The recent Zika virus (ZIKV) outbreak in Brazil has been directly linked to increased cases of microcephaly in newborns. Current evidence indicates that ZIKV is transmitted vertically from mother to fetus. However, the mechanism of intrauterine transmission and the cell types involved remain unknown. We demonstrate that the contemporary ZIKV strain PRVABC59 (PR 2015) infects and replicates in primary human placental macrophages, called Hofbauer cells, and to a lesser extent in cytotrophoblasts, isolated from villous tissue of full-term placentae. Viral replication coincides with induction of type I interferon (IFN), pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. Our results suggest a mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting placental cells and disrupting the placental barrier.

Here we demonstrate that primary human HCs, and to a lesser extent CTBs, are permissive to productive infection by a contemporary strain of ZIKV, closely related to the strains currently circulating in Brazil. Upon infection, HCs are modestly activated and produce IFN-α and other pro-inflammatory cytokines. Analysis of antiviral gene expression shows upregulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) transcription as well as downstream antiviral effector genes, indicating that ZIKV induces an antiviral response in HCs and CTBs. Our results suggest that ZIKV gains access to the fetal compartment by infecting and proliferating in the cells of the placenta.

Vertical transmission of ZIKV from an infected mother to the developing fetus in utero reflects tropism for placental cells. This organ is a target for a number of viruses by direct and contiguous infection of the cell layers, virion passage through a breach, or cell-associated transport. Examples include rubella, cytomegalovirus, herpes simplex, HIV-1, hepatitis B and C virus, and parvovirus B19 (). The placenta is characterized by contact between the maternal blood and fetal chorionic villi. Each villus is lined by trophoblasts, which encase the fetal blood supply and placental macrophages (Hofbauer cells [HCs]). Several studies have confirmed HCs are targets of viral infection in vivo () and in vitro (). In contrast, syncytiotrophoblasts (differentiated cytotrophoblasts [CTBs]) have been shown to be resistant to infection by a wide range of viruses (). A recent study showed that syncytiotrophoblasts also appear to be resistant to infection by phylogenetically related, historic ZIKV strains at early times following infection (24 and 48 hr post-infection [hpi]) ().

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has rapidly spread to over 30 countries in the Americas and causes illness with symptoms of fever, rash, joint pain, and conjunctivitis (). ZIKV is transmitted through several routes, including mosquito bites, sexual contact, and blood transfusion (). Most notably, ZIKV can be vertically transmitted from an infected mother to the developing fetus in utero, resulting in adverse pregnancy outcomes that include fetal brain abnormalities and microcephaly, a condition characterized by a reduction in head circumference that is often associated with delayed or arrested brain development (). The mechanism by which ZIKV crosses the placenta to establish infection in the developing fetus is not well understood. Recent studies have identified ZIKV RNA in amniotic fluid and fetal and newborn brain tissue (), and ZIKV-specific IgM antibodies have been detected in newborn cerebrospinal fluid (). Additionally, ZIKV antigen was found in the chronic villi of a human placenta from a mother who gave birth to an infant with microcephaly, and ZIKV RNA has been isolated from placental tissue of mice infected with ZIKV (). Finally, a recent study detected ZIKV antigen in placental tissue from a mother diagnosed with ZIKV disease (). In particular, ZIKV antigen was detected in placental macrophages and histiocytes in the intervillous space.

The kinetics of the antiviral response are complex and variable, and we observed donor-to-donor variation in induction of antiviral gene expression. Of note, HCs from donor 2, which exhibited the highest viral loads, and donor 5, which exhibited the lowest viral loads, induced similar levels of antiviral effector genes by 96 hpi, although genes in donor 2 were induced at a faster rate ( Figure 4 ). This may reflect the higher rate of replication and viral output by HCs from this donor ( Figure 1 ). There is likely a multifactorial rationale for why viral load does not correlate with antiviral gene expression that likely encompasses differences in individual genetics and the antagonistic capabilities of the virus.

