ZIKV-002 macaques challenged with ZIKV MR766 are protected from heterologous reinfection with ZIKV-FP.

<p><b>A.</b> Study timeline with dates of primary and secondary, heterologous ZIKV challenges. Samples were collected daily from 0 to 10 dpi, and then weekly thereafter until secondary challenge (denoted by ticks along the timeline). Challenge stocks were derived from the East African and French Polynesian virus strains detailed above the timeline. <b>B.</b> Plasma viral loads, shown as vRNA copies/mL for each of the macaques challenged with 1 x 10⁶ (solid green line), 1x 10⁵ (solid orange line), or 1 x 10⁴ (solid blue line) PFU/mL of ZIKV MR766 challenge stock from the date of primary challenge through 10 days post heterologous challenge with ZIKV-FP. For comparison of plasma viral loads between ZIKV strains, solid light grey lines depict the plasma viral load trajectories for animals that were challenged with the same dose of ZIKV-FP and then rechallenged with homologous ZIKV-FP [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005168#pntd.0005168.ref022" target="_blank">22</a>]. <b>C.</b> Oral swab and <b>D.</b> pan urine viral loads.</p

An N-linked glycosylation site in envelope is rapidly selected in vivo.

<p>Envelope sequences from the three animals were sequenced at three days post infection, and from two of the animals at day six post infection. A Muscle alignment of the translated sequences was generated in Geneious. Dots represent identity to the consensus sequence. Dashes represent deletions. Capital letters represent amino acids. Only regions of the E protein with sequence variants are depicted. <b>A.</b> E protein amino acid positions 136–178. The frequencies of the deletion and the restored deletion are shown below each of the stock sequences, with the indicated site boxed. Amino acid variant frequencies matching the variant sites in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005168#pntd.0005168.g001" target="_blank">Fig 1A</a> are shown. The gray ellipse above the sequence alignment represents the 150 loop of the E protein [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005168#pntd.0005168.ref020" target="_blank">20</a>]. <b>B.</b> E protein amino acid positions 271–313. <b>C.</b> E protein amino acid positions 361–450. There were two additional nonsynonymous variants at greater than 5% in animal 562876 at day three, and the frequency of the amino acid variants from the other animals and time points are shown below each sample.</p

Summary of virus stocks and culture history.

<p>All Zika virus strains are the MR 766 prototype strain derived from the virus that was isolated from a sentinel rhesus monkey in Zika Forest, Entebbe, Uganda in April 1947[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005168#pntd.0005168.ref006" target="_blank">6</a>]. All have undergone extensive mouse brain passage. The MR766 challenge stock was created for nonhuman primate natural history studies and was derived from the CDC virus. Challenge virus was prepared by inoculation of CDC virus onto a confluent monolayer of C6/36 mosquito cells and a clarified harvest of the culture medium was collected nine days post infection.</p

East African ZIKV MR766 envelope sequences often contain an in-frame deletion of an N-linked glycosylation site and are heterologous with respect to Asian ZIKV.

<p>The amino acid sequences of the Envelope protein for six ZIKV MR766 Genbank sequences were aligned to the consensus amino acid sequences of the three ZIKV MR766 stock viruses (Chal Stck, CDC Stock, and WRCEVA stock) using a Muscle alignment in Geneious. Dots represent identity to the consensus sequence. Dashes represent deletions. Only sections of the E protein with variations are shown, all other parts of the E protein showed no variation. Capital letters represent amino acids. The frequencies of the deletion and the restored deletion are shown below each of the stock sequences. Genbank reference sequence AY632535 had two amino acids that were different from the other reference sequences. The frequency of reads with these amino acid variants as determined by deep sequencing are shown below each of the stock sequences. <b>A.</b> Envelope protein amino acid region 136–178. The gray ellipse above the sequences represent the 150 loop of the E protein [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005168#pntd.0005168.ref020" target="_blank">20</a>]. <b>B.</b> Envelope protein amino acid region 271–313.</p

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques

<div><p>Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10–12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.</p></div

Complete blood counts (CBCs) and serum chemistries for pregnant macaques infected with ZIKV.

<p>Animals were infected with 10⁴ PFU of ZIKV. Animals infected in the first or third trimesters are represented by color coding (<b>A</b>) as presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006378#ppat.1006378.g001" target="_blank">Fig 1</a>. All animals had CBC analysis performed on EDTA blood and chemistry analysis performed on serum at -7, -3, 0, 1–10 and additional indicated dpi. <b>B.</b> AST blood chemistries, <b>C.</b> ALT serum chemistries, <b>D.</b> CK serum chemistries, <b>E.</b> WBC counts, <b>F.</b> % lymphocytes, <b>G.</b> red blood cell (RBC) counts.</p

Fetal growth following ZIKV infection.

<p>Growth curves of femur length (FL), biparietal diameter (BPD), and head circumference (HC) obtained from fetal ultrasound images throughout gestation are presented as individual lines or symbols with specific colors as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006378#ppat.1006378.g001" target="_blank">Fig 1</a>. (<b>A</b>) FL, (<b>B</b>) BPD (<b>C</b>) and HC were determined for the fetuses in this study and plotted against data from Tarantal [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006378#ppat.1006378.ref035" target="_blank">35</a>], which is presented as the mean (solid black line) and 1, 2, and 3 standard deviations from the mean as grey lines above and below the mean. The data from the last month of pregnancy are also presented as a magnified view of the scatter of individual data points on the right. Representative ultrasound images of FL, BPD, and HC are also shown at the right.</p

Fetal growth as assessed by predicted gestational ages.

<p>The predicted gestational age (pGA) as described by Tarantal [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006378#ppat.1006378.ref035" target="_blank">35</a>] from each of the pregnancies is plotted against the actual day of gestation estimated from breeding activity and animal menstrual records. The pGA was derived from the average of BPD+FL (dashed lines), or the HC (solid lines). <b>A</b> (animal 827577) and <b>B</b> (animal 660875), first trimester infection. <b>C</b> (animal 357676) and <b>D</b> (animal 598248), late second/early third trimester infection.</p

Study layout and viral RNA burden in pregnant rhesus fluids.

<p>(<b>A</b>) Schematic representation of the timeline of infection, sampling for maternal viral burden, and experimental cesarean section, for all animals in the study. Animals received a ZIKV challenge in the first or late second/early third trimesters of pregnancy, and blood and other fluid samples were collected according to the schedule indicated in detail in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006378#ppat.1006378.s001" target="_blank">S1 Fig</a>. (<b>B</b>) ZIKV viral load in pregnant macaque fluids. Viral RNA loads (vRNA copies/ml) measured in plasma, urine, saliva, and amniotic fluid presented individually for the four pregnant animals. The day post-inoculation is indicated below each graph, and gestational age (days) for each animal is indicated above (term = 165±10 days). Limit of assay quantification is 100 copies/mL. Limit of detection is 33 copies/mL. Colors for individual animals are continued through the rest of the Figures, including the Supplementary Figures.</p

Immunohistochemical localization of ZIKV in fetal [and maternal] tissues.

<p>(<b>A</b>) Immunofluorescent staining for ZIKV NS2B (red) and macrophage marker CD163 (green) in fetal axillary lymph node with a high vRNA burden. The white scale bar = 100 μm. (<b>B</b>) H&E stained near section of the tissue presented in 9A. (<b>C</b>) Nonspecific immunostaining with control isotypes for ZIKV NS2B and CD163.</p