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

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

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

Molecularly barcoded Zika virus libraries to probe <i>in vivo</i> evolutionary dynamics

<div><p>Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a “synthetic swarm” whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects <i>in vivo</i>, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time <i>in vivo</i> to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.</p></div

Ocular and uteroplacental pathology in a macaque pregnancy with congenital Zika virus infection

<div><p>Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.</p></div

Vector competence of <i>Aedes aegypti</i> following peroral exposure to ZIKV-BC-1.0-infected pregnant macaque 4 days post inoculation.

<p>Vector competence of <i>Aedes aegypti</i> following peroral exposure to ZIKV-BC-1.0-infected pregnant macaque 4 days post inoculation.</p

Number of viral templates used to characterize the full genome sequences of the two ZIKV stocks.

<p>Number of viral templates used to characterize the full genome sequences of the two ZIKV stocks.</p

Amniotic fluid (AF) markers confirm rupture of membranes and contamination of pan-collected urine.

<p>(A) An AmniSure^® test, which measures PAMG-1 protein, was performed on pan urine collection (28 dpi, 45 dpi, 49 dpi) and AF (28 dpi) from the pregnant animal. Nonpregnant control animal urine and pregnant animal AF are included as controls. (B) Relative pixel density of the Amnisure^® test strip test band and control band. (C) Amniotic fluid protein IGFBP-1 ELISA. Body fluids from the pregnant animal (pan urine collection 28, 42, 45, 49 dpi and AF 28 dpi), nonpregnant negative control male and female urine samples, amniotic fluid from a control pregnancy were evaluated for the presence of IGFBP-1. Dashed lines indicate the upper and lower limits of quantitative accuracy of the assay. In Panels B and C, white bars denote body fluids from the experimental animal and grey bars denote control fluids from other animals in the colony.</p

Maternal and fetal necropsy images.

<p>(A) The uterus was removed in entirety from the abdominal cavity of the dam using sterile instruments and a syringe was used to aspirate the purulent fluid from inside the uterine cavity. (B) The fetus was removed from the uterus and was covered in thick fibrinous material. Placental discs 1 (C) and 2 (D) were covered in the same thick fibrinous maternal that covered the fetus.</p