To evaluate the antiviral potential of HCs and CTBs, we examined the expression of several antiviral effector genes. We observed increased expression of IFNA transcripts as early as 24 hpi in HCs ( Figure 4 A), concordant with increased IFNα secretion ( Figure 3 ). While we did not observe IFNβ secretion, we detected an increase in IFNB1 transcripts over time-matched mock cells as early as 24 hpi ( Figure 4 A), suggesting possible discordance between transcript levels and translation/secretion of IFNβ (). In contrast, both IFNA and IFNB1 were induced at low levels in CTBs ( Figure S3 B). We next measured expression of the RLRs, a family of PRRs known to recognize flavivirus RNA and induce production of type I IFNs and pro-inflammatory cytokines (). Expression of DDX58 (RIG-I), IFIH1 (MDA5), and DHX58 (LGP2) transcripts is induced above time-matched, mock-infected HCs across all donors by 72 hpi and remains highly expressed through 96 hpi ( Figure 4 B). RLR expression corresponds to kinetics of virus replication, suggesting that RLRs are induced in response to ZIKV infection of HCs. In CTBs, RLR transcription is modestly induced, and both IFIH1 and DHX58 return to near basal levels by 96 hpi, though DDX58 expression remains slightly elevated through 96 hpi ( Figure S3 B). We also evaluated expression of several antiviral genes produced downstream of the RLR and type I IFN signaling axes and found that RSAD2, IFIT1, IFIT2, IFIT3, and OAS1 were all induced by 72 hpi in HCs and remained elevated through 96 hpi ( Figure 4 C). In CTBs, these genes were modestly induced through 72 hpi ( Figure S3 B), likely corresponding to the low level of viral replication during this time period ( Figure 1 ). By 96 hpi, a time point at which we observed productive virus replication, these cells also initiate an antiviral immune response. Importantly, we observed low levels of IFNA and ISG expression in mock-infected HCs and CTBs, likely induced by the cell isolation procedure, which may limit the percent of infected cells we see in our in vitro system. Taken together, these results show that both HCs and CTBs respond to ZIKV infection through initiation of antiviral signaling pathways.

HCs from five donors were infected with ZIKV (PR 2015) at an MOI of 1, and antiviral gene expression determined by qRT-PCR. Gene expression data are represented as fold change relative to time-matched, mock-infected controls (gene expression normalized to GAPDH − ΔΔCmethod). Individual donors are depicted as separate bars, organized from donor 1 to donor 5, within each time point block. Viral titers determined in Figure 1 are represented as a separate heat map below each group of genes. (A) shows type I IFNs, (B) shows RIG-I-like receptors, and (C) shows antiviral effector genes. hpi, hours post-infection.

When cells are infected with virus, pattern recognition receptors (PRRs) within the cell recognize the viral genetic material and trigger a potent innate immune response to control viral replication and spread. Upon binding viral RNA, PRRs initiate signaling cascades that result in the production of type I interferons (IFNs) and pro-inflammatory cytokines, and expression of antiviral effector genes that serve to limit virus replication. In order to further assess the immunostimulatory potential of HCs, we measured pro-inflammatory cytokines and chemokines in supernatants from infected cells by multiplex bead array. Following ZIKV infection, we observed increased IFNα secretion, but not IFNβ or IFNλ1 (IL-29; Figure 3 Table S1 ). We also found increased secretion of the pro-inflammatory cytokine IL-6 and chemokines MCP-1, involved in monocyte infiltration, and IP-10, involved in recruitment of activated effector T cells. Though these cytokines were induced in all five donors, there were individual differences in the magnitude of production. Donor 2, which had the highest viral load at 48 and 72 hpi ( Figure 1 A), tended to exhibit the highest overall levels of IFN-α, IL-6, MCP-1, and IP-10; however, donor 2 was not consistently the lead producer of cytokines over mock-infected controls. Of note, donor 5, which had the lowest viral load at 48 and 72 hpi, did not consistently show the lowest levels of cytokines, but did exhibit reduced induction over mock-infected controls at 72 hpi. No discernable patterns could be confidently drawn with CXCL-8, MIP-1α, MIP-1β, or IL-1RA. In contrast to HCs, we observed limited induction of type I IFN, IL-6, and IP-10, and no detectable type III IFN in CTBs at the time points assessed ( Figure S3 A; Table S2 ). Donor 1, while slightly less permissive to viral infection and replication ( Figure S1 ), did not have correspondingly lower levels of cytokine production compared to donors 2 and 3. We did observe, however, that donor 1 tended to have reduced production of cytokines over mock-infected control cells at 72 hpi. These findings demonstrate that HCs are capable of initiating an inflammatory response to ZIKV infection.

