceptible help identify suitable cellular models for investigating potential to infection. These findings suggest mechanisms for microcephaly and therapeutic interventions. Although development of a vaccine other pathologic features of infants with congenital ZIKV infection could provide a long-term solution to the current ZIKV epidemic, that are not explained by neural stem cell infection alone, such as there remains an unmet clinical need to identify drugs that can calcifications in the cortical plate. Furthermore, we find that blocking limit or prevent the consequences of congenital infection. A recent the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a screen of a subset of Food and Drug Administration (FDA)- glial cell line nearly abolished infection. Finally, we evaluate 2,177 approved compounds against ZIKV in hepatic cells identified compounds, focusing on drugs safe in pregnancy. We show that the several anticancer, antimicrobial, antiparasitic, and antifungal macrolide antibiotic azithromycin reduced viral proliferation and virus- drugs with anti-ZIKV activity (10). Another screen, based on hu- induced cytopathic effects in glial cell lines and human astrocytes. Our man neural progenitor cells, identified an antifungal drug and characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis Significance for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic. Zika virus (ZIKV) is a mosquito-borne flavivirus that has rapidly spread through the Americas and has been associated with fetal Zika virus | cortical development | azithromycin | microcephaly abnormalities, including microcephaly. To understand how micro- cephaly develops, it is important to identify which cell types of correlation between congenital exposure to the mosquito- the developing brain are susceptible to infection. We use primary Aborne and sexually transmitted Zika flavivirus (ZIKV) and human tissue to show that radial glia and astrocytes are more the increased incidence of severe microcephaly suggests a causal susceptible to infection than neurons, a pattern that correlates with relationship between ZIKV infection and neurodevelopmental expression of a putative viral entry receptor,AXL.Wealsoperform abnormalities (1, 2). However, the mechanisms of infection and a screen of Food and Drug Administration-approved compounds, specifically which cell populations are vulnerable to ZIKV during with an emphasis on drugs known to be safe in pregnancy. We the course of human brain development remain unclear. Major identify an antibiotic, azithromycin, that reduces viral proliferation insights have been drawn from in vitro models of human brain in glial cells, and compare its activity with daptomycin and sofos- buvir, two additional drugs with anti-ZIKV activity. development and primary mouse tissues. In the developing mouse brain, ZIKV has been shown to infect radial glia and neurons (3), Author contributions: H.R., E.D.L., C.A., K.A.K., M.T.L., A.A.P., T.J.N., A.R.K., and J.L.D. whereas studies in human pluripotent stem cell (hPSC)-derived designed research; H.R., E.D.L., C.A., K.A.K., M.T.L., C.S.-E., W.R.M.L., J.S., and C.M.-B. neural cells have highlighted widespread infection and apoptosis performed research; R.K. and E.M.U. contributed new reagents/analytic tools; H.R., E.D.L., of neural progenitor cells (4, 5). Because these models do not fully C.A., K.A.K., M.T.L., C.S.-E., W.R.M.L., J.S., C.M.-B., and T.J.N. analyzed data; and H.R., E.D.L., C.A., K.A.K., M.T.L., A.A.P., T.J.N., A.R.K., and J.L.D. wrote the paper. recapitulate the developmental events and cell types present Reviewers: N.S., Yale University School of Medicine; and P.-Y.S., University of Texas during human brain development, these results may not faithfully Medical Branch. represent ZIKV-induced pathology in vivo. The authors declare no conflict of interest. During human brain development, radial glial cells, the neural Freely available online through the PNAS open access option. stem cells, give rise to diverse types of neuronal and glial cells, 1H.R. and E.D.L. contributed equally to this work. including neurons, oligodendrocytes, and astrocytes, in a tempo- 2To whom correspondence may be addressed. Email: [email protected] or rally controlled pattern. We reasoned that identifying cell types [email protected]. that are especially vulnerable to viral infection would facilitate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. studies of the viral life cycle, including entry mechanisms and host 1073/pnas.1618029113/-/DCSupplemental. 14408–14413 | PNAS | December 13, 2016 | vol. 113 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1618029113 Downloaded by guest on September 30, 2021 several scaffold compounds for further development (11). How- antiviral activity may quickly narrow the search for drugs that could ever, the majority of compounds with anti-ZIKV activity from these mitigate the effects of congenital ZIKV infection. screens are contraindicated or of unknown safety during pregnancy. Here we assessed ZIKV cell tropism in the developing human Furthermore, two promising candidates that might be safe during brain and performed a drug screen on relevant cell types targeted by pregnancy, daptomycin and sofosbuvir, showed variable effective- the virus with an emphasis on drugs known to be safe in pregnancy. ness by cell type (7, 10, 12). Combining unbiased screens of ap- We found that radial glia and, later in development, astrocytes were proved compounds with comparisons of top candidates with known especially vulnerable to ZIKV infection. By screening FDA-approved Overview of the Infection Distribution of Cell Types Infected Infected Cells by ZIKV A ENV/SOX2 BCD SATB2 ENV merge CP Pia SP CP maturingm CP CP neuron SATB2+ OSVZ SP SP VZ newborn 0 100 % of ENV+ cells neuron SATB2+ OSVZ 80 radial glia SOX2+ OSVZ 40 microglia VZ IBA1+ % of ENV+ cells VZ OSVZ radial glia VZ 0 SOX2 SATB2 SOX2+ Enriched Infection in Germinal Zone, 15 pcw SOX2 GFAP E SOX2 ENV ENV GFAP SOX2 F SOX2/ENV 15 pcw OSVZ oRG Cell Infection G NS5 ENV NS5/ENV Fig. 1. Tropism of ZIKV for radial glia in the developing human brain. Human cortical organotypic brain slices were infected with ZIKV-BR and cultured for 72 h. (A and OSVZ B) Low-magnification overview of ZIKV infection de- tected by ENV (green) within the cortex. (A) ENV staining was analyzed with respect to region and cell type. CP, Viral Replication Viral cortical plate; OSVZ, outer subventricular zone; SP, subplate; VZ, ventricular zone. (Scale bar, 100 μm.) (B) High magnifi- H SOX2 SATB2 ENV merge cation of A. Notably, ENV staining (arrowheads) appears to be preferentially enriched in theVZandOSVZ.(Scalebars, + 20 μM.) (C) Quantification of ENV cells by region (Top)and cell type (Bottom)at13to14pcw.n = 2; mean ± SD [SI Materials and Methods; an error bar is not shown where it is shorter than the line thickness (Top,CP;Bottom, SOX2)]. (D) Schematic summary of cell types observed to be sus- ceptible to ZIKV infection (green) in the developing human 13 pcw OSVZ brain during midneurogenesis. (E) High-magnification Neuron Infection view of a ZIKV-infected radial glial cell in the OSVZ MICROBIOLOGY (arrow).
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