tabata petitt et al

Cell Host & Microbe

Article

Zika Virus Targets Different PrimaryHuman Placental Cells, SuggestingTwo Routes for Vertical TransmissionTakako Tabata,1,3 Matthew Petitt,1,3 Henry Puerta-Guardo,2 Daniela Michlmayr,2 Chunling Wang,2 June Fang-Hoover,1

Eva Harris,2,* and Lenore Pereira1,*1Department of Cell and Tissue Biology, School of Dentistry, University of California, San Francisco, San Francisco, CA 94143, USA2Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA 94720-3370, USA3Co-first author*Correspondence: [email protected] (E.H.), [email protected] (L.P.)http://dx.doi.org/10.1016/j.chom.2016.07.002

SUMMARY

Zika virus (ZIKV) infection during pregnancy is linkedto severe birth defects, but mother-to-fetus trans-mission routes are unknown. We infected differentprimary cell types from mid- and late-gestation pla-centas and explants from first-trimester chorionicvilli with the prototype Ugandan and a recently iso-lated Nicaraguan ZIKV strain. ZIKV infects primaryhuman placental cells and explants—cytotropho-blasts, endothelial cells, fibroblasts, and Hofbauercells in chorionic villi and amniotic epithelial cellsand trophoblast progenitors in amniochorionicmem-branes—that express Axl, Tyro3, and/or TIM1 viralentry cofactors. ZIKV produced NS3 and E proteinsand generated higher viral titers in amniotic epithelialcells from mid-gestation compared to late-gestationplacentas. Duramycin, a peptide that binds phos-phatidylethanolamine in enveloped virions and pre-cludes TIM1 binding, reduced ZIKV infection inplacental cells and explants. Our results suggestthat ZIKV spreads from basal and parietal deciduato chorionic villi and amniochorionic membranesand that targeting TIM1 could suppress infection atthe uterine-placental interface.

INTRODUCTION

Zika virus (ZIKV) is a mosquito-borne flavivirus that was relativelyobscure until outbreaks in Yap and French Polynesia in 2007 and2013, respectively, and the 2015–2016 pandemic expandingrapidly from Brazil across the Americas brought it to worldwideattention. In the United States, 691 imported cases and 11 casesof confirmed sexual transmission have been reported (http://www.cdc.gov/zika/geo/united-states.html). ZIKV is linked to se-vere birth defects and Guillain-Barre syndrome (Cao-Lormeauet al., 2016; Sarno et al., 2016), and in February of 2016, theWorld Health Organization declared the Zika pandemic a PublicHealth Emergency of International Concern (http://www.who.int/mediacentre/news/statements/2016/emergency-committee-

zika-microcephaly/en/). However, little is known about thecausal mechanisms. Mounting evidence indicates that infectionin early gestation can lead to miscarriage, stillbirth, intrauterinegrowth restriction (IUGR), and microcephaly, a malformation ofthe fetal brain (Brasil et al., 2016; Mlakar et al., 2016); however,infection of the mother in the second or third trimester and pro-longed viremia could contribute to fetal abnormalities (Brasilet al., 2016; Driggers et al., 2016). ZIKV has been detected inbrain glia and neurons, placenta, and amniotic fluid of babieswith microcephaly, further linking infection during pregnancy tocongenital disease (Mlakar et al., 2016; Rasmussen et al.,2016). How ZIKV infects the placenta and reaches the fetalcompartment is unknown.In early gestation, trophoblasts from chorionic villi of the

placenta develop into twomajor cell types, syncytiotrophoblasts(STBs), which cover the villus surface, and cytotrophoblasts(CTBs). Villus CTBs proliferate and switch from an epithelial toan endothelial phenotype, differentiate, invade the uterine wall,and remodel uterine arteries (Zhou et al., 1997). Chorionic villi an-chor the placenta to the uterus and channel blood from circula-tion to the maternal blood space. To maintain immune toleranceto the hemiallogeneic placenta, natural killer cells, macrophages,and dendritic cells emigrate to the basal decidua, attracted bychemokine-receptor networks (Red-Horse et al., 2001). Oppo-site the basal decidua, where chorionic villi are anchored, amuch larger portion of the uterine wall is lined by the parietaldecidua. By 15 weeks gestation, the parietal decidua is in con-tact with the chorionic membrane, which is fused to the amnioticmembrane lined on the fetal side by amniotic epithelial cells(AmEpCs) (Benirschke and Kaufmann, 2000). Trophoblast pro-genitor cells (TBPCs) in the chorion differentiate into invasiveCTBs that migrate into the parietal decidua and attach theamniochorionic membranes to the uterus (Genba!cev et al.,2015). The parietal decidua contains maternal blood vesselsand lymphatic vessels and functions as a paraplacental ex-change organ that filters fluid from maternal circulation via thechorion and contributes tomaintenance of equilibrium in the fetalcompartment. As pregnancy advances and the fetus grows, thechorionic surface of the amniochorionic membrane adjoins theparietal decidua across almost the entire uterine surface.Flaviviruses bind to a variety of surface molecules that serve

as entry mediators or cofactors (Perera-Lecoin et al., 2013).Recently, dengue virus (DENV)was shown to bind the TAM family

Cell Host & Microbe 20, 155–166, August 10, 2016 ª 2016 Elsevier Inc. 155

of tyrosine kinase receptors—Tyro3, Axl, andMertk—which clearapoptotic cells (Meertens et al., 2012) and regulate innate im-mune functions (Lemke and Rothlin, 2008; Rothlin et al., 2007).TAM is activated by ligands that bind phosphatidylserine (PS) inmembranes of apoptotic cells and can form bridges between vi-rions and TAM. DENV also binds TIM1, a member of the T cellimmunoglobulin and mucin domain protein family that regulatesinnate and adaptive immune functions and cell survival (Freemanet al., 2010). Axl and Tyro3 and, to a lesser extent, TIM1 serve asentry cofactors for DENV (Meertens et al., 2012; Perera-Lecoinet al., 2013), and it was demonstrated that TIM1 directly bindsPS and phosphatidylethanolamine (PE) in the virion envelope ofdengue, West Nile, and Ebola viruses (Jemielity et al., 2013;Richard et al., 2015). A recent ZIKV isolate was shown to infecthuman dermal fibroblasts, epidermal keratinocytes, and imma-ture DCs, with DC-SIGN, Axl, Tyro3, and TIM1 facilitating entryand Axl playing a major role (Hamel et al., 2015).

