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

ZIKV-002 tracks data in 34 datasets over 480 time points. Data is present for 3 Participants.

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ZIKV-002: Infection of three rhesus macaques with Uganda 1947 Zika virus 

Primary objectives

  • Assess the infectivity of a Zika virus isolate from Africa 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 heterologous, Asian-lineage Zika viruses

Study design

Three Indian-origin rhesus macaques will be challenged subcutaneously with different doses of African lineage Zika virus (Uganda 1947). 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 10E4 PFU of French Polynesian Zika virus.

Result summary

This study started Monday, March 7, 2016


  • 562876 challenged with 10E6 PFU Zika virus/R.macaque-tc/UGA/1947/MR766-3329
  • 405734 challenged with 10E5 PFU Zika virus/R.macaque-tc/UGA/1947/MR766-3329
  • 295022 challenged with 10E4 PFU Zika virus/R.macaque-tc/UGA/1947/MR766-3329

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



(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.)

Key data: plasma viral loads 

Key data: urine viral load 

Neutralizing Antibody Titers 

MR766 Uganda 1947 ZIKV challenge stock comparison 

[Download a Geneious Pro 9.1.2 archive containing all the files used in this analysis]

After sequencing the ZIKV-002 challenge stock and noting differences from the Genbank MR766 sequence (including a 12nt in-frame deletion) we obtained two additional MR766 isolates. One comes from UTMB and the second is a seed stock from CDC from which we expanded the stock we used in ZIKV-002. All three stocks were deep sequenced by isolating viral RNA, preparing double-stranded cDNA, and then fragmenting the ds-cDNA with Nextera reagents. Libraries were deep sequenced on an Illumina miSeq.

1. Comparison of Genbank MR766 sequences

In our initial analysis of the ZIKV-002 challenge stock, we compared sequence reads with NCBI Genbank sequence LC002520 [Genbank]. This sequence was deposited in Genbank in 2014 and is the only full-length genome in Genbank that is found with the query 'Zika MR766' as of 10 April 2016. As I prepared this analysis, I noticed that there are five additional full-length polyprotein sequences of this virus stock in Genbank, but these are only obtained by searching with the query 'Zika "MR 766"':

  • HQ234498.1
  • KU720415.1
  • NC_012532.1
  • AY632535.2
  • DQ859059.1

To assess the similarity of these sequences to one another, I did a MUSCLE alignment (Geneious default parameters) of all 6 MR766 sequences alongside the Asian-lineage ZIKV PF/2013 challenge stock consensus sequence:

and made a tree with the Geneious Tree Builder

Looking at the alignment, a few observations are obvious:

  • The sequence with accession DQ859059.1 looks nothing like the others at the nucleotide level. I would be cautious about using this as a reference for any MR766 studies unless its relationship with MR766 is clarified by the group that submitted it.
  • Both the frameshift and 12nt in-frame deletion observed in the ZIKV-002 challenge stock are present in a subset of the Genbank MR766 sequences
  • There are two pairs of identical sequences:
    • KU720415.1 and HQ234498.1
    • NC_012532.1 and AY632535.2 (NC_012532.1 is the NCBI RefSeq and is derived from AY632535.2)

These consensus level changes undoubtedly mask variants present within each virus preparation.

[Download Geneious Pro files used in this analysis]

2. Which Genbank reference is most similar to ZIKV-002 challenge stock?

Aligned Genbank MR766 sequences with MUSCLE, this time including the ZIKV-002 challenge stock consensus sequence defined previously. Made tree as described above. The closest Genbank sequence to the ZIKV-002 challenge is HQ234498.1 as shown below. Note that the biggest difference between these sequences is the 12nt deletion in ZIKV-002's challenge stock that is not represented in HQ234498.1:

3. Comparison of three MR766 isolates to HQ234498.1

I used the Zequencer workflow to map reads from three MR766 isolates using HQ234498.1 as the reference. Each of the virus isolates has variants relative to the reference, as shown below:

I plotted the frequency of these variants in

There are three high frequency variants relative to the reference. The first is a synonymous SNP at nt 993. The second is the aforementioned in-frame deletion beginning at nt 1327. It is present in at least 73% of reads in each of the three sequenced isolates. The third is another synonymous SNP at nt 10237.

4. What happens to these variants in vivo?

Shelby generated sequence data from one of the ZIKV-002 animals 2 days post-challenge. I mapped these reads against the HQ234498.1 reference using the Zequencer reference. Note that these reads were generated by fragmenting five overlapping RT-PCR amplicons instead of by fragmenting ds-cDNA.

Interestingly, the deletion present in the stock isolates is not present (or present in < 5% of stock sequences). 

