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


ZIKV-001 tracks data in 32 datasets over 463 time points. Data is present for 3 Participants.

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ZIKV-001: Infection of three rhesus macaques with French Polynesian Zika virus 

Primary objectives

  • Assess the infectivity of a Zika virus isolate from French Polynesia at different doses in rhesus macaques
  • Measure concentration of Zika virus RNA in plasma, urine, CSF, saliva, and feces
  • Determine whether immunity elicited by Zika virus infection protects from subsequent re-infection with genetically similar, Asian-lineage Zika viruses

Study design

Three Indian-origin rhesus macaques will be challenged subcutaneously with different doses of French Polynesian Zika virus. For each of the first 10 days, samples will be collected daily for intensive virologic analyses. From days 11-28, samples will be collected 2-3x per week. After 28 days, the animals will be rested for approximately 6 weeks before being re-challenged with 10E6 PFU of French Polynesian Zika virus.

Result summary

All three macaques were successfully infected with Zika virus. Plasma viremia peaked at more than 1e6 viral RNA copies / mL in two of the three animals. Plasma viremia resolved by approximately 10 days post-infection we just saw a blip at day 16 in one animal. In two of three animals, viral RNA was detected in urine slightly longer than in blood. Viral RNA was also detected in saliva from all three animals.

Participants

  • 826226 challenged with 10E6 PFU Zika virus/H.sapiens-tc/FRA/2013/FrenchPolynesia-01_v1c1
  • 393422 challenged with 10E5 PFU Zika virus/H.sapiens-tc/FRA/2013/FrenchPolynesia-01_v1c1
  • 912116 challenged with 10E4 PFU Zika virus/H.sapiens-tc/FRA/2013/FrenchPolynesia-01_v1c1

Clinical and Assay Data

Real-time editorial study commentary

Viral challenge stock analysis

Viral RNA quantification

  • Plasma viral loads Chart
  • Pan urine viral loads Chart
  • Cystocentisis viral loads Chart
  • CSF viral loads Chart
  • Oral swab viral loads Chart

Clinical

Immunology

(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.)


Featured data: plasmablast expansion following Zika virus infection 

This data is courtesy of Josh Eudailey, Tony Moody, and Sallie Permar from the Human Vaccine Institute and Department of Pediatrics, Duke University Medical Center:

Three rhesus macaques (M. mulatta) of Indian origin were inoculated with varying doses of a Zika virus isolated in 2013 from an infected individual in French Polynesia.  Subsequently PBMCs from these NHPs were isolated at 4 timepoints post-infection (days 3, 7, 11, 14) to study the kinetics of the primary humoral immune response during acute infection, focusing specifically on the expansion of the antibody-synthesizing plasmablast population. This population was defined by excluding lineage cells (NK cells, T Cells, B Cells, monocytes, plasmacytoid, and dendritic cells) and selecting for HLA-DR+/CD80+ expression, markers known to be expressed on macaque plasmablasts and their human counterparts (Silveira et al. 2015; Wrammert et al. 2008). This population has been shown to contain antibody-secreting cells but may also contain other cell populations, and so the frequency of these cells observed by flow cytometry may be an overestimate of the circulating plasmablast population (Silveira et al. 2015). NHP 826228 was administered the highest dose (10E6 PFU) of Zika virus and had the highest frequency of circulating plasmablasts at Day 7, whereas NHP 912116 received the lowest dose (10E4) and that animal had its highest frequency of circulating plasmablasts at Day 11.  These preliminary findings may prove useful in the context of vaccine development to determine if the early plasmablast response is capable of eliciting protective Abs against emerging strains of Zika virus. Due to a recently observed association between Zika infected mothers giving birth to infants with microcephaly and other potential viral-associated neurologic effects, it will be critically important to understand Zika virus immunity in mothers infected prior to or during pregnancy to determine whether a vaccine or other intervention may be able to prevent adverse fetal outcomes.

  Attached Files  
   
 Screen Shot 2016-03-12 at 1.09.53 PM.png
 Screen Shot 2016-03-12 at 1.09.42 PM.png
 Plasmablast Figures.pptx
 ZIKV-001-plasmablasts.xlsx


Neutralizing Antibody Titers 

Messages 
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Phew!
david h oconnor 2016-03-15
We are now through the first 28 days of ZIKV-001. The animals will be rested for six weeks before being rechallenged. This will let us assess whether the immune responses elicited by primary challenge are protective. If they are, then in the future we hope to systematically deplete individual lymphocyte subsets (e.g, CD8 T cells) to define the precise immune responses that are mediating control.