In the present study, we took advantage of our previous workon human cytomegalovirus (HCMV) infection in the developingplacenta and primary cells expressing viral receptors (Fisheret al., 2000; Maidji et al., 2007; Tabata et al., 2007, 2015; Zydeket al., 2014) to understand how ZIKV infection and transmissionfrom mother to fetus occur. We hypothesized that, like HCMV,hematogenous spread of ZIKV entails infection of cells atsites in the uterus perfused by maternal blood—decidua andplacenta—and by amniotic fluid, which bathes the amnioticmembrane and fetus. To test this possibility, we isolated differentprimary cell types from mid- and late-gestation placentas andestablished explants from first-trimester chorionic villi, and wefound that placental cells and explants were highly infectablewith the prototype Ugandan ZIKV strain and new 2016 ZIKVisolates from the current epidemic in Nicaragua. Cell typessusceptible to ZIKV replication expressed Axl, Tyro3, and TIM1cofactors to varying degrees, and immunohistological andimmunoblot analyses of placental biopsy specimens and pri-mary cells confirmed that expression varied by cell type, donor,differentiation state, and gestational age. Notably, TIM1 wasconsistently and highly expressed throughout the basal deciduaand parietal decidua, chorionic villi, and amniochorionic mem-branes surrounding the fetus, and in AmEpCs that were produc-tively infected and released high virus titers. Duramycin, a cyclic19-aa peptide that specifically binds PE, potently blocked ZIKVinfection in all primary placental cells and chorionic villus ex-plants, indicating that TIM1 plays a critical role in infection andsuggesting strategies for clinical management.

RESULTS

ZIKV Infects Primary Human Placental Cells from Mid-and Late Gestation and Chorionic Villus Explants fromEarly GestationTo determine whether ZIKV infects primary cells from the humanplacenta, isolatedAmEpCs frommid- and late-gestation amnioticmembranes, TBPCs from chorion, andHPFs andCTBs fromcho-rionic villi were isolated from donors (n = 20). These cells, alongwith a primary human umbilical vein endothelial cell (HUVEC)line from umbilical cord, were infected with prototype ZIKV strainMR766 and, at 72 hr post-infection, were immunostained withmonoclonal antibodies (MAbs) to E glycoprotein (Figure 1Ai–v)

and nonstructural protein NS3 (Figure 1Avi–x), which is notsecreted and is widely used as a marker for flavivirus replication.AmEpCs (Figure 1Ai, ii, vi, and vii) were highly susceptible toZIKV, as indicated by expression of E and NS3, while infectionof TBPCs (Figure 1Aiii and viii), HPFs (Figure 1Aiv and ix), HUVECs(Figure1Avandx), andCTBs (FigureS1, availableonline) occurredin single cells and/or discrete foci. Productivity of primary cellinfectionwas indicatedby releaseof infectiousprogenyquantifiedby plaque and focus-forming assays (FFAs) (Table S1). Most pro-ductive were AmEpCs, which released high titers (105–107) at lowMOI (0.001) ascomparedwithTBPCs,HPFs,HUVECs,andCTBs,which were infected at higher MOI (1–2).Having found that primary AmEpCs from mid- and late-gesta-

tion placentas were susceptible to ZIKV infection, these cellswere infected with clinical isolates Nica1-16 and Nica2-16 inparallel with MR766. Nica1-16 and Nica2-16 were isolated inNicaragua in January 2016; full-length sequencing confirmedtheir close relationship to the Brazil 2015–2016 strains. LikeMR766, Nica1-16 readily infected AmEpCs at low MOI, ex-pressed E (Figure 1Bi and ii) and NS3 (Figure 1Biii and iv), andproduced high titers at 3, 5, and 7 dpi (days post-infection) (TableS1). Interestingly, AmEpCs from mid-gestation placentas pro-duced 6- to 8-fold higher titers than those from late-gestationplacentas at 5–7 dpi when infected with MR766 (Figure 2A),Nica1-16 (Figure 2B), or Nica2-16 (Figure 2C). Similarly, ZIKVrelease from Nica1-16-infected AmEpCs titered on humanARPE-19 cells was !4-fold greater in mid-gestation than inlate-gestation cells at 5–7 dpi (Figure 2D).We next cultured chorionic villus explants from first-trimester

placentas (n = 4), infected with MR766 or Nica1-16, and immu-nostained for ZIKV E and NS3 at 3 dpi (Figure 1Ci and ii). Lowmagnification (middle) showed E and NS3 proteins in a focusof proliferating villus CTBs (cytokeratin [CK] and Ki67 positive).Approximately 15 CTBs near the villus core were infected, andboth strong and weak staining were observed, suggesting cell-to-cell spread (Figures 1Ci and ii and S2H–S2S). ZIKV-infectedCTBs expressed CK, but did not express Ki67, indicating theywere no longer proliferating (Figures 1Ciii, S2J, S2M, S2P, andS2S). Invasive CTBs were also infected, expressed E and NS3proteins, and continued to express CK (Figure 1Di, ii, iv, and v)and MHC class I HLA-G, a differentiation antigen (Figure 1Diiiand vi). ZIKV proteins were also detected in villus CTBs andnumerous small foci with two to four infected cells (FiguresS2N–S2S). Detailed examination of the explants indicated thatSTBs did not produce ZIKV NS3 protein and were not infected,although E glycoprotein was detected in small cytoplasmic ves-icles (data not shown). In villus explants infected with Nica1-16,Hofbauer cells in the villus core were infected and expressed Eand NS3 proteins (Figure 1Ei, ii, iv, and v) and CD68 (Figure 1Eiiiand vi). Likewise, MR766 infected Hofbauer cells in villus ex-plants (Figures S2B–S2G). Titers of infectious progeny releasedfrom chorionic villus explants infected with MR766 or Nica1-16were quantified in ARPE-19 cells and ranged from 2 3 103

PFU (plaque-forming units) to 1 3 105 PFU.Together, these results show that different types of primary

cells from mid- and late-gestation placentas are permissive toinfection with prototype and contemporary ZIKV strains. Further,in the tissue environment of early gestation chorionic villus ex-plants, proliferating villus CTBs in proximal cell columns were

156 Cell Host & Microbe 20, 155–166, August 10, 2016

infected, after which proliferation was arrested. Differentiating/invasive CTBs and Hofbauer cells in the villus core were alsokey targets of infection.