There are four variants detected in the 2 day sequence. The first is a synonymous SNP at nt2544 that is present in 39% of reads. It is not detected at >5% in any of the three sequenced stock isolates. The second is synonymous SNP at nt 4785 that is in 38% of reads and is also not in any of the three sequenced stock isolates. The third is a synonymous SNP at nt 5071 that is in 58% of the reads. It is in the ZIKV-002 challenge stock, but only at 10%. The fourth is a variant at nt 9475 that is in 40% of reads, but is not present in any of the stock isolates. 

In other words, within two days of infection, the circulating virus sequence is amino acid identical to the HQ234498 reference sequence. 

Anyone who is using viruses termed ZIKV MR766 needs to carefully examine the sequence composition of their stocks. Multiple viruses all termed MR766 may have different sequences and biological properties. In the case of the MR766 we are using in our studies, there is a deletion in the challenge stock that is strongly selected against quickly in vivo. There is a remote possibility that the use of unbiased vs. amplicon sequencing is biasing the detection of this and other variants, but based on our experience using these approaches with other viruses consider this unlikely. The amino acid sequence of viruses in the ZIKV-002 macaque at day 2 post-infection is identical to the amino acid sequence of the HQ234498 reference sequence, which will be used as the reference sequence for subsequent comparisons. 

  Attached Files  
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 17301 challenge stock sequences vs. HQ234498 reference sequence (1).png
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ZIKV-002 Challenge Stock Summary 

Virus stock info: Zika virus/R.macaque-tc/UGA/1947/MR766-3329

ZIKV strain MR766 (GenBank:LC002520), originally isolated from a sentinel Rhesus monkey on 20 April 1947 in Zika Forest, Entebbe, Uganda with 149 suckling mouse brain passages and two rounds of amplification on Vero cells, was obtained from Brandy Russell (CDC, Ft. Collins, CO).  Virus stocks were prepared by inoculation onto a confluent monolayer of C6/36 mosquito cells.

Harvest Date: 15 February 2016        Titer:  6.77 log10 PFU/ml

Unlike the challenge stock for ZIKV-001, where the consensus sequence of the challenge stock was identical to the Genbank sequence of the parental virus, there is one significant consensus-level change between the challenge stock and the Genbank sequence from ZIKV MR766. There are also several synonymous sites where variation is fixed in the challenge stock relative to the Genbank sequence. I do not know whether this is due to sequence changes in the source ZIKV MR766 virus that we received and amplified or whether these changes accumulated during in vitro expansion of the virus to prepare the challenge stock.

Analysis method

To map and call variants, I used a modified version of the Zequencer workflow I developed to analyze ZIKV-001 data. This version of the workflow:

- removes duplicate reads using the bbmap script

- trims low quality sequence and removes adapter sequences from the ends of reads

- filters out reads shorter than 150bp after trimming

- maps reads to the reference sequence using the bbmap algorithm in local alignment mode, using the normal sensitivity preset

- calls variants supported by >5% of reads with a p-value < 10e-60 and a minimum strand bias P value of 10e-5 when exceeding 65% bias

This version of the workflow does not rely on any external plug-ins and can be run using only integrated plug-ins available in Geneious Pro 9.1.2.

Download the workflow

Assessment of challenge stock variants

The most interesting region of variability involves a sequence at position 1430-1441 (relative to Genbank LC002520) where the majority of sequences have a 4 amino acid in-frame deletion. Reads that do not have the deletion have two non-synonymous nucleotide changes in the same region. 

There are a number of putative variants in the 5' and 3' UTRs:

5' UTR

3' UTR

All variants

Here is a table of all of the variants observed in the challenge stock at >5% along with their predicted impact on protein function. Variants at sites within the region encoding the polyprotein are highlighted in yellow. Note that the only non-synonymous variants predicted to impact amino acid sequence are located in the same location as the deletion that is present in many reads. In other words, some reads have a deletion while others have two non-synonymous substitutions. 