HCs from five donors were infected with ZIKV (PR 2015) at an MOI of 1, or mock infected. Cytokine levels in the supernatants were determined by multiplex bead array. All values are represented in pg/mL and shown with a connecting line between ZIKV-infected samples (48 and 72 hpi) and their respective donor- and time-matched, mock-infected samples. See also Table S1

Next, to determine if ZIKV-infected HCs are poised to interact with T cells, we measured cell surface expression of the co-stimulatory molecules CD80, CD86, and MHC II. In ZIKV-infected HCs from all three donors, we observed minimal upregulation of both CD80 and CD86 as compared to time-matched, mock-infected cells between 48 and 72 hpi ( Figures 2 B and 2C). Consistent with enhanced virus replication, ZIKV infection of HCs from donor 2 led to upregulation of both CD80 and CD86 by 72 hpi. Additionally, significant upregulation of MHC II was only observed with donor 2 between 48 and 72 hpi ( Figure 2 D). Overall, there appears to be donor-to-donor variability in terms of upregulation of co-stimulatory molecules; however, enhanced virus replication led to greater activation of HCs. These data suggest that ZIKV infection has the potential to program HCs for antigen presentation and T cell priming.

Of note, percent infectivity and infectious virus production did not necessarily correspond to viral RNA levels ( Figures 1 and 2 A). Specifically, while donors 1 and 2 had a 6-fold difference in cellular infectivity at 48 hpi and a consistent 1-log-fold difference in infectious virus release between 24 and 96 hpi, both had similar viral RNA levels present at 48 and 72 hpi. Differences in infection between donor 1 and 2 may be explained by an enhanced rate of genome replication within HCs from donor 2, noted by an early increase in viral RNA at 24 hpi in donor 2, but not donor 1 ( Figure 1 ). Overall, we observed variable levels of viral RNA at 24 and 48 hpi, despite similar levels of viral RNA at early (3 hpi) and late (48 and 72 hpi) time points, further supporting differential rates of genome replication between donors. Indeed, while donors 1, 3, and 4 had similar production of infectious virus at all time points assessed, notable differences in viral RNA levels were observed at 48 hpi between these donors ( Figure 1 ). Furthermore, while donor 5 showed minimal production of infectious virus, we observed comparable RNA levels to the more permissive donors, further highlighting discordance between genome replication and release of infectious virus. Together, these results suggest that different donors may have the capacity to differentially regulate ZIKV replication and may be restricting replication at different stages of the viral life cycle.

To assess ZIKV replication in HCs at the single-cell level, flow cytometry was utilized to detect intracellular expression of viral E protein. Consistent with peak production of viral RNA and infectious virus ( Figure 1 ), we detected 0.8%–6.8% and 0.4%–3.0% infected HCs at 48 and 72 hpi, respectively ( Figure 2 A). Minimal background staining was observed in donor- and time-matched uninfected cells and in ZIKV-infected cells stained with an IgG isotype control ( Figure S2 B). Consistent with our FFA findings, HCs isolated from donor 2 were the most permissive to infection, with an average of 5.6% and 2.3% infected cells at 48 and 72 hpi, respectively. This is consistent with infected cell counts observed by immunofluorescence microscopy ( Figure 1 E). In contrast to recent studies with neuronal progenitor cells (), we did not observe a significant loss of viability during ZIKV infection through 96 hpi ( Figure S2 C), suggesting that these cells may be more resistant to virus-induced cell death or that ZIKV (PR 2015) is a less cytopathic virus in HCs.

