STx of ZIKV from AIR mice to Ifnar1 −/− mice

In a previous study, analysis of ZIKV infected AIR mice at the clinical time point, 14–17 dpi, showed virus infection of the brain and testes by in situ hybridization (ISH) and immunohistochemistry (IHC) staining12. To determine if virus was present in the testes of AIR mice prior to, or at the onset of clinical disease, testes were analyzed for infectious virus by plaque assay. Virus titers peaked at 12 dpi with a slight decrease by approximately ½ a log by 16 dpi (Fig. 1A). ISH and IHC in the testes of infected AIR mice demonstrated infection and replication primarily in germinal spermatogonia and primary spermatocytes and to a lesser extent sertoli stromal cells, which was not observed in IgR mice (Fig. 1B). Thus, infected AIR male mice have infectious virus in the testes early during infection.

Figure 1 ZIKV infection in the testes of AIR mice. (A) Plaque-forming units (PFU) of ZIKV in testes of infected normal mouse IgG-treated Rag1 −/− (IgR) and AIR treated mice at 8, 12 and 16 dpi, detected as described in the methods. No virus was detected in the testes of IgR-treated mice. Statistical analysis was completed by One-way ANOVA, with Tukey post-test. *P < 0.05, **P < 0.01. (B) ISH labeling for (top panels) genomic RNA, (middle panels) replicative RNA intermediates and immunohistochemical labeling for ZIKV (bottom panels) in the testes of infected IgR and AIR mice. Full size image

To determine if ZIKV could be transmitted sexually, ZIKV-infected male AIR mice were mated with naïve Ifnar1 −/− females between 8–12 dpi or 12–16 dpi (Fig. 2A). As a control for incidental transmission, Ifnar1 −/− female mice were co-housed with ZIKV-infected AIR female mice. Following mating, males were removed and Ifnar1 −/− females were weighed every other day as a measure of clinical disease. Of the 11 Ifnar1 −/− females mated with the AIR mice at 8–12 dpi, two became pregnant and gained weight throughout the experiment (Supplementary Figure 1A). Four mice (36.4%) lost more than 20% of starting body weight indicating clinical disease (Fig. 2B,C: black lines, Ψ indicates euthanized). Another four mice (36.4%) had initial weight loss beginning between 7–9 dpi, but regained weight by the experimental endpoint (red lines). These mice never lost more than 10% of starting body weight. One mouse did not lose weight (dotted green line). Thus, 8 of 11 (72%) Ifnar1 −/− female mice bred with ZIKV-infected AIR male mice that did not become pregnant showed some form of weight loss, suggesting sexual transmission of ZIKV. In contrast, none of the Ifnar1 −/− mice co-housed with female AIR mice lost weight (Fig. 2B,C: dashed blue lines).

Figure 2 STx of ZIKV in AIR mice. (A) In two replicate experiments, AIR male and female mice were infected with ZIKV intraperitoneally (i.p.). At 8 dpi, these male and female AIR mice were mated/co-housed with Ifnar1 −/− female mice for 4 days at the times indicated. (B,C) Ifnar1 −/− female mice were then monitored for pregnancy and weight loss for 20 days post mating (dpm). Data are shown as individual percent starting body weight of Ifnar1 −/− females bred with AIR mice. Plots are color-coded to indicate weight loss groups. Ψ indicates mice euthanized due to clinical disease. Two mice became pregnant during these experiments and are shown in Supplementary Figure 3. (D–F) At 20 dpi, (D) brain, (E) spleen and (F) plasma from Ifnar1 −/− mice were analyzed for viral RNA or NAb. (D–E) quantitative real-time (qRT) PCR analysis of individual mice for viral RNA in (D) brain and (E) spleen. Dotted line indicates sensitivity level of assay for each tissue. (F) Inhibitory dilution of neutralizing antibody (NAb) for each animal. No NAb titer was detected for the mouse that was mated with no weight loss (green symbol) or co-housing control mice. One mouse could not be tested in the mated (clinical) group due to lack of plasma sample. Full size image