-dave
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Plasmablasts
david h oconnor 2016-03-12
Thanks to the hard work of Sallie Permar and colleagues at Duke University, we now have plasmablast data from the ZIKV-001 animals. This defines how quickly antibody producing plasmablasts expand, which will be useful because it tells scientists which timepoints are best for isolating monoclonal antibodies responding to Zika virus.



-dave
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Citation
david h oconnor 2016-03-11
We received a question about how to cite this data. If you use this data in a manuscript, you can cite it like this:



1. Zika experimental science team (2016) ZIKV-001: Infection of three rhesus macaques with French Polynesian Zika virus . Available: https://dholk.primate.wisc.edu/project/dho/public/Zika/public/ZIKV-001-public/begin.view? Accessed 11 March 2016.



Thanks!



-dave
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Citation
david h oconnor 2016-03-11
We received a question about how to cite this data. If you use this data in a manuscript, you can cite it like this:



1. Zika experimental science team (2016) ZIKV-001: Infection of three rhesus macaques with French Polynesian Zika virus . Available: https://dholk.primate.wisc.edu/project/dho/public/Zika/public/ZIKV-001-public/begin.view? Accessed 11 March 2016.



Thanks!



-dave
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Aviremic
david h oconnor 2016-03-09
At the latest timepoint, none of the ZIKV-001 animals had virus detectable by qRT-PCR. We will follow these animals closely until next Monday and then rest them for about six weeks. Then we will try to re-infect them with the same virus. This will tell us whether the immune responses mounted to the first infection confer protection from re-infection with the same virus.



Shelby also has some sequence data from this study to share. A teaser -- there's something peculiar going on in the NS1 gene (we don't know what it means yet, but it is interesting).



-dave
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Simmering
david h oconnor 2016-03-03
Day 17 viral loads are now available. Interestingly, two of the three animals have a blip - one has detectable viral RNA in plasma and the other has detectable viral RNA in urine. This suggests that even after more than two weeks, the infections are not fully resolved. It is possible virus continues to replicate at higher levels in other fluids/tissues.



-dave
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Oops, part 3
(1 response) david h oconnor 2016-03-03
Once again, I'm trying to update the data and am running into problems. Am working to fix as quickly as I can.



-dave
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Zika virus RNA in saliva
tcf 2016-03-02
Today we retrospectively tested saliva samples collected from the 3 infected monkeys by oral swabs. Interestingly, all 3 animals have detectable Zika virus RNA in their saliva at multiple times after infection. This is consistent with previous reports that have detected Zika virus in saliva from infected humans. A few notes about this.



1. As we've noted before, our test detects viral RNA only. (vRNA concentrations are expressed per ml of swab eluate.) We therefore do not know whether there is *infectious* Zika virus in saliva, and if so, how much there is. We will soon determine that using plaque assays. But these results raise the possibility that saliva contains infectious virus.



2. We detect very little viral RNA in oral swabs collected during the first 5 days of infection. This could reflect the real kinetics of virus shedding in saliva, but we suspect it may be an artifact: for the first 5 days of this study, oral swabs were collected into RNALater. Thereafter we began collecting swabs in viral transport medium (VTM), realizing that this would be a better choice for preserving infectious virus. We will continue to collect swabs into VTM from now on.



3. Interestingly, animal 912116 showed the most prolonged shedding of virus in saliva, and in fact was still positive in saliva at day 10. This animal received the lowest dose of Zika virus inoculum (1 x 10^4 pfu) and also shed virus for the longest amount of time in urine among the 3 animals in this pilot.



4. The quantification of viral RNA in oral swabs is more difficult than for other fluids. The exact amount of starting material is unknown and may contain residual cells. Therefore we feel confident saying there is viral RNA in these samples, but the data values should be interpreted with caution, as changes in vRNA concentration could result from differences in starting material and not necessarily "true" changes in the specimens.



-Tom and Dave
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Expanding data sharing
david h oconnor 2016-03-02
I've been surprised at how popular this information is - many thanks to everyone who is reading, and, in many cases, contributing advice and suggestions for improvement. One question I've been asked is why, if scientists favor making data available, more scientists don't do it. There are a lot of reasons, and there is no single right or wrong answer. But one of the obstacles certainly is that a lot of groups don't have the expertise to run a website like this one.