(B–D) Surface expression of (B) CD80, (C) CD86, and (D) MHC II was determined by flow cytometry. Data are represented as median fluorescence intensity (MFI). Horizontal bars indicate the mean of four technical replicates. Representative histograms are provided (right panels). hpi, hours post-infection. See also Figure S2

(A) HCs from three donors were infected with ZIKV (PR 2015) at an MOI of 1, or mock infected. Percentages of infected cells at 48 and 72 hpi were determined by intracellular viral E protein staining and flow cytometry (left panels). Horizontal bars indicate the mean of four technical replicates.

In contrast, we observed minimal viral replication in CTBs at early times post-infection (3–72 hpi; Figure S1 A, available online). Of note, we found evidence of productive infection at 96 hpi, with all three donors exhibiting approximately 5-fold increase in viral load between 72 and 96 hpi, suggesting that CTBs may support productive virus infection, albeit at lower levels compared to HCs. We observed concurrent increases in viral RNA in all three donors between 72 and 96 hpi as well ( Figure S1 B). Most notably, we detected persistent viral RNA in CTBs at all time points through 72 hpi, further suggesting ZIKV infects and replicates in CTBs with delayed kinetics. Collectively, these findings demonstrate that HCs are permissive to ZIKV infection and represent a key target cell of ZIKV infection within the placenta.

To determine whether human placental cells are permissive to ZIKV infection, we isolated primary HCs and CTBs from villous tissue of full-term placentae and infected them with ZIKV (MOI 1). In this study, we used a low cell-culture-passaged and sequence-verified ZIKV strain, PRVABC59 (PR 2015), isolated from the sera of an infected patient in Puerto Rico in December 2015. This strain is closely related to the epidemic strains circulating in the Americas that have been linked to in utero ZIKV infection (). Through multiple virologic assays, we demonstrate that HCs, and to a lesser extent CTBs, are permissive to productive ZIKV infection ( Figure 1 ). Following infection of HCs, we performed a focus forming assay (FFA) on Vero cells and observed a steady decline in viral titers from 3 hpi through 24 hpi that was immediately followed by log phase virus growth through 72 hpi ( Figure 1 A). Notably, we observed donor-to-donor variation in viral kinetics and magnitude among HCs isolated from five donors. For donor 2, we detected an approximately 35-fold increase in virus in the supernatant between 3 and 48 hpi. In contrast, donor 5 showed about a 2.5-fold increase in virus in the supernatant between 48 and 96 hpi. We confirmed infection of HCs with viral qRT-PCR ( Figure 1 B) and immunofluorescence microscopy ( Figures 1 C–1E). In HCs, viral RNA was substantially increased in all donors by 48 or 72 hpi, reflecting an increase in virus release into the supernatant ( Figure 1 A). Furthermore, we detected viral envelope (E) protein, which localized to distinct, perinuclear regions within infected HCs ( Figures 1 C and 1D). This pattern may be indicative of viral localization to the endoplasmic reticulum (ER), or ER-associated vesicles, a staining pattern consistent with virus assembly (). Finally, we observed between 4.9% and 7.2% infected cells by immunofluorescence staining using a pan-flavivirus antibody ( Figure 1 E).

(A) HCs from five donors were infected with ZIKV (PR 2015) at an MOI of 1, and viral titers in supernatants determined by FFA. Viral inoculum for all donors was 1 × 10 6 ffu/mL. Data are represented as the mean of four technical replicates ± SD (top). Representative FFA staining (bottom). ffu, focus forming units.