Additional Ifnar1 −/− females were mated with the same AIR males in Exp. 1, but a later point (12–16 dpi) to determine if virus could also be transmitted at later stages of infection. Interestingly, none of these Ifnar1 −/− females lost weight (Supplementary Fig. 1C). Furthermore, they did not have detectable virus in any tissue examined (data not shown). One mouse failed to gain weight throughout the experiment (Supplementary Fig. 1C, green symbols) and one mouse did become pregnant and had normal weight gain (Supplementary Fig. 1C, red symbols). Thus, ZIKV-infected AIR male mice can sexually transmit virus to Ifnar1 −/− female mice, but transmission occurs primarily during the early 8–12 dpi time point.

Detection of ZIKV RNA and NAb in non-clinical Ifnar1 −/− females following STx

To confirm ZIKV infection in female Ifnar1 −/− mice, brain and spleen tissue was assayed for ZIKV RNA by qRT PCR and plasma viremia and neutralizing antibody (NAb) was measured (Fig. 2D–F). Clinical Ifnar1 −/− mice that lost greater than 20% starting body weight (black symbols) had high levels of viral RNA in brain and spleen tissue, correlating with ZIKV infection (Fig. 2D,E). Interestingly, Ifnar1 −/− mice that showed initial weight loss (red symbols) had detectable viral RNA in spleens, albeit 10–100 fold lower than clinical mice (Fig. 2E). Virus was not detected in spleens from the no weight loss Ifnar1 −/− mouse or from control mice (data not shown). In correlation with detectable virus, Ifnar1 −/− mice with clinical disease or initial weight loss had high levels of NAbs against ZIKV (Fig. 2F). In contrast, the mouse with no weight loss (Fig. 2C, dotted green line) and co-housing control mice (Fig. 2B,C dashed blue line) had no ZIKV NAb (Fig. 2F), suggesting they were never infected. Additionally, one of the two pregnant mice from these experiments (Supplementary Fig. 1B) had a detectable NAb response. None of the mice analyzed had viremia at the time of sampling (data not shown). Together, these findings suggest that even in mice that did not develop severe clinical disease, ZIKV infection was detectable as indicated by initial weight loss and viral RNA in the spleen. The presence of NAb was also suggestive of infection, although not conclusive as exposure to viral antigen may also initiate a response.

Ifnar1 −/− mice bred to infected AIR males at 12–16 dpi did not lose weight (Supplementary Fig. 1C), however, the mouse that failed to gain weight (Supplementary Fig. 1C,D green symbols) and the mouse that became pregnant (Supplementary Fig. 1C,D red symbols) had detectable NAb indicating they were infected at a low titer or were exposed to ZIKV antigen. Collectively, these experiments suggest that STx occurs with high frequency in this model at the early time point (9 of 13 mice at 8–12 dpi, including pregnant mice), and less frequently at the later time point (2 of 6 mice at 12–16 dpi). Interestingly, some of the Ifnar1 −/− mice infected with virus by STx controlled virus replication after initial weight loss, indicating that this route of infection can be suppressed in the absence of type I IFNs.

ZIKV infection of vaginal tract correlates with development of clinical disease

We next analyzed whether ZIKV could be detected in the vaginal tract following STx. 12 naïve female Ifnar1 −/− mice were mated with ZIKV-infected AIR mice at 8–12 dpi. Vaginal swabs taken at 0, 5, 9–10 and 12–14 days post mating (dpm) were analyzed by real-time PCR to determine if virus could be detected in the vaginal tract. At 5 dpm, four mice were positive for virus (Table 1), with two swabs showing detectable viral RNA (+), and two swabs having viral RNA equivalent to 103–104 PFUs (++). By 9–10 dpi, 3 of these 4 mice remained positive for virus in the vaginal swabs and showed signs of clinical disease (Fig. 3A, black, red lines). An additional two mice were positive for virus at 9–10 dpm, but did not shown clinical signs until 12 dpm (Fig. 3A, blue lines). One mouse that did not develop clinical disease, but had a detectable NAb response (Table 1, XZ186-2) experienced weight loss at 11 dpm but recovered (Fig. 3B, red squares). All other mated Ifnar1 −/− female mice did not demonstrate remarkable weight loss (Fig. 3B). Thus, positive vaginal swabs were a strong indicator for development of clinical signs with 6 of 7 mice that had positive swabs developing clinical disease (Table 1). These data also show that STx of ZIKV is associated with detectable virus in the vaginal tract, which strongly correlated with the development of clinical disease.