Primary Placental Cells Express Potential Cofactors forZIKV InfectionWe hypothesized that Axl, Tyro3, and TIM1 could be cofactorsfor ZIKV infection in the human placenta and that their expres-

A

C

D

E

B Figure 1. Prototype Ugandan and Nicara-guan 2016 ZIKV Strains Infect Primary CellTypes from Human Placentas(A) Primary human placental cells were infected

with ZIKV MR766 and immunostained for E (i–v)

and NS3 (vi–x) proteins at 3 dpi. (i, ii, vi, and vii)

Primary amniotic epithelial cells (AmEpCs) at mid-

and late gestation were infected at MOI 0.1. (iii and

viii) Trophoblast progenitor cells (TBPCs) (P15),

(iv and ix) human placental fibroblasts (HPFs), and

(v and x) human umbilical vein endothelial cells

(HUVECs) (P5) were infected at MOI 1.

(B) Primary AmEpCs from mid- and late gestation

were infected with ZIKV Nica1-16 at MOI 0.001

and immunostained for E glycoprotein (i and ii) and

NS3 protein (iii and iv) at 3 dpi.

(C) (i–iii) Chorionic villus explant (8.4 weeks) in-

fected with ZIKV MR766 (1 3 106 PFU) and

immunostained for E and CK (i), NS3 and CK (ii),

and NS3 and Ki67 (iii) in adjacent sections at 3 dpi.

Left and right panels: higher magnification images.

Arrowheads, cells expressing viral proteins; ar-

rows, cells expressing Ki67. Images representa-

tive of n = 4.

(D) Chorionic villus explant (8 weeks) infected

with ZIKV MR766 (1 3 106 PFU) and immuno-

stained for E and CK (i), NS3 and CK (ii), and MHC

class I HLA-G (differentiation antigen) and CK (iii).

(E) (i–vi)Chorionic villus explant (8.4weeks) infected

with Nica1-16 (5 3 103 PFU) and immunostained

for E and CK (i and iv), NS3 and CK (ii and v) and

CD68 (Hofbauer cell/macrophage marker) and CK

(iii and vi) in adjacent sections at 2 dpi. Villus core

Hofbauer cells/macrophages (circled) were posi-

tive for NS3 and E. Nuclei, blue (DAPI). VC, villus

core. See also Figures S1 and S2 and Table S1.

sion could bemodulated during cell isola-tion and culture in vitro. To this end,immunoblotting was used to measureexpression of these cofactors in CTBs,amniotic membranes, isolated AmEpCs,and low-passage TBPCs, HPFs, andHUVECs.In CTBs isolated from mid-gestation

placentas, cofactor expression variedconsiderably during differentiation from0 to 96 hr (Figure 3A). Axl was either upre-gulated or not expressed, while Tyro3levels were generally low. In contrast,TIM1 showedmoderate or strong expres-sion that declined over time. In late-gestation CTBs, TIM1 expression wasstrong and detected throughout differen-

tiation, in contrast to Axl and Tyro3 (Figure 3A). Titers fromMR766- and Nica1-16-infected CTBs were about 100-fold lowerthan titers from mid-gestation CTBs (Table S1). TIM1 wasstrongly produced in trophoblast cell lines (JEG-3 and HTR8)that are highly infected by ZIKV (Bayer et al., 2016; Miner et al.,2016), but Axl was not expressed by JEG-3 cells (Figure 3B). Inintact amniotic membranes from late gestation, little or no Axlwas detected (Figure 3C); however, in cells newly isolated from

Cell Host & Microbe 20, 155–166, August 10, 2016 157

these membranes and subsequently at passage 0 (P0), faster-migrating forms of Axl were present, and a highmolecular weightform was strongly upregulated at P1. Tyro3 was initially weaklyexpressed but in some cases strongly upregulated after isolation(Figure 3C). In contrast, TIM1 was consistently and stronglyexpressed in both intact amniotic membranes and isolatedAmEpCs from all donors (Figure 3C). Next, we comparedcofactor expression in AmEpCs from mid- and late-gestationplacentas (Figure 3D). Axl was produced by AmEpCs fromeleven of thirteen donors, but two mid-gestation donors madefaster-migrating forms, and Tyro3 was not expressed in four do-nors (Figure 3D; Table S3). Uniformly robust TIM1 expressionwas found in AmEpCs from all donors. In TBPCs isolated fromearly and mid-gestation chorionic membranes, Axl and Tyro3were weakly expressed in early gestation and strongly in lategestation, whereas TIM1 expression was robust throughout (Fig-ure 3E). In HPFs and HUVECs, Axl was strong, Tyro3 varied, andTIM1 expression was moderate (Figure 3F).

We next used immunofluorescence to visualize the subcellularlocalization of Axl and TIM1. Axl was concentrated at plasmamembranes and perinuclear sites in mid-gestation AmEpCs;however, abundant perinuclear and vesicular staining wasdetected in late gestation (Figure 3G). TIM1 was concentratedin plasma membranes in mid-gestation AmEpCs, but diffusenuclear and cytoplasmic staining increased in late gestation (Fig-ure 3H). Immunostaining of first-trimester chorionic villus ex-

plants showed that TIM1 was expressed in villus CTBs andHofbauer cells (CD68 positive) (Figure S2A). In summary, immu-noblot and immunofluorescence studies showed that TIM1, apotential cofactor for ZIKV infection, was expressed in CTBand trophoblast cell lines and strongly in amniotic membranesand in primary AmEpCs isolated from all mid- and late-gestationplacentas and in early gestation chorionic villi. In contrast, Axland Tyro3 expression differed by cell type and donor and wasmodulated in culture and as cells differentiated in vitro.

