Study Overview 
Investigator: O'Connor and Osorio Grant: NIH P51 to WNPRC and NIH R01 supplement 3R01AI116382-01A1S1 to O'Connor


ZIKV-003 tracks data in 32 datasets over 558 time points. Data is present for 2 Participants.

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ZIKV-003: Infection with French Polynesian Zika virus during the first pregnancy trimester 

Primary objectives

  • Assess whether fetal development is impacted by maternal infection with French Polynesian Zika virus during the first pregnancy trimester
  • Measure concentration of Zika virus RNA in amniotic fluid and virus transmission to the fetus
  • Pilot methods for studying Zika virus infection during pregnancy

Study design

A single pregnant Indian-origin rhesus macaque (approximately 25-30 days post-conception) will be challenged subcutaneously with 10E4 PFU French Polynesian Zika virus. Natural history of Zika virus will be evaluated as in ZIKV-001. Additionally, we will quantify virus in amniotic fluid at multiple timepoints and monitor fetal development by ultrasound imaging.

Result summary

A pregnant rhesus macaques was infected with Zika virus on March 7, 2016

Participants

  • 827577 challenged with 10E4 PFU Zika virus/H.sapiens-tc/FRA/2013/FrenchPolynesia-01_v1c1
Real-time editorial study commentary

Clinical and Assay Data

Viral challenge stock analysis

Viral RNA quantification

  • Plasma viral loads Chart
  • Pan urine viral loads Chart
  • Oral swab viral loads Chart
  • Amniotic Fluid viral loads Chart

Clinical

Immunology

Ultrasounds Data

Fetectomy Tissues Data | Histopathology

(Note: When more than one plot is available, scroll down to see all plots. Click 'TestId' in the bottom right corner to show/hide specific plots.)