Table 1 Detection of virus in vaginal swabs of Ifnar1 −/− mice mated to ZIKV-infected AIR males. Full size table

Figure 3 Detection of virus in vaginal tissues following STx. (A–B) Weights of (A) clinical and (B) non-clinical mice described in Table 1. Data are shown as individual percent starting body weight, similar to that shown in Fig. 2B–C. (A) Mouse in red symbol/line (XZ188-2) was determined to be pregnant at the time of clinical disease. (C–E) ISH and (F) IHC of (C,E) uterus and (C,D,F) placental tissue from XZ188-2. (C) Low magnification of uterus, placenta and fetus from XZ188-2 stained for genomic RNA (brown). Red line is drawn to indicate fetal tissue, which was not positive for either genomic or intermediate viral RNA. (D–E) Higher magnification of (D,Di) placental and fetal or (E,Ei) uterus tissue showing detection of ZIKV (D,E) genomic RNA or (Di,Ei) intermediate RNA. Intermediate sections were stained in adjacent sections to those used for genomic RNA. (F) Placenta tissue from XZ188-2 showing viral protein (NS5) as well as active Caspase 3 staining (Casp 3) in localized areas of the placenta. Full size image

Pregnancy associated with STx of ZIKV can lead to infection of uterine and placenta tissue

Of the 30 Ifnar1 −/− females mated to ZIKV-infected AIR males, four mice became pregnant (Supplementary Fig. 1A,C). Three of the four pregnant mice had NAb against ZIKV (Supplementary Fig. 1B,D), but only one mouse, (XZ188-2, Exp. 3) showed signs of clinical disease. Analysis of the uterus from this mouse at the time of clinical disease showed detectable genomic and intermediate viral RNA in cells in the uterus (Fig. 3C,E,Ei), and placenta (Fig. 3C,D,Di,). IHC analysis also demonstrated numerous apoptotic cells (Fig. 3F) in areas of virus infection in the placenta. Fetal tissue was also found (Fig. 3C,D, red line), but there was no clear evidence of infection (Fig. 3C,D,Di). Two of the other pregnant mice had detectable NAb to ZIKV, despite a lack of clinical disease, indicating that they were at least exposed to virus antigen (Supplementary Fig. 1B–D). However, further analysis of these mice indicated no viremia and that viral RNA was not detected in the brain or spleen of the animals or in the placenta or brain of the resulting pups (data not shown). Thus, in the relatively few cases of pregnancy from ZIKV-infected AIR male mice, there was no clear demonstration of VTx. However, there was evidence of infection of both the uterus and the placenta. Therefore, ZIKV infection in AIR male mice leads to sufficient levels of virus in the testes that results in STx of virus and can result in infection of maternal tissues, including reproductive tissues.

VTx of ZIKV to fetuses in AIR female mice

In addition to the potential of VTx transmission of virus to the fetus following STx, VTx of ZIKV can also occur following peripheral infection of ZIKV. To examine what effect IFN suppression and deficiency of the adaptive immune response had on VTx following peripheral infection, we infected female AIR mice impregnated by Rag1 −/− males with ZIKV at 7 dpm (Fig. 4A). Pregnant, ZIKV-infected AIR mice had similar weight gains as pregnant, ZIKV-infected IgR mice (Fig. 4B). Non-pregnant AIR mice started showing initial weight loss at ~12–13 dpi, with a 20% loss in weight (clinical disease) by 17 dpi (Fig. 4B). Thus, ZIKV-infection did not appear to dramatically affect pregnancy-induced weight gain in AIR mice, allowing for analysis of how ZIKV affects fetal development without complications of severe weight loss in the dam.