Potential Cofactors for ZIKV Infection Are Present at theUterine-Placental InterfaceAxl was reported to play a major role in ZIKV infection in humankeratinocytes (Hamel et al., 2015). To determine whether Axl waspresent in the decidua, placenta, and fetal membranes, biopsyspecimens from mid-gestation were co-stained for Axl and thecellular proteins vimentin (fibroblasts), cytokeratin (CTB), CD68(Hofbauer cells/fetal macrophages), and CD31 (endothelial cellsin blood vessels). In basal decidua, Axl was expressed indecidual cells (Figure 4A) and a subset of deeply invasiveCTBs (Figure 4B). In chorionic villi of the adjacent placenta, Axlwas present in Hofbauer cells/macrophages (Figure 4C), endo-thelial cells in blood vessels (Figure 4D), and HPFs in the villuscore (Figure 4E). In amniochorionic membranes, Axl wasexpressed in AmEpCs and TBPCs (Figure 4F). Interestingly,the intensity of Axl expression depended on location, especiallyin invasive CTBs in decidua (Figures 4A and 4B), Hofbauer cells,and HPFs (Figures 4C and 4E), suggesting responses to the tis-sue environment. Modulation of cofactors was also shown byimmunoblot analysis of differentiating CTBs from various donorsthat upregulated Axl as they invaded extracellular matrix in vitro(Figure 3A).To determine whether potential cofactors for ZIKV infection

were expressed in vivo, we surveyed a library of biopsy speci-mens from 30 mid- and late-gestation placentas for Axl, Tyro3,and TIM1. Multiple tissue sections were screened from amnio-chorionic membranes, chorionic villi of the placenta, basaldecidua, and parietal decidua. Detailed examination indicatedthat expression of specific cofactors depended on the donor,location of cells in tissues, and gestational age (Figure 5; TableS2). The strength of Axl signal varied and was absent fromsome mid-gestation and all late-gestation amniotic membranes(Figures 5A, 5D, 5G, and 5P) and smaller chorionic villi (Figure 5J);however, basal decidua and parietal decidua were positive (Fig-ures 5M and 5S; Table S2). Tyro3was present in amniotic epithe-lium (Figures 5B, 5E, 5H, and 5Q) and in basal and parietaldecidua (Figures 5N and 5T), but not in chorionic villi (Figure 5K).In contrast, TIM1 was strongly and consistently expressed in alltissues of mid- and late-gestation placentas—amniotic epithe-lium (Figures 5C, 5F, 5I, and 5R), STBs, Hofbauer cells, andHPFs in chorionic villi (Figure 5L); decidual cells and invasiveCTBs in basal decidua (Figure 5O); and TBPCs in chorion—and was dramatically induced in the population of differentiatingCTBs that invade the parietal decidua (Figure 5U).Table S3 summarizes results of immunoblot and immunostain-

ing experiments in isolated cells and biopsy specimens of pla-centas from mid- and late gestation. TIM1 was consistentlyand strongly expressed on cells throughout the uterine-placentalinterface in basal and parietal decidua, chorionic villi, and

A B

C D

mid GA

late G

A104

105

106

FFU

/ml

**

mid GA

late G

A104

105

106

FFU

/ml

**

FFU

/ml

mid GA

late G

A105

106

107

**

mid GA

late G

A105

106

107

FFU/

ml

*

Figure 2. Viral Titers from ZIKV-Infected AmEpCs from Mid-gesta-tion Are Higher Than Those from Late GestationAmEpCs isolated from mid- (21.2 and 22.6 weeks) and late (38.6 and

40.2 weeks) gestation were infected with (A) MR766 at MOI 0.001, (B and D)

Nica1-16 at MOI 0.001, and (C) Nica2-16 at MOI 0.009. Viral titers were

determined by FFA at 3 dpi (A) and 5 and 7 dpi (B and C). (D) Viral titers at 5 and

7 dpi were quantified in ARPE-19 cells and stainedwithMAb 4G2 at 28 hpi. GA,

gestational age. n = 4 from 2 independent experiments; *p < 0.05, **p < 0.01,

unpaired t test. See also Table S1.


amniochorionic membranes surrounding the fetus. In contrast,Axl and Tyro3 expression varied by donor, gestational age,and tissue andwasmodulated in culture and differentiating cells.

TIM1 Inhibitor Blocks ZIKV Infection across PlacentalCell Types and Reduces Infection in Chorionic VillusExplantsHaving found that ZIKV infects primary cells from chorionic villiand amniochorionic membranes that express Axl and TIM1,we considered they could function as cofactors. Although Axlwas variably expressed and absent from amniotic membranes

in late gestation (Figures 5A, 5D, 5G, and 5P; Tables S2 andS3), we tested the effects of the Axl-specific tyrosine kinase in-hibitor R428 on ZIKV infection. At the highest concentrationtested (3.0 mM), R428 reduced infection of AmEpCs by MR766and Nica2-16 by !40%–50% (Figures 6A and 6B).Since TIM1 was consistently made by all permissive cell types

and was broadly expressed at the uterine-placental interface byall donors (Figures 5C, 5F, 5I, 5L, 5O, 5R, and 5U; Tables S2 andS3), we performed infection-blocking experiments using dura-mycin, a cyclic peptide that binds PE and inhibits infection byDENV, a close relative of ZIKV, in cells that express TIM1 (Richard

AxlDAPI

A

TBPC wk: 7.3 15.6

AmEpC D5 AmEpC D109

D1 D2 D3 D4 D5 D6 D7 D108 D109

TIM1DAPI

HPF HUVEC

0 16 96

CTB-D202 0 16 96

CTB-D203

Axl

TIM1

Actin

Tyro3

hr: 0 16 96

CTB-D201 CTB-D207 0 96

AmEpC D5 AmEpC D109

C AmEpC D110

Inta

ctIs

olat

edP0 P1 In

tact

Isol

ated

P0 P1 Inta

ctIs

olat

edP0 P1

AmEpC D111 AmEpC D112

Late gestation Mid-gestation

AmEpCD

E F G

Axl

TIM1

Actin

Tyro3

Axl

TIM1

Actin

Tyro3

Axl

TIM1

Actin

Tyro3

H

50µm

20µm

Mid-gestation

0 16 96 CTB-D220

Late-gestation

JEG3

HTR8

B

Axl

TIM1

Actin

Tyro3

TrophoblastCell Lines

Figure 3. TIM1 Is Consistently Expressed in Primary Placental Cells, While Axl and Tyro3 Expression Varies According to Cell Type, Differ-entiation State, and Individual Donor(A–F) Expression of cofactors by immunoblotting in (A) differentiating CTBs isolated frommid-gestation (n = 4) and late-gestation placentas (n = 1); (B) trophoblast

cell lines JEG-3 and HTR8; (C) cells from three late-gestation amniotic membranes, including intact membranes, freshly isolated AmEpCs before plating, and

confluent P0 and P1 cells; (D) AmEpCs (P1) from mid- and late-gestation placentas; (E) TBPCs from early and mid-gestation (P15) placentas; and (F) HPFs and

HUVECs (P5).