Discussion

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Honein M.A. Projecting month of birth for at-risk infants after Zika virus disease outbreaks. The present data demonstrate that primary HCs and CTBs isolated from full-term placentae are permissive to productive ZIKV infection by a contemporary strain currently circulating in the Americas. We also found that HCs respond to infection by triggering antiviral defense programs in the absence of overt cell death. In this limited study of five donors, we observed individual variability in kinetics and magnitude of virus replication, inflammation, and antiviral gene expression, likely reflecting differences in individual genetics (). Though unlikely given the low number of cell passages PR 2015 has undergone, it is possible that minor cell culture adaptations or quasi-species may also be playing a role in donor-to-donor variability. These observations suggest that donors may have the capacity to restrict ZIKV at different stages of the viral replication cycle. This may also relate to observed differences in intrauterine transmission efficiency, where more susceptible HCs from a pregnant mother may support higher levels of virus replication and subsequent spread to the developing fetal nervous system. Additionally, it will be important in future studies to characterize when HCs and CTBs are most susceptible to ZIKV infection (i.e., first, second, or third trimester). Recent projections from the CDC based on data from Brazil indicate that virus infection during the first trimester or early in the second trimester of pregnancy is temporally associated with the observed increase in infants born with microcephaly ().

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Coyne C.B. Type III interferons produced by human placental trophoblasts confer protection against Zika virus infection. A recent study reported that primary syncytiotrophoblasts isolated from full-term placentae are resistant to ZIKV infection through a potential mechanism involving type III IFN-mediated antiviral immunity (). Similarly, in CTBs we observed a lack of productive virus replication through 48 hpi; however, we did observe persistent viral RNA through 72 hpi. By 96 hpi, we observed low-level virus replication as well as induction of antiviral effector genes, suggesting that ZIKV infects and persists in CTBs but is efficiently controlled at early times post-infection. Additionally, while Bayer et al. were able to identify IFN-λ (type III IFN) in the supernatant of uninfected syncytiotrophoblasts, we did not detect the presence of IFN-λ in the supernatants of ZIKV-infected HCs or CTBs. The discordance between these two studies may be attributed to differences in time points assessed and viral isolates used in each study (FSS13025 and MR766 as compared to PR 2015).

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et al. Notes from the field: evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses—Brazil, 2015. What are the possible mechanisms by which ZIKV crosses the placental barrier and infects HCs? One explanation is that ZIKV may initially infect trophoblasts and productively replicate and disseminate locally within the placenta to involve HCs, which then support more efficient ZIKV replication than CTBs. An alternative hypothesis is that non-neutralizing, cross-reactive antibodies bind ZIKV and traffic across the placenta, through a neonatal Fc-receptor-mediated mechanism, to infect placental macrophages. ZIKV crossing the placenta and replication in/release from HCs likely result in viral dissemination through the cord blood with subsequent infection of neural progenitor cells. At this time, it is uncertain whether maternal macrophages are infected or play a role in allowing ZIKV to cross the placental barrier. However, a recent report has directly identified the presence of viral antigen through immunohistochemistry in the placenta from a mother with an infant who developed ZIKV-related fetal anomalies (). Of note, ZIKV antigen was detected within the chorionic villi and not in the maternal decidua. Based on these findings, it does not appear that decidual macrophages are key players in ZIKV transmission at the placenta.

HCs are likely programmed to limit inflammation following virus infection, a mechanism that is consistent with the immune-tolerant environment of the placenta and which would support higher infection of HCs compared to maternal macrophages. An alternative hypothesis is that the relative paucity of effector cells in the placenta that would otherwise readily kill infected macrophages (e.g., CD8+ T cells) contributes to a permissive environment for ZIKV infection and replication in HCs. Altogether, our data support the notion that HCs represent a key target cell within the placenta. These findings stress the importance of developing antiviral therapies directed against ZIKV replication within placental cells as a means to reduce vertical transmission in the mother-infant dyad and the incidence of adverse pregnancy outcomes and fetal abnormalities.