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.
- 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
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.
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.)
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[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"':
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 plot.ly:
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.
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17301 challenge stock sequences vs. HQ234498 reference sequence (1).png
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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.
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 dedupe.sh 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:
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.
|Name||Type||Minimum||Maximum||Length||Amino Acid Change||CDS Position||Coverage||Protein Effect||Variant Frequency||Variant P-Value (approximate)
|A||Polymorphism||10||10||1|| || ||51|| ||76.50%||7.80E-95
|CGC||Polymorphism||15||17||3|| || ||55 -> 59|| ||69.5% -> 72.7%||1.20E-99
|T||Polymorphism||22||22||1|| || ||150|| ||28.70%||1.30E-87
|C||Polymorphism||27||27||1|| || ||263|| ||44.90%||5.80E-254
|A||Polymorphism||28||28||1|| || ||300|| ||13.70%||6.60E-65
|G||Polymorphism||28||28||1|| || ||300|| ||25.70%||1.50E-143
|A||Polymorphism||29||29||1|| || ||300|| ||25.70%||1.50E-143
|C||Polymorphism||29||29||1|| || ||300|| ||13.70%||7.50E-61
|T||Polymorphism||32||32||1|| || ||311|| ||24.80%||3.60E-142
|CGC||Polymorphism||35||37||3|| || ||392 -> 396||19.4% -> 19.6%||4.00E-133
|C||Polymorphism||45||45||1|| || ||655|| ||11.60%||2.70E-143
|A||Polymorphism||326||325||0|| ||220||12,516||Frame Shift||6.60%||0
| ||Polymorphism||1,430||1,441||12||TVND -> ||1,324||17679 -> 18169||Deletion||79.8% -> 82.0%||0
|T||Polymorphism||1,