(G and H) Representative images of Axl and TIM1 staining in AmEpCs frommid- (left) and late-gestation (right) placentas (n = 6). See also Figure S2 and Table S3.


et al., 2015). ZIKV MR766 was incubated with increasing con-centrations of duramycin and added to AmEpCs. Immunofluo-rescence assays revealed a striking reduction in ZIKV NS3-ex-pressing cells in the range of 0.2 mM to 1 mM duramycin; fewNS3-expressing cells were detected after treatment withR0.2 mM duramycin (Figure 6C). Quantification of NS3-express-ing AmEpCs showed that duramycin potently blocked ZIKVinfection in cells from mid- (22.6 weeks) and late- (40.2 weeks)gestation placentas (Figure 6Di). Likewise, when duramycin-treated MR766 was added to placental TBPCs, HPFs, andHUVECs, infection was reduced by >50% with 0.1 mM and>95% with 0.2 mM duramycin, even at 10-fold higher MOI (Fig-ure 6Dii). Similar results were obtained when AmEpCs (Figure6Diii) and invasive CTBs (data not shown) were infected withNica1-16 treated with duramycin. Titration of infectious progenyreleased frombothmid- and late-gestation AmEpCs showed that0.2–1 mMduramycin significantly reduced virus titers (Figure 6E).

To determine whether duramycin affects ZIKV infection in thetissue environment, chorionic villus explants from first-trimesterplacentas were infected with untreated (n = 2) or 1 mM duramy-

cin-treated (n = 2) MR766 (13 106 PFU) 2 hr after plating, and in-fectious progeny were titered in ARPE-19 cells. Titers werereduced from 1 3 105 PFU/mL in the explants infected with un-treated ZIKV to 4 3 103 and 0 PFU/mL in the explants infectedwith duramycin-treated virus. Together, these studies showthat duramycin blocks ZIKV infection of numerous primaryplacental cell types from mid- and late gestation, whereas theAxl inhibitor was far less effective in blocking infection. More-over, duramycin reduces ZIKV infection in chorionic villusexplants from early gestation placentas.

DISCUSSION

In this study, we show that ZIKV prototype strain MR766and recent ZIKV isolates from the current Zika pandemicinfect numerous human primary cells—AmEpCs, CTBs, TBPCs,HPFs, and HUVECs—isolated from mid- and late-gestation pla-centas and produce infectious progeny. In explants of chorionicvilli from first-trimester placentas, ZIKV infected proliferating vil-lus CTBs, invasive CTBs, and Hofbauer cells in the villus coreand expressed E and NS3 proteins, indicating viral replication.Of note, ZIKV-infected CTBs no longer proliferated. Virus titerswere dependent on cell type and gestational age, as mid-gesta-tion AmEpCs produced significantly higher titers than late-gesta-tion cells. Analysis of biopsy specimens revealed sites at theuterine-placental interface with strong and consistent TIM1expression in cells where maternal blood perfuses the placenta.These include the basal decidua and adjacent chorionic villi (Fig-ure 7, left) and the parietal decidua, lining most of the uterus andadjacent to amniochorionic membranes surrounding the fetus(Figure 7, right). Expression of other potential cofactors, Axland Tyro3, varied by donor, location of susceptible cell typeswithin biopsy specimens, and gestational age. Infection of iso-lated placental cells was potently inhibited by duramycin, a 19-aa cyclic peptide that specifically binds PE in the virion envelopeand blocks infection of the closely related DENV (Richard et al.,2015). Duramycin blocked ZIKV infection of primary placentalCTBs that contact maternal blood circulation and AmEpCs inamniotic fluid and reduced infection in the tissue environmentof early gestation chorionic villus explants, potentially by pre-cluding binding of ZIKV to TIM1, a cofactor we showed wasuniversally expressed across the uterine-placental interfacethroughout gestation. In contrast, an inhibitor of Axl, reportedto be a cofactor for ZIKV in dermal and epidermal cells (Hamelet al., 2015), modestly reduced infection, suggesting a second-ary contribution in placental cells.Figure 7 shows a model of the uterine-placental interface and

identifies the type and location of primary cells infected in vitro,suggesting sites of viral replication and identifying placentaland paraplacental routes of ZIKV transmission to the fetus. Thebasal decidua, the first zone of contact between the uterusand adjacent placenta, develops as placental CTBs invade theuterine wall and remodel blood vessels. The opposed parietaldecidua, the second zone of contact between the uterusand fetal membranes, constitutes a considerably larger surfacewhere maternal and fetal zones remain in contact from mid-gestation to delivery (Genba!cev et al., 2015). Our findings sug-gest that in the parietal decidua, ZIKV could disseminate fromuterine blood vessels to invasive CTBs to fetal membranes,

A B

DC

FE

Figure 4. Axl Is Strongly Expressed at the Uterine-PlacentalInterfaceImmunofluorescence staining of frozen sections frommid-gestation placentas

for Axl (green) and cell-type markers (red).

(A) Axl and vimentin in cells of the basal decidua.

(B) Axl and cytokeratin (MAb 7D3) in invasive CTBs.

(C) Axl co-localizes with CD68 (red), a marker of Hofbauer cells and macro-

phages.

(D) Axl in CD31-positive vascular endothelial cells in a villus core.

(E) Axl in villus core partially overlaps with vimentin, a marker of HPFs.

(F) Axl in the amniotic epithelium and cells of the mesenchymal portions of the

amnion and chorion, where TBPCs reside. Cytokeratin 7 (CK7) staining iden-

tifies CTBs lining the surface of the chorionicmesenchyme. DAPI, blue. Images

representative of n = 4. See also Figure S2 and Table S3.


infecting TBPCs in the chorionic membrane and then AmEpCs inthe amniotic membrane. Progeny virions released into amnioticfluid could infect susceptible cells in fetal skin (Fujita et al.,

1991; Kubo et al., 2009), analogous to DENV infection of theskin (Hamel et al., 2015; Wu et al., 2000). Recent reports supportboth placental and paraplacental routes of ZIKV transmission,

A B C

D E F

G H I

J K L

M N O

P Q R

S T U

Figure 5. Axl and Tyro3 Vary by Site and Gestational Age, Whereas TIM1 Is Consistently ExpressedAxl, Tyro 3, and TIM1 expression in human placenta and fetal membranes was examined by immunohistochemical staining of fixed, paraffin-embedded placental

biopsies from mid- and late gestation (n = 26).