Neutralizing Antibody Titers 

Key data: plasma viral loads 

Key data: urine viral load 

Ultrasounds post infection 
FilterGrid ViewsReportsChartsGroupsExportPrintPagingView Specimens
Participant ID
Date
Comments
File Name
 8275772016-03-07Preinfection ultrasound. Fetus is in the middle. One placental disk (lower) is outlined for area estimation. The other disk you can see above and to the left of the fetus.Placenta disk 2 d0.jpg
8275772016-03-14day 7 post infection031416_day_7_postinfection.jpg
8275772016-03-16Ultrasound image 9 days postinfection. The growing fetus is indicated by a yellow arrow. Fetus and placentas are looking healthy.031616_d9_postinfection.jpg
8275772016-03-18day 11 post infection031816_d11_postinfection.jpg
8275772016-03-21day 14 post infection032116_d14_postinfection.jpg
8275772016-03-23day 16 post infection3.23.16_d16_post-infection_fetal_brain.jpg
8275772016-03-23day 16 post infection3.23.16_d16_post-infection_head_measurements.jpg
8275772016-03-23day 16 post infection3.23.16_d16_post-infection_placental_discs.jpg
8275772016-03-23day 16 post infection3.23.16_d16_post-infection_unannotated.jpg
8275772016-03-23day 16 post infection3.23.1_d16_post-infection_fetal_head_circumference.jpg
8275772016-03-28longitudinal view of ZIKV-003 d21 post infection032816_21d_postinfection_827577.jpeg
8275772016-03-30HC_BPD827577_3.30.0030_HC_BPD.jpg
8275772016-03-30FL_Placenta827577_3.30.0031_ZIKV_003_FL_Placenta.jpg
8275772016-03-30Summary827577_3.30.0033_ZIKV_003_Summary.jpg
8275772016-04-01Image of ZIKV-003 fetal head. Day 25 post infection.040116_d25_post_infection_827577.jpeg
8275772016-04-08Sagittal view of fetal head 32 days post infection, approximately 80 days of gestation4.8.16_827577_A1.jpg
8275772016-04-12blood flowZIKV003_4.12.16_blood_flow.jpg
8275772016-04-12cisterna magnaZIKV003_4.12.16_cisterna magna.jpg
8275772016-04-12fetusZIKV003_4.12.16_fetus.jpg
8275772016-04-12head circumferenceZIKV003_4.12.16_head_circumference.jpg
8275772016-04-12heart rateZIKV003_4.12.16_heart_rate.jpg
8275772016-04-12measurementsZIKV003_4.12.16_measurements.jpg
8275772016-04-12nuchal translucencyZIKV003_4.12.16_nuchal translucency.jpg
8275772016-04-12placentaZIKV003_4.12.16_placenta1.jpg
8275772016-04-12placenta 2ZIKV003_4.12.16_placenta2.jpg
8275772016-04-16 4.19.16_HC_BPD.jpg
8275772016-04-19 4.19.16_Plac.jpg
8275772016-04-19 data_summary_041916.jpg
8275772016-04-19HC_BPDIM-0001-0001_ZIKV_003_4_19_2016_HC_BPD.jpg
8275772016-04-19FLIM-0001-0031_ZIKV_003_4_19_2016_FL.jpg
8275772016-04-26 ZIKV_003_4.26.16 _BPD-HC.jpg
8275772016-04-26CerebellumZIKV_003_4.26.16_cerebellum.jpg
8275772016-04-26Femur LengthZIKV_003_4.26.16_femur_length.jpg
8275772016-04-26HeartbeatZIKV_003_4.26.16_heartbeat.jpg
8275772016-04-26PlacentaZIKV_003_4.26.16_placenta.jpg
8275772016-04-26SummaryZIKV_003_4.26.16_summary.jpg
8275772016-04-26VentricleZIKV_003_4.26.16_Ventricle.jpg
8275772016-05-03Femur LengthZIKV_003_5.3.16_femur_length.jpg
8275772016-05-03 ZIKV_003_5.3.16_HC-PBD.jpg
8275772016-05-03Heart RateZIKV_003_5.3.16_heart_rate.jpg
8275772016-05-03PlacentaZIKV_003_5.3.16_placenta.jpg
8275772016-05-03SummaryZIKV_003_5.3.16_summary.jpg
8275772016-05-03VentricleZIKV_003_5.3.16_Ventricle.jpg
8275772016-05-10HeartbeatZIKV_003_5.10.16_IM-0001-0001.jpg
8275772016-05-10Femur LengthZIKV_003_5.10.16_IM-0001-0006.jpg
8275772016-05-10BPD/HCZIKV_003_5.1.0.16_IM-0001-0011.jpg
8275772016-05-17HCZIKV_003_827577_IM-0001-0003.jpg
8275772016-05-17HeartbeatZIKV_003_827577_IM-0001-0007.jpg
8275772016-05-17FemurZIKV_003_827577_IM-0001-0008.jpg
8275772016-05-17PlacentaZIKV_003_827577_IM-0001-0009.jpg
8275772016-05-17SummaryZIKV_003_827577_IM-0001-0011.jpg
8275772016-05-17BPDZIKV_003_IM-0001-0001.jpg
8275772016-05-24BPDZIKV_003_5_24_2016_BPD_IM-0001-0001.jpg
8275772016-05-24FLZIKV_003_5_24_2016_FL_IM-0001-0004.jpg
8275772016-05-24HCZIKV_003_5_24_2016_HC_IM-0001-0002.jpg
8275772016-06-07BPD/HCIM-0001-0002_ZIKV_003_BPD_HC.jpg
8275772016-06-07Heart RateIM-0001-0005_ZIKV_003_HeartRate.jpg
8275772016-06-07FLIM-0001-0009_ZIKV_003_FL.jpg
8275772016-06-07PlacentaIM-0001-0012_ZIKV_003_placenta.jpg
8275772016-06-07SummaryIM-0001-0014_ZIKV_003_summary.jpg
8275772016-06-14Heart RateIM-0001-0001_ZIKV_003_6_14_HeartRate.jpg
8275772016-06-14Femur LengthIM-0001-0003_ZIKV_003_6_14_FL.jpg
8275772016-06-14BPD/HCIM-0001-0007_ZIKV_003_6_14_BPD_HC.jpg
8275772016-06-14SummaryIM-0001-0008_ZIKV_003_6_14_summary.jpg
8275772016-06-21Heart RateIM-0001-0001_ZIKV_003_6_21_HeartRate.jpg
8275772016-06-21Femur LengthIM-0001-0003_ZIKV_003_6_21_FL.jpg
8275772016-06-21PlacentaIM-0001-0008_ZIKV_003_6_21_placenta.jpg
8275772016-06-21PlacentaIM-0001-0013_ZIKV_003_6_21_placenta.jpg
8275772016-06-21SummaryIM-0001-0016_ZIKV_003_6_21_summary.jpg
8275772016-07-01BPD_HCIM-0001-0005_ZIKV_003_7_1_2016_BPD_HC.jpg
8275772016-07-01Heart RateIM-0001-0010_ZIKV_003_7_1_2016_Heartrate.jpg
8275772016-07-01FLIM-0001-0012_ZIKV_003_7_1_2016_FL.jpg
8275772016-07-01PlacentaIM-0001-0015_ZIKV_003_7_1_2016_PLACENTA.jpg
8275772016-07-01SummaryIM-0001-0018_ZIKV_003_7_1_2016_Summary.jpg