Enter LabKey Software (http://www.labkey.org). Their LabKey Server platform is what I use to make our data accessible. There are many tools available as part of the platform that makes it relatively easy to share text, files, and study data. Building on the success of this site, we are now working together with LabKey to create a repository where others who are working on Zika virus can put their results in real-time. This will also make it easier to find the results from many labs in one easy-to-find place. If you're interested, please contact me and I'll put you in touch with the appropriate people at LabKey.



Thanks!



-dave
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Oops, part 2
david h oconnor 2016-03-01
For a short while, public access was accidentally disabled. It should be fixed now. Again, please bear with us as we reorganize the site so we can share more data when we infect more animals next week.



-dave
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Oops
david h oconnor 2016-03-01
We are trying to prepare the public portal for the second set of challenges which will happen on Monday, but we are having some technical difficulties. Plots should be available again as soon as we fix this.



-dave
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Day 14
david h oconnor 2016-02-29
Today nearly all fluids in all three animals were undetectable by qRT-PCR. As sampling moves to every 2-3 days, our measurements will lose some resolution.



Though beginning next Monday we will be challenging three more macaques with African (Uganda 1947) ZIKV to compare to Asian ZIKV infection.



-dave
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Blip
david h oconnor 2016-02-27
Day 11 showed a blip in plasma viral loads in one of the three animals. Perhaps our conclusion of cleared plasma viremia was premature.



-dave
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NK cell responses to ZIKV through day 9
david h oconnor 2016-02-25
From Eva:



"I think the green shaded data have interesting changes. What we see is a typical acute infection phase with NK cells being the primary responders as a first line of defense. We could confirm that with the increased absolute count of circulating proliferation marker (Ki-67)+ NK cells."



-dave (on behalf of Eva)
 NK through day 9.xlsx 
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Plasma viremia cleared
david h oconnor 2016-02-25
Today is the last day of daily sampling. The animals will now be sampled 2-3x a week until 28 days post-infection. Then the animals will be rested for about six weeks before being rechallenged to see if immune responses against the initial virus protect from subsequent, homologous reinfection.



As of today, plasma viremia is gone from all three animals, but this doesn't mean there won't be blips or recrudescence. We are also planning on resampling the CSF in about a week once we are more confident viral loads are really negative. Still virus in urine from one animal.



Once again, I want to thank everyone on the team who rallied together to collect these results every day for the last 10 days. Especially Gabrielle and Andrea in Tom's lab, who did the qRT-PCR, and Mariel, Meghan, Mustafa, and Dawn in my lab who coordinated sample processing. The Wisconsin National Primate Research Center staff, including Buddy, Nancy, Heather, and Jennifer did a great job of closely monitoring the animals and ensuring their well-being.



-dave
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Near full-length challenge stock sequence
david h oconnor 2016-02-25
Matt Aliota correctly noted that the challenge stock sequences I posted did not have complete UTR sequences. I derived a new consensus sequence from the challenge stock by mapping against a reference that has full-length UTRs. Erwin Camacho from the University of Sucre annotated the updated consensus sequence. We are still missing about 30bp from the end of the 3' UTR, but the 5' UTR is intact. These files in Geneious and Genbank format are attached.



-dave
 ZIKV PF_2013_ZIKV-001-challenge-stock.geneious  PF_2013_ZIKV-001-challenge-stock.geneious.gb 
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Interpreting variation in the ZIKV-001 challenge stock
david h oconnor 2016-02-23
There are 8 nucleotide sites where more than 5% of the reads in the challenge stock were variant (see attached file). Shelby, Dawn, and I initially did not think this was very much variation, but discussions with others suggest perhaps for a flavivirus stock, it is.



-dave
 ZIKV-001-challenge-stock-variants.csv 
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Zequencer and ZIKV-001 challenge stock sequencing
david h oconnor 2016-02-23
We have completed a preliminary analysis of the ZIKV-001 challenge stock. Briefly, viral RNA was prepared from an aliquot of the challenge stock. Double-stranded cDNA was synthesized and fragmented to produce an Illumina sequencing library. Sequencing was performed on an Illumina miSeq.

To analyze the data reproducibly, Shelby and I developed a Geneious Pro 9.1.1 beta workflow, termed the Zequencer, that maps datasets containing ZIKV to a reference genome and calls variants. More details about the Zequencer, as well as installation and usage instructions are located in "Link 1" below. Before going to Link 1, PLEASE be sure to download the Zequencer files, as listed in "Link 2" below.