(A and D) Axl immunostaining in two mid-gestation amnions. Amnion 1 stains positively (A) and amnion 2 is negative (D).

(B, C, E, and F) Mid-gestation amnions stain positively for Tyro 3 (B and E) and TIM1 (C and F).

(G–O) Three zones of the maternal-placental interface from three placentas: amniotic epithelium on fetal side, bathed in amniotic fluid (G–I); chorionic villi of

placenta with fetal blood vessels and villus core covered by STBs in maternal blood (J–L); and basal decidua containing invasive CTBs from the placenta (M–O).

(P–U) Interface of the amniochorion with parietal decidua from three placentas, including the amniotic epithelium (P–R) and the chorion, CTB layer, and parietal

decidua, containing invasive CTBs (S–U). Images are representative of 26 donors (Table S2). Donor numbers are indicated in the lower right corner. See also

Table S2.


including (1) persistence of ZIKV in maternal blood during preg-nancy, (2) high viral load in fetal membranes and viral RNA in am-niotic fluid, (3) limited placental damage with high viral titers infetal brain, (4) more cases of symptomatic congenital ZIKV infec-tions with microcephaly in early gestation as compared with lategestation, and (5) a narrow window after primary infection untilfetal anomalies develop, suggesting direct infection of the fetus(Brasil et al., 2016; Calvet et al., 2016; Driggers et al., 2016).

Regarding the first zone of contact between floating chorionicvilli in the maternal blood space and anchoring villi in the basaldecidua (Figure 7, left panel), it was recently reported that ZIKVfails to infect syncytialized CTBs from late gestation due tosecretion of type III interferon (Bayer et al., 2016). Althoughthe cofactor TIM1 is strongly expressed in chorionic villi, weobserved that STBs are spared from infection, suggesting innate

A B

C

D

E

Figure 6. Axl and TIM1 Inhibitors Block ZIKVInfection Differentially in Primary PlacentalCells(A and B) Axl kinase inhibitor reduces infection of (i)

MR766 and (ii) Nica2-16 (both MOI 0.014) in AmEpCs

(40.2 weeks). (A) Representative immunofluores-

cence images stained for NS3, and (B) quantification of

relative infection (%) (mean ± SD) of two replicates of

each condition from two independent experiments.

**p < 0.01, unpaired t test.

(C and D) TIM1 inhibitor (duramycin) potently reduces

ZIKV MR766 and Nica1-16 infection of various

placental cell types.

(C) Representative immunofluorescence images of

ZIKV MR766 infection (MOI 0.3) in AmEpCs (22.6 and

40.2 weeks). NS3 in green.

(D) (i) Relative infection with ZIKV MR766 of AmEpCs

(22.6 and 40.2 weeks; MOI 0.3) and (ii) HPFs, TBPCs,

and HUVECs (MOI 3). n = 2 for HPFs and TBPCs and

n = 1 for HUVECs. (iii) Relative infection of AmEpCs

with Nica1-16; n = 3.

(E) Virus titers from ZIKV MR766-infected AmEpCs

treatedwithduramycinat 3dpi, quantifiedbyFFA. n=2.

In (D) and (E), **p < 0.01, ***p < 0.001, one-way ANOVA

with Dunnett’s post-test.

immune responses protect the villus surface(Savidis et al., 2016). Nonetheless, we identi-fied ZIKV infection with both NS3 and Eexpression in foci of proliferating CTBs nearthe villus core and invasive CTBs expressingHLA-G. In countries with endemic DENVinfection, it is likely that pre-existing maternalantibodies cross-reactive with ZIKV but withlow neutralizing activity could promote infec-tion of immune cells bearing Fcg receptors(FcgRs) for IgG, as reported for DENV (Balsi-tis et al., 2010; de Alwis et al., 2014). Theseobservations suggest a potential role for anti-body-mediated transcytosis of IgG-ZIKVimmune complexes by the neonatal Fc re-ceptor expressed in STBs and infectionof underlying CTBs, as reported for HCMV(Maidji et al., 2006). ZIKV dissemination inthe villus core could result in enhanced infec-

tion of FcgR-expressing Hofbauer cells, reported to be targets ofZIKV infection in the placenta (Quicke et al., 2016). Here, wefound that fetal Hofbauer cells in the villus core of explants in-fected with ZIKV expressed E glycoprotein and NS3 protein,consistent with viral replication, suggesting that virion uptakecan result in infection of fetal macrophages. Infection of cellsbearing FcRs in basal and parietal decidua could potentially beenhanced by immune complexes of ZIKV with weakly neutral-izing DENV cross-reactive antibodies or antibodies that developafter primary maternal ZIKV infection.Our results have potential clinical relevance for congenital

ZIKV infection and implications regarding disease outcome.We found that AmEpCs from mid-gestation placentas producehigher virus titers than late-gestation cells, suggesting fetuses in-fected earlier in gestation are exposed to more virus for a longer


time during pregnancy. Infection in early and mid-gestation viabasal decidua and chorionic villi could disseminate virus to fetalcirculation at a critical stage in brain development, when highviral load increases the risk of malformations. We observedthat ZIKV infected numerous clusters of proliferating CTBs inintact villus explants, but ZIKV-infected cells stopped prolifer-ating, and virus replicated in invasive CTBs. High viral titerswere released that could potentially spread infection to Hofbauercells, placental fibroblasts, and endothelial cells in the villuscore and fetal circulation. Moreover, the patterns of infectionobserved suggest downstream structural defects: calcificationof the villus surface, truncation of developing cell columns thatthen fail to attach to the uterine wall, and a reduced populationof invasive CTBs, which would impair remodeling of uterineblood vessels, leading to IUGR, which is associated withcongenital ZIKV infection.Regarding potential cofactors of ZIKV infection, immunoblot

and immunohistochemical analysis of isolated placental cellsand amniotic membranes, and biopsies of placental and fetalmembranes revealed that TIM1 was expressed consistentlyand uniformly, regardless of individual donor and gestationalage, and was maintained during differentiation. In contrast, wefound that Axl and Tyro3 cofactors varied substantially acrossdonors, gestational age, and differentiation state. The consistentand strong expression of TIM1 on fetal membranes in mid- andlate-gestation placentas and on numerous cell types at the uter-ine-placental interface and early gestation chorionic villus ex-