Messages 
Showing: all messages
Re-breeding follow up
lgomes 2017-08-22
827577 participant: birth on 7-27-2017 and baby seems to be fine.
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Consistency
david h oconnor 2016-03-15
In a nice bit of reproducibility, the pregnant animal had detectable virus in urine after a several day delay, exactly as we observed in ZIKV-001. Plasma viremia appears to have peaked. Viral loads from vaginal and oral swabs should be available later in the week.



-dave
view message
Successful infection
david h oconnor 2016-03-09
The ZIKV-003 macaque is infected. Plasma viremia is climbing through day 2 (today). The next viral loads will be run in a batch on Monday (I think).



-dave
view message
Summary Comparison of Female Reproductive System in Human and the Cynomolgus Monkey (Macaca fascicularis)
david h oconnor 2016-03-09
How similar are macaque and human pregnancies? The attached file has a useful comparison (h/t Ted Golos). The source document can be found at: http://tpx.sagepub.com/content/36/7_suppl/171S.full.pdf+html and I believe it is available without a subscription.



In terms of timing, the average macaque pregnancy is about 160 days long.



-dave
 Toxicol Pathol-2008-Van Esch-171S-2S.pdf 
view message
Visualizing Zika virus infection
david h oconnor 2016-03-07
ZIKV-003 is the first time we have infected a pregnant macaque with Zika virus. We are, once again, hoping to make our data available in real-time. I am aware that sharing ultrasound imagery of developing macaques can elicit stronger emotions than looking at relatively sterile charts and tables. It certainly did for me and our staff who saw the first ultrasound today. These are not experiments I expected to perform as part of my career as an infectious disease researcher, but once again, as with HIV/AIDS, my lodestone is that a relatively small number of animals used in research may have an important role in preventing human suffering due to Zika virus infection. I feel like I remember every pregnant woman I saw on the street when Dawn and I visited Rio de Janeiro two weeks ago. The idea that some of them may have their pregnancies and families threatened by Zika virus is heartbreaking.



I want to thank those who have viewed our data so far and have chosen not to use it out of context to further arguments against using animals in research. I hope that even those who disagree with me, and our research, acknowledge that we have common ground in trying to make data as available as quickly and as widely as possible to minimize the number of animals that need to be used in research.



-dave
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What is the source of virus causing prolonged viremia? 

Pregnant macaque 827577 has been viremic for >28 days as of this writing (compared to ~10 days in non-pregnant macaques). Her viral loads will be measured again today at day 35. Identifying the source of the virus leading to this prolonged viremia is an important question. There are two basic explanations we have considered:

  • 827577 is immunosuppressed due to pregnancy and does not mount an effective immune response against Zika virus. Thus, the virus is able to persist longer than in non-pregnant, immunocompetent macaques
  • During initial viremia of 827577, Zika virus infected the fetus. Zika virus is replicating in the fetus and being shed back into 827577's bloodstream

These results are still preliminary, but two lines of experimental evidence support the second explanation.

1. Sustained virus detection in 827577 is confined to the blood

In other Zika virus infected macaques, virus is detected in urine beginning a few days after it is detected in blood. Resolution of viruria happens at about the same time as virus can no longer be detected in blood. In 827577, urine is cleared with typical kinetics, even though plasma viremia is sustained. If 827577 was immunosuppressed, we would expect to see continued shedding in multiple fluids due to incomplete immunologic containment.