Figure 4 VTx of ZIKV in AIR mice. (A) Rag1 −/− mice were bred for 3 days and infected with 104 ZIKV i.p. at 7 dpm. Mice were treated i.p. with 1 mg of either normal mouse IgG or αIFNAR1 antibody on −1, 1, 3, 7 and 11 dpi and followed for weight loss and for delivery of pups. (B) Percent of starting body weight of ZIKV infected IgR or AIR non-pregnant or pregnant mice. (C) qRT PCR analysis of brain tissue from (left graph) fetal or (right graph) neonatal mice from pregnant mice in (B). The different symbol shapes/colors indicate individual litters. Full size image

To determine if VTx occurred in this model, three litters from IgR mice and eight litters from AIR pregnant mice (Fig. 1B) were analyzed for VTx of ZIKV. Two litters from IgR and four litters from AIR mice were analyzed prior to birth at 11–13 dpi (approximately embryonic day18-19) and one and four litters, respectively, were analyzed post birth (14–17 dpi, post-birth days 1–3). Visually, all fetuses/pups were unremarkable in terms of overall size and head size. Three pups per litter were used for histological analysis and the remaining pups used for quantitative real-time (qRT) PCR detection of viral RNA. Analysis of brain tissue showed 3 of 26 (12%) of the fetuses (Fig. 4C) and 9 of 17 (53%) of the born pups (Fig. 4C), were positive for ZIKV RNA. Interestingly, not all fetuses/pups from the same litter were positive for virus (Fig. 4C, pups in same litter designated with same symbol). In total, 11 of 43 (26%) brains from fetuses/pups were positive for ZIKV RNA (Fig. 4C). These results indicate that ZIKV can be transmitted from dam to fetus in this model, but that the occurrence of VTx varies between fetuses in the same dam.

Virus detection in CNS and lymph nodes of fetuses following VTx

Histological and morphological analysis of brains from fetuses/pups demonstrated grossly normal development (Fig. 5A). However, viral RNA (Fig. 5B,C) and antigen (NS5) (Fig. 5E,F) were found in multiple areas of the developing CNS including the neocortical ventricular zone, superior colliculus, pretectum, tegmentum, thalamus, medulla and cervical spinal cord (Fig. 5A–C and data not shown). ZIKV CNS infection was not widespread, but found typically in single or small groups of cells (Fig. 5A–C and E,F). Co-staining demonstrated ZIKV infection was in early neuroprogenitor Sox2+ cells (Fig. 5E) and not mature NeuN+ neurons (Fig. 5F). Thus, VTx of ZIKV in AIR mice resulted in focal infection of ZIKV in neuroprogenitor cells of the CNS.

Figure 5 ZIKV infection in pups/fetuses from infected pregnant AIR mice. (A) Low magnification image of ZIKV ISH labeled section with nuclear counterstain of a whole-mount fetus (~E18) and associated placenta from an infected, pregnant AIR mouse. Notice the intense labeling in the placenta on the left side of the image. Areas outlined in boxes correspond to high magnification images of ZIKV positive cells in the (B,C) pretectum of the brain and the (D) pharyngeal/cervical region of the embryo. Dark signal associated with the eye is the developing retinal pigment epithelium and not ZIKV RNA as control animals also contained this signal (not shown). The scale bar shown in (A) pertains to (A–D). (E,F). IHC of medulla tissue from a neonatal pup. Tissue was labeled with antibodies against ZIKV NS5 (green) and co-stained with (E) Sox2 (magenta) or (F) NeuN (magenta). DAPI stain was used to visualize cellular nuclei. Scale bars are labeled. Full size image