Zequencer instructions, Link 1: https://goo.gl/pMQMxb

Zequencer files, Link 2: https://bitbucket.org/dhoconno/zequencer.git

Here are the links to sequencing data from the challenge stock:

Raw FASTQ

  1. SampleSheet-R1-25616.fastq.gz
  2. SampleSheet-R2-25616.fastq.gz
Read mapping
  1. BAM file
  2. BAI file
Fasta consensus (it is the same as the Genbank reference KJ776791)
  1. FASTA consensus
Variants
  1. VCF file
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But is it infectious?
tcf 2016-02-22
Just a quick note about our viral detection methods. Here we are using quantitative RT-PCR to detect RNA, the Zika virus genetic material, in blood and other specimens from infected monkeys. We use this method because it is rapid and very sensitive, but it is important to remember that this method cannot tell what proportion of viruses detected are actually infectious. We may find, for example, that viral RNA is detectable in urine, but not much of that virus is actually "live" and capable of infecting new cells. We will attempt to isolate viruses from blood and other specimens to address this issue.



-Thomas F.
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Days 5 and 6
david h oconnor 2016-02-22
Over the weekend, the animal challenged with the highest dose apparently cleared his plasma viremia, though he, as well as the other two animals, all had detectable virus in urine on Sunday (day 6).



Apologies that a few of the immunophenotyping plots aren't working right and that the sequence data isn't posted yet. Will try to fix ASAP.



-dave
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Challenge virus deep sequencing
david h oconnor 2016-02-20
This morning Shelby and Dane received deep sequencing results from the challenge virus. The consensus sequence matches the Genbank sequence. There is one non-synonymous site with ~40% variation and a number of sites with >5% variation. I hope to post raw data as gzip-compressed FASTQ files and read mapping/variant calling in Geneious format later today. Since Dawn and I are leaving for Brazil tomorrow morning and there's a lot going on, it may take a little longer than I'd like.



-dave
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Immunophenotyping under construction
david h oconnor 2016-02-19
We are loading a new and improved version of the immunophenotyping data. It should be available soon.



-dave
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Day 4 viral loads
david h oconnor 2016-02-19
The most interesting note about today's viral loads is the first detection of virus in pan urine. We've been collecting it every day of the study and it was totally negative until today. We also collected and tested CSF for the first time today. There is a possibility of minor carryover from blood, but 2/3 animals had detectable CSF viral loads.



-dave
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Day 3 viral loads
david h oconnor 2016-02-18
Thanks to Andrea Weiler and Tom Friedrich, day 3 viral loads are now available. The macaque challenged with 10E4 PFU now has the highest viral load, while the one challenged with 10E6 seems to be post-peak viremia. Also, virus was detected at very low levels in the animal infected with 10E4 PFU for the first time.



-dave
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Immunophenotyping
david h oconnor 2016-02-17
For the last two days, Mustafa and I have worked with Eva Rakasz to get immunophenotyping data loaded and charted. Hopefully it is useful.



-dave
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Day 2 viral loads
david h oconnor 2016-02-17
We now know the day 1 viral loads were not due to residual inocula, since loads increased in all three animals between days 1 and 2 post-infection. The animal given the highest dose has a viral load of 1.74x10^6 vRNA copies / mL. It will be interesting to see if the challenge dose also affects timing of peak viremia.



-dave
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Dose dependence on viral load
david h oconnor 2016-02-17
The day one plasma viral loads are now loaded. The animal challenged with the highest dose of virus (10E6 PFU) has the highest viral loads, while the one challenged with the lowest dose of virus (10E4 PFU) has the lowest. While not unexpected, it suggests that viral dynamics may be modulated by challenge dose, though of course additional data will provide better resolution.



-dave
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Successful infection
david h oconnor 2016-02-16
All three animals were viremic with detectable plasma viral loads at day 1 post-infection. This establishes that experimental infections of Indian rhesus macaques with this challenge stock are possible.



-dave
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Mild rash at injection site
david h oconnor 2016-02-16
Veterinarians reported a mild rash at the injection site in two of the three animals challenged yesterday. The animals with mild rash were infected with the two lower doses of Zika virus. The animal infected with the highest dose of Zika virus did not have a rash.



-dave
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Successful challenge
david h oconnor 2016-02-15
This morning three Indian rhesus macaques were challenged subcutaneously with French Polynesian Zika virus. Baseline blood, urine, feces, and saliva samples were collected immediately preceding infection. I expect baseline complete blood counts to appear later this afternoon or tomorrow morning. The first viral loads from day 1 post-infection should be available late Tuesday or early Wednesday.



-dave
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