NameTypeMinimumMaximumLengthAmino Acid ChangeCDS PositionCoverageProtein EffectVariant FrequencyVariant P-Value (approximate)
APolymorphism10101  51 76.50%7.80E-95
CGCPolymorphism15173  55 -> 59 69.5% -> 72.7%1.20E-99
TPolymorphism22221  150 28.70%1.30E-87
CPolymorphism27271  263 44.90%5.80E-254
APolymorphism28281  300 13.70%6.60E-65
GPolymorphism28281  300 25.70%1.50E-143
APolymorphism29291  300 25.70%1.50E-143
CPolymorphism29291  300 13.70%7.50E-61
TPolymorphism32321  311 24.80%3.60E-142
CGCPolymorphism35373  392 -> 39619.4% -> 19.6%4.00E-133
CPolymorphism45451  655 11.60%2.70E-143
APolymorphism3263250 22012,516Frame Shift6.60%0
CPolymorphism1,0991,0991 99319,229None82.00%0
 Polymorphism1,4301,44112TVND -> 1,32417679 -> 18169Deletion79.8% -> 82.0%0
TPolymorphism1,4311,4311T -> I1,32518,049Substitution18.80%0
CPolymorphism1,4431,4431I -> T1,33718,126Substitution16.50%0
TPolymorphism1,4441,4441 1,33818,155None82.60%0
CPolymorphism1,6691,6691 1,56319,889None99.70%0
CPolymorphism4,5194,5191 4,41339,374None99.20%0
TPolymorphism5,1775,1771 5,07122,582None10.20%0
CPolymorphism5,5125,5121 5,40625,111None99.80%0
APolymorphism7,0847,0841 6,97822,290None12.00%0
APolymorphism7,5317,5311 7,42523,541None11.40%0
TPolymorphism7,7417,7411 7,63522,615None8.10%0
TPolymorphism10,13810,1381 10,03232,760None11.00%0
CPolymorphism10,34310,3431 10,23725,396None81.30%0
CTPolymorphism10,61110,6122  5489 -> 555134.7% -> 34.9%0
CCAPolymorphism10,61510,6151  5,416 35.10%0
APolymorphism10,61810,6181  5,111 37.40%0
TCPolymorphism10,62010,6212  5066 -> 507437.6% -> 37.7%0
CTGPolymorphism10,62510,6273  4877 -> 490539.4% -> 39.6%0
TPolymorphism10,63510,6351  4,210 46.40%0
CAPolymorphism10,63710,6382  4145 -> 414847.00%0
APolymorphism10,64010,6401  4,051 48.20%0
CTPolymorphism10,64210,6432  3972 -> 397849.0% -> 49.1%0
GPolymorphism10,64510,6451  3,951 49.40%0
TTCPolymorphism10,65310,6553  3579 -> 363249.4% -> 50.0%0


  Attached Files  
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ELISPOT Results 

We performed IFNg-ELISPOT at 4dpi, 10dpi, and 14dpi using peptide pools spanning the NS5 peptide for the African lineage zika virus. Each pool was comprised of 10 overlapping 15mer peptides. Each peptide pool was run in duplicate, as well as a Concanavalin A (ConA) positive control and a non-stimulated negative control. Data was baseline corrected by subtracting the average negative control values from each response. A threshold of 10.0 SFC/100,000 cells was set as the minimum value to be considered a positive T cell response.

295022       562876       405734

Shared responses were observed in two animals with a shared MHC haplotype, which suggests that these peptide responses may be restricted the shared Mamu-A*006 allele. elispot_summary

  Attached Files  

Showing: all messages
Prolonged shedding in urine
david h oconnor 2016-03-17
As in ZIKV-001, we are detecting virus in urine after it is no longer detectable in plasma. In ZIKV-001, this only lasted for a few days. We should know early next week how long the ZIKV-002 animal with viruria (virus in urine) continues to shed.

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Lower viral laods
david h oconnor 2016-03-15
With a little more data, it is now clear that the animals challenged with African lineage Zika virus have consistently lower plasma viremia than animals infected with Asian lineage Zika virus. Moreover, only one of the three animals in this study have any detectable virus in urine, while all three animals given Asian lineage virus had detectable virus in urine. If experience with HIV is any guide, higher concentrations of virus in various fluids can be proportional to transmission risk.

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ZIKV-002 challenge virus sequence information
david h oconnor 2016-03-11
I just posted our initial analysis of the ZIKV-002 challenge sequence. Of interest to others who are using this African lineage virus, we noted a four amino-acid deletion in the majority of sequences in a region of envelope. It is possible this occurred during in vitro expansion of the source virus, but it is also possible this deletion is present in the source virus. At this time, we have no way to assess when the deletion arose or what, if any, impact it will have on virus replication. We would urge others using their viruses to sequence them. If you have a challenge stock that will be used in an animal study, Dr. Shelby O'Connor ( will sequence it free of charge as a service provided by the Wisconsin National Primate Research Center's Genetics Services Unit.

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david h oconnor 2016-03-09
All three animals challenged with African lineage Zika virus are now infected. As before, the amount of virus in the blood one day after infection is proportional to the challenge dose. Also similar to our first study, virus is not detected in the urine early during infection even when it is present in blood.

Andrea notes that viral loads at day 1 and 2 are slightly lower than what we observed in ZIKV-001.

[UPDATE] - after looking more closely, this difference is more striking than I initially noticed. All three of the animals challenged with African ZIKV have lower viral loads than any of the three animals challenged with Asian ZIKV. If this trend holds, and it is still very, very preliminary, it could provide a reason why the consequences of Asian/American ZIKV infections are more severe than what was previously observed in Africa.

Information about the challenge virus should be available later tonight.

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First viral loads later today or tomorrow morning
david h oconnor 2016-03-08
Plasma viral loads will be determined on samples collected for each of the next ten days. The first question we will answer with today's data is whether animals were successfully infected.

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ZIKV-002 begins Monday
david h oconnor 2016-03-03
We will be starting ZIKV-002 on Monday March 7, 2016.
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