Analysis of a whole fetuses/pups showed virus RNA outside the CNS in two of 11 animals, one with infection of the pharyngeal/cervical region (Fig. 5A) and another in a lymph node rostral to the salivary gland (Supplementary Fig. 2A–D). Further analysis using ISH primers that bind replicative RNA intermediates of ZIKV also detected positive signal in the lymph node (Supplementary Fig. 2C), indicating active virus replication and not just phagocytosis of virus particles. IHC for Iba1+ macrophages showed strong staining of this region, correlating with the identification as a lymph node (Supplementary Fig. 2D). Although only two of 11 animals had detectable virus outside the CNS, these findings do demonstrate that VTx can result in infection of other fetal tissues besides the CNS.

ZIKV infection of maternal placental tissue

One of the barriers to fetal infection by ZIKV may be the placenta. Placental tissue from all fetal tissue were analyzed by real-time PCR (Fig. 6A) or IHC and ISH (Fig. 6B–D). Additionally, three placentas were recovered from pups at birth (Fig. 6A) and were used for RNA analysis. All placental tissue from ZIKV-infected AIR mice, regardless if taken pre- or post-birth, were positive for ZIKV RNA, while placental tissue from ZIKV-infected IgR controls were negative (Fig. 6A). ISH and IHC of adjacent sections of placental tissue showed that genomic (Fig. 6B) and replicative intermediates (Fig. 6C) of ZIKV RNA as well as ZIKV NS5 protein (Fig. 6D) were associated with spongiotrophoblast cells of the maternal junctional zone of the placenta (Fig. 6B–D, outlined in 6D). In contrast, virus was largely excluded from fetal placental tissues, with only a few positive signs of ZIKV protein (Fig. 6D and d, white arrows). However, viral RNA was observed in direct apposition to fetal capillary endothelial cells of the labyrinth zone (Fig. 6B–C, inserts). These data suggest active infection and viral replication in areas directly adjacent to the fetal blood supply which is consistent with trans-placental VTx in mice11.

Figure 6 ZIKV infects the placenta in pregnant AIR mice. (A) qRT PCR analysis for ZIKV RNA in placental tissues from pregnant, ZIKV infected IgR and AIR mice. Red symbols represent placental tissues from fetal mice, while blue symbols are from newborn pups. (B–D) Staining of serial placenta sections from an ~E18 pup from an AIR mouse for (B) ZIKV genomic RNA and (C) replicative RNA intermediates by ISH or (D) ZIKV NS5 protein (green) and Iba1(magenta) by IHC. (D) NS5 labeling is found primarily within the maternal junctional zone (delimited by yellow lines) with a few exceptions (white arrow). Arrows in the insets demonstrate positive ZIKV RNA signal in areas directly opposed to the fetal blood. (b,c) White arrow in (d) represents NS5 staining in fetal placenta. Scale bars are shown as the length of the side of the boxes used to indicate insets (b,c) or are directly shown in the image (D). Full size image

VTx of ZIKV is not associated with breakdown of placental barrier

One potential explanation for the VTx of ZIKV in some fetuses and not others within the same dam could be the breakdown of the maternal/fetal barrier in the placenta. To directly examine if this barrier was breached in fetuses with ZIKV infection, we injected three pregnant dams with Evans Blue dye at embryonic day 17–18. Placentas and fetuses from these dams were analyzed for Evans Blue leakage into the umbilical cord and fetus. Interestingly, no Evans Blue dye was detected in the umbilical cord or fetuses by gross histological examination, while the placenta showed clear Evans Blue staining (Fig. 7A,B) compared to untreated controls (Fig. 7C,D). IHC analysis of fetal CNS tissues from some of these fetuses detected ZIKV NS5 staining in Sox2+ cells in the neocortical ventricular zone (Fig. 7E), suggesting that virus infection of the fetus occurred in the absence of placental barrier breakdown. Thus, ZIKV is vertically transmitted in AIR mice, but this may be an active process between maternal and fetal cells, rather than a passive leakage due to breakdown of the placental barrier.