2. Viral variant compartmentalization

We recently obtained viral sequence from days 2, 7, and 21 post-infection in 827577 [note: we also have day 5 sequence but have reason to believe this sample may have been accidentally switched with another sample on the same sequencing run]

For those interested in the raw data, as well as the Zequencer output files:

Day 0 (challenge stock) R1_FASTQ | R2_FASTQ | BAM (BAI - FASTA) | VCF

Day 2 R1_FASTQ | R2_FASTQ | BAM (BAI - FASTA) | VCF

CAUTION! CAUTION! CAUTION! Day 5 R1_FASTQ | R2_FASTQ | BAM (BAI - FASTA) | VCF CAUTION! CAUTION! CAUTION!

Day 7 R1_FASTQ | R2_FASTQ | BAM (BAI - FASTA) | VCF

Day 21 R1_FASTQ | R2_FASTQ | BAM (BAI - FASTA) | VCF

High quality variants (>5% of reads; p<10e-60; strand-bias 10e-5 when exceeding 65% bias) were identified in each dataset relative to the challenge stock consensus sequence [Geneious | Genbank] using the Zequencer workflow in Geneious Pro 9.1.2.

There are 9 high quality variants at day 7. I tabulated the frequencies of these exact same variants at the other timpeoints like this:

daysTrack NameMinimumChangeVariant FrequencyAmino Acid ChangeProtein Effect
0Variants: 827577-inocula (without duplicates)3,219T -> C1.40%L -> SSubstitution
2Variants: 827577-Day2 (without duplicates)3,219T -> C1.60%L -> SSubstitution
7Variants: 827577-Day7 (without duplicates)3,219T -> C18.80%L -> SSubstitution
21Variants: 827577-Day21 (without duplicates)3,219T -> C0.04%L -> SSubstitution

 If a particular variant is not detected at all at a given timepoint, the variant frequency is set to 0.00%, like this:

daysTrack NameMinimumChangeVariant FrequencyAmino Acid ChangeProtein Effect
0Variants: 827577-inocula (without duplicates)4,502G -> C39.80%E -> QSubstitution
2Variants: 827577-Day2 (without duplicates)4,502G -> C32.30%E -> QSubstitution
7Variants: 827577-Day7 (without duplicates)4,502G -> C9.20%E -> QSubstitution
21 4,502 0.00% synthetic variant

 I then plotted the frequency of high-quality variants at day 7 throughout infection of 827577. Each colored line in the following plot represents a separate high quality variant. Most of the high quality variants detected at day 7 were also present at day 2. Strikingly, none are present at >0.3% of reads at day 21. So these variants, which were enriched during the first 7 days of replication in 827577 all essentially disappear in the virus sequenced at day 21.

I repeated the analysis using the high quality variants detected at day 21. The reciprocal occurs - variants observed at day 21 are almost entirely absent from the day 7 virus. If the day 21 viremia was due to a continuation of the infection from day 7, this would be highly unlikely (we are working with colleagues to put a number on exactly how unlikely). But this is exactly what you might expect if during early maternal viremia, a small number of viruses infect the fetus and replicate as a compartmentalized subpopulation.

This is still a preliminary analysis. There are a few important caveats:

  • We did not have complete genome coverage in all datasets. Therefore we limited our analysis to nucleotides 308-4597 where we have high quality coverage in all the datasets.
  • The day 5 dataset was excluded because it appears to have been switched with another sample on the same instrument run. Even though the results presented here make sense, we are wary of any sequencing experiment with known sample mix-ups
  • Each sample was only sequenced in a single replicate. We would like to perform duplicate or triplicate technical replicates, but sample availability is limiting
  • More timepoints will provide better variant resolution. Day 28 and 35 timepoints, as well as selected earlier timepoints are in the process of being sequenced

Nonetheless, the two complementary types of data both pointing to fetal infection and shedding into the mother are provocative. 

  Attached Files  
   
 Asian-lineage ZIKV PF 2013 challenge stock consensus sequence.geneious
 Zequencer-workflow.geneiousWorkflow
 Asian-lineage ZIKV PF 2013 challenge stock consensus sequence.gb
 17302-02-day-7-plot.png
 17302-03-21-days-plot.png