plants, together with the susceptibility of proliferating andinvasive CTBs and Hofbauer cells to infection, suggests thatTIM1 may be an important cofactor for ZIKV infection. Thisconcept was supported by our results showing that duramy-cin—a cyclic peptide inhibitor that exclusively binds PE in thevirion envelope, thus presumably precluding TIM1 binding ofPE in the virion envelope—inhibits ZIKV infection of TIM1-ex-pressing cells at the uterine-placental interface. In addition, atrophoblast cell line that does not express Axl is highly infectedby ZIKV (Bayer et al., 2016; Miner et al., 2016), suggesting thatAxl is not essential for infection. However, whether duramycin’seffect on ZIKV infection is mediated solely by TIM1 remains to beshown and is the subject of ongoing experiments. Duramycinalso blocks infection of dengue, Ebola, andWest Nile viruses (Ri-chard et al., 2015), reported to bind TIM1 as a cofactor for virionentry (Kondratowicz et al., 2011; Moller-Tank et al., 2013), sug-gesting utility of duramycin as a broad-spectrum antiviral thatcould have clinical application for congenital ZIKV. Interestingly,a TIM1 polymorphism is associated with susceptibility to severehepatitis A virus infection in certain populations (Kim et al., 2011),but other forms predispose toward inflammation associated withasthma and allergy (Gao et al., 2005).Whether genetic variants ofTIM1 expressed by pregnant women infected with ZIKV areassociated with severe congenital disease remains to be deter-mined. Additionally, whether Axl, which we found varies inexpression by donor, gestational age, and tissue, plays a rolein ZIKV infection during pregnancy is unclear given the limited

Figure 7. A Model of Uterine-Placental Interface Indicating ZIKV Infection of Numerous Cell Types and Possible Routes of Transmission(Left) Cross-section of the pregnant uterus and anchoring villus with CTBs that remodel blood vessels that channel maternal blood to the placenta surface. The

basic structural unit is the chorionic villus, composed of a stromal fibroblast core with fetal capillaries and Hofbauer cells/macrophages. During early devel-

opment, villus CTBs detach from the basement membrane and either fuse to form STBs, which cover floating villi, or proliferate and aggregate into a column of

extravillus CTBs that differentiate and invade the basal decidua and uterine wall (invasive CTBs). These anchoring villi establish physical connections between

mother and fetus through attachment of CTB cell columns. (Right) The amniochorionic membrane circumscribes the fetal compartment, extending from the

margins of the placenta to surround the fetus, with AmEpCs in direct contact with amniotic fluid. At 15–20 weeks gestation, as the amniotic sac enlarges, the

amniochorionic membranes fuse and contact the parietal decidua, and a subset of CTBs derived from TBPCs in the chorion invade the parietal decidua lining

the uterine wall (invasive CTBs). ZIKV infects primary placental cells that strongly express TIM1 (red). These include proliferating villus CTBs in proximal cell

columns; invasive CTBs, Hofbauer cells, HPFs, and HUVECs in chorionic villi; and TBPCs and AmEpCs in fetal membranes, defining a continuum of susceptible

cells that represent two routes of ZIKV transmission from the basal decidua to chorionic villi and fetal circulation (left), and parietal decidua to amniochorionic

membranes, amniotic fluid, and the fetus (right). Modified from Pereira et al. (2005).


effect of an inhibitor (Holland et al., 2010) against infection in pri-mary placental cells.

In summary, we have shown that ZIKV replicates in primaryhuman placental cells from mid- and late gestation and villusexplants from first-trimester human placentas, suggestingplacental and paraplacental routes of transmission, and thatinfection of these cells is inhibited by the cyclic peptide duramy-cin. Further, we suggest that these ex vivomodel systems can beused to elucidate the molecular mechanisms of ZIKV infectionand test antiviral drugs.

EXPERIMENTAL PROCEDURES

Human Tissue SamplesThis study was approved by the Institutional Review Board of the University of

California, San Francisco (UCSF). Tissue was collected from term deliveries

and elective terminations at UCSF Medical Center. For tissue analysis,

placenta membrane biopsies from 30 donors were fixed in 4% paraformalde-

hyde (PFA) and frozen in optimal cutting temperature compound (OCT; Tissue

Tek), as described previously (Tabata et al., 2012), or fixed in 10% formalin,

embedded in paraffin, and sectioned for immunohistochemistry and histology

(McDonagh et al., 2006; Pereira et al., 2014).

Primary Human Cells, Cell Lines, and Cell CultureAmEpCs were isolated from early (16.5–23.6 weeks; n = 10) and late (38.6–

41.0 weeks; n = 6) gestational-age fetal membranes and cultured according

to published methods (Miki et al., 2005) with minor modifications. AmEpCs

were cultured on fibronectin (Sigma-Aldrich) in DMEM/F12 (GIBCO) with

20 ng/mL epidermal growth factor (EGF; R&D Systems), 10% fetal bovine

serum (FBS; Invitrogen), nonessential amino acids, 55 mM2-mercaptoethanol,

and Antibiotic-Antimycotic (Thermo Fisher Scientific). Only P1 cells 100% im-

munopositive for cytokeratin 19 were used. TBPCs from human chorion (7.3

and 15.6 weeks), provided by Susan Fisher (UCSF), were cultured on gelatin

in DMEM/F12 (1:1) with 10 ng/mL basic fibroblast growth factor (bFGF; R&D

Systems), 10 mM SB431542 (Tocris Bioscience), and 10% FBS (Genbacev

et al., 2011). TBPCs 100% immunopositive for cytokeratin (MAb 7D3) (Damsky

et al., 1992) and GATA4 were used up to P15. CTBs were isolated from

second-trimester (15.6–21.1 weeks; n = 7) and third-trimester placentas

(37.2 weeks; n = 1), as previously described (Fisher et al., 2000; Tabata

et al., 2007), and cultured on Matrigel (BD Biosciences) in serum-free medium:

DMEM/F12 (1:1) with 2% Nutridoma (Roche Diagnostics), 1% sodium pyru-

vate, 1% HEPES, penicillin/streptomycin (P/S), and gentamycin (UCSF Cell

Culture Facility) (Fisher et al., 2000). Human placental fibroblasts (HPFs;

8 weeks) were grown in DMEM/M199 (4:1) with amino acids and 10% FBS.

