Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication

Abstract

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

Figure 1. Phylogenetic characterization of ZIKV-FLR and demonstration of its replication in primary human trophoblasts.

(A) Phylogenetic tree of full genome nucleotide sequences of all 77 currently available ZIKV strain complete genome or polyprotein CDS assemblies (accessed June, 2016); ZIKV-FLR strain is shown bolded in blue and annotated as Colombia_H_sapiens_2015_1. Sequences were aligned using MAFFT v7.0 (Katoh; ref. 23) with the accurate (L-INS-i) setting. PAUP* v4.0 (Swofford; ref. 24) automated model selection was used to identify optimal likelihood parameters based on the best AICc. A Neighbor Joining tree was constructed and the tree was visualized using FigTree v1.4.2 (Rambaut; http://tree.bio.ed.ac.uk/software/figtree/). Leaf nodes were labelled as country_host_year of collection. Methods and accession numbers are provided in Supplementary Materials. In panels B and C, primary human trophoblasts infected with ZIKV-FLR strain were analyzed at 5 days post-infection for the presence of viral replicative complexes (B) and envelope glycoprotein expression (C) by immunofluorescence, in three different unrelated placental donors. (B) Viral dsRNA was detected in primary human trophoblasts using the J2 monoclonal antibody and AF568-conjugated secondary anti-mouse IgG; the J2 antibody is not specific for ZIKV, but only detects viral replicative complex. Both mock-infected trophoblasts and those infected with UV-inactivated 1 × 10⁵ ZIKV (left two panels) failed to demonstrate evidence of dsRNA viral replicative complexes (red labeling). By contrast, presence of replicative viral complexes were observed in primary human trophoblast cultures on day 5 post infection among donors infected with 1 × 10⁵ RNA copies of ZIKV (red labeling on right three panels, with three separate unrelated donor trophoblasts). (C) Viral E-glycoprotein expression, using the 4G2 monoclonal antibody and FITC-conjugated secondary anti-mouse IgG (green labeling) in parallel cultures to panel B: mock-infected cells, UV-inactivated 1 × 10⁵ ZIKV, and 1 × 10⁵ ZIKV in three separate unrelated donor trophoblasts.

Figure 2. Quantitative data demonstrating primary human placental trophoblasts are permissive for ZIKV infection and replication, but not DENV infection nor replication.

(A) In the initial two experiments, trophoblasts were isolated from two unrelated donors (donor 1, donor 2) and plated at 1 × 10⁶ cells/well. On day 5 (donor 1) or day 4 (donor 2) after isolation, now differentiated trophoblasts were infected with ZIKV FLR. (B) Zika and dengue virus growth curves in primary trophoblasts, after infection with 1 × 10⁵ RNA copies per virus. All wells contained 1 × 10⁶ cells, except for one donor, which contained 1 × 10⁵ cells. The average of 16 ZIKV and 12 DENV infections are projected (see Fig. S1). (C) Zika and dengue viral growth curves, by day of trophoblast cell infection post isolation (i.e., ex vivo culture). No differences with ZIKV inoculated cultures were observed 3 days after trophoblast differentiation had occurred (comparing 3 (solid lines), 4 (dashed lines) or 5 (dotted lines) days post isolation). (D) ZIKV infection is dependent upon active viral replicons, as UV treated ZIKV RNA was not detectable 72 hours post infection (p < 0.01, ANOVA with Bonferroni post-hoc).

Figure 3. Putative ZIKV cell entry receptors are expressed in syncytialized primary human trophoblasts.

(A) Select viral receptor targets were measured on day 5 post isolation. Cultured primary trophoblasts exhibit AXL, DC-SIGN, TYRO3 and TIM-1, but not L-SIGN transcription as assessed by RT-PCR detection of human transcript message. Isolated reverse transcribed mRNA was amplified using intron/exon junction spanning primers with a GAPDH as a loading control. For DC SIGN and L- SIGN, primers were designed to capture the multiple transcript variants. Discovered major bands are shown comprising RefSeq transcript variant 1 for DC-SIGN, and transcript variants 1, 8, 9 and 10 (larger band), and variants 7 and 11 (smaller band) for L-SIGN. Isolated buffy coat, A549 cells, isolated primary human hepatocytes and whole human liver were used as controls. (B) Immunofluorescence labeling for the expression and cellular localization of the Axl presumptive ZIKV entry receptor. Representative images of day 5 uninfected trophoblasts were imaged following antibody probing with goat polyclonal antibody and Alexa Fluor^® 647 labeled anti-goat secondary antibody for detection (i). Z-stacks of 20X immunofluorescence images were deconvolved and maximum projected. Areas were abstracted (ii-iii) to visualize cellular localization at the cell membrane. Panel iii is indicated by the boxed area.

Figure 4. Direct visualization of ZIKV viral RNA in primary human placental trophoblasts by single molecule RNA FISH.

(A) Vero cells mock (left) or infected with 10⁵ RNA copies with ZIKV-FLR strain (right) were probed with specific fluorescently-labeled oligo panels to ZIKV RNA at 5 dpi to demonstrate the specificity of the RNA FISH probe set (red). (B) Primary human placental trophoblasts on post-isolation day 4 were also mock (left panels) or infected with ZIKV-FLR strain (right) at 1 × 10⁵ RNA copies, and were fixed at 5 dpi. Images on the right demonstrate the positive strand of ZIKV in infected cells compared to mock controls (left). Select images from six separate experiments captured at 60X/1.42 NA, deconvolved and maximum projected are shown. All staining in both Vero and trophoblast cultures is cytoplasmic, with appearance of overlay reflective of dimensionality.

Figure 5. Significant decrease in the level of the TLR7/8 ligand binding miR-21 miRNA, but not C19MC miRNA species following ZIKV infection in primary human trophoblasts.

Exosomal RNA was isolated from primary trophoblasts of ten donors following ZIKV infection with 1 × 10⁵ viral copies, or mock infected controls. RNA was then isolated from trophoblast cultures 3–5 days post infection. TaqMan qPCR assays were employed for species-specific miRNA quantification. Significant differences in miRNAs were observed for miR-21 (decreased ~1.5 fold (0.68 ± 0.2 SD), p = 0.001), while transcripts of the C19 miRNA cluster were not significantly changed by ZIKV infection. Fold change in miRNA species were calculated by the delta delta Ct method, normalizing first to U6 and then mean delta Ct of mock infected controls. Data was filtered for outliers as designated by a Q of 0.01. Significance was determined using t-tests, with designation of significance annotated by c (c significance p = 0.001).