Trophoblast cell lines JEG-3 and HTR8, a gift fromMichael S. Diamond (Wash-

ington University in St. Louis), were grown in DMEM/F12 and RPMI 1640 with

10% FBS. HUVECs were grown in EBM-2 with EGM-2 SingleQuots (Lonza).

Cells 98% immunopositive for von Willebrand factor (Novocastra Labora-

tories), P4–P6, were used. Human adult retinal pigment epithelial cells

(ARPE-19) (Dunn et al., 1996) were grown in DMEM (GIBCO) with 10% FBS.

BHK-21 clone 15 cells (gift from S. Kliks) were grown in MEM with 5% FBS,

nonessential amino acids, 2 mM L-glutamine, and P/S (GIBCO). Aedes

albopictus C6/36 cells (gift from Ralph Baric, University of North Carolina,

Chapel Hill) were grown in MEM-a with 2% FBS and 1% P/S (GIBCO) at

32"C with 5% CO2.

ZIKV Strains and Infection In VitroStocks of prototype (Uganda 1947, MR766; resequenced, GenBank:

KX421193), a kind gift from Michael S. Diamond (Washington University in

St. Louis), and Nica1-16 and Nica 2-16 strains at low passage were propa-

gated in C6/36 cells. Cell-free supernatants were harvested 3 dpi, aliquotted,

and stored at #80"C. Nica1-16 and Nica 2-16 were isolated from ZIKV-in-

fected patients by the National Virology Laboratory, Ministry of Health, Man-

agua, Nicaragua, using C6/36 cells. Full-length sequencing by MiSeq

confirmed them to be closely related to Brazilian 2015–2016 strains (GenBank:

KX421195 and KX421194). Cells were infected with MR766 at MOI 0.001–3

and maintained up to 10 days. AmEpCs (21.2, 22.6, 38.6, and 40.2 weeks)

were infected with Nica1-16 or Nica2-16 at MOI 0.001–0.009. CTBs were in-

fected with MR766 (MOI 1–2) 16 hr after isolation and plating on Matrigel

and maintained for 3 days (Yamamoto-Tabata et al., 2004).

Placental Villus ExplantsNormal human placentas (7.5–8.4 weeks; n = 4) were obtained from elective

termination of pregnancy (AdvancedBioscienceResources). Chorionic villusex-

plants were isolated and cultured as anchoring villi as described previously (Ta-

bata et al., 2012). Explants were cultured on Millicell-CM inserts (0.4 mm pore

size, Millipore) coated with Matrigel in DMEM/F12 (1:1) with 10% FBS, P/S,

and amino acids. Two or 24 hr after attachment to Matrigel, explants were in-

fectedwithZIKVMR766 (13106PFU/explant) orNica1-16 (53103PFU/explant)

andmaintained for 2–3 days before fixation and embedding (Tabata et al., 2012).

ZIKV TitrationInfectious virions were titered by plaque assay (PA), as described previously

(Diamond et al., 2000) with minor modifications, or FFA on BHK-21 cells.

Focus-forming units (FFUs) were stained with anti-envelope (E) MAb 4G2

(Henchal et al., 1982), anti-mouse IgG-HRP (Jackson Laboratories) secondary

antibody, and TrueBlue (KPL) substrate. Titers were also determined by rapid

immunofluorescence-based infectivity assay in human ARPE-19 cells, as

previously described for HCMV (Zydek et al., 2014), using anti-E antibody.

Antibodies and ReagentsMAb 4G2 was obtained from ATCC, and anti-DENV non-structural protein 3

(NS3) MAb E1D8 was generated by the E.H. laboratory (Balsitis et al., 2009).

See the Supplemental Information for additional details.

Immunofluorescence StainingSee the Supplemental Information for details.

Immunohistochemical StainingSections from 30 donors were stained with hematoxylin and eosin or immuno-

stained (Pereira et al., 2014). Antigen retrieval was performed for all antibodies

using 10 mM sodium citrate (pH 6.0) under high pressure and heat.

ImmunoblottingSee the Supplemental Information for details.

Inhibitor StudiesFor Axl kinase inhibition, confluent AmEpCs grown in 24-well plates were incu-

bated with R428 (0.03–3.0 mM; ApexBio) for 1 hr at 37"C, 5% CO2, then

replaced with 250 mL of R428-containing medium with virus (MR766 or

Nica2-16 at MOI 0.014). After 2 hr at 37"C, 250 mL medium was added to

each well, and plates were incubated overnight. Medium was then replaced,

and cells were incubated 1–2 days and fixed (4% PFA) for immunostaining

with anti-NS3 antibody. Total and NS3-positive cells were counted, and the

percentage of cells infected was determined. For duramycin inhibition, virus

was incubated in 250 mL medium containing duramycin (0.04–1.0 mM;

Sigma-Aldrich) for 1 hr, and was then added to confluent cells. After 2 hr

at 37"C, 5% CO2, 250 mL medium was added to each well, and cells were

incubated, fixed, and analyzed as above.

Statistical AnalysisSee the Supplemental Information for details.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

two figures, and three tables and can be found with this article online at

http://dx.doi.org/10.1016/j.chom.2016.07.002.

AUTHOR CONTRIBUTIONS

Conceptualization, L.P., E.H., and T.T.; Methodology and Investigation, T.T.,

H.P.-G., M.P., D.M., C.W., and J.F.-H.; Writing, L.P., E.H., T.T., M.P.,

H.P.-G., and D.M.; Funding Acquisition and Supervision, L.P. and E.H.


ACKNOWLEDGMENTS

We thank Saira Saborio and Dr. Angel Balmaseda at the National Virology Lab-

oratory of the Nicaraguan Ministry of Health and the entire study team of the

Nicaraguan Pediatric Dengue Cohort Study. We are grateful to Magelda Mon-

toya and Alejandro Ramirez for their technical assistance. This work was sup-

ported by grants from the NIH Institute for Allergy and Infectious Diseases:

R01AI046657 (L.P.), P01AI106695 (E.H.), and R01AI124493 (E.H.).

Received: June 18, 2016

Revised: July 7, 2016

Accepted: July 8, 2016

Published: July 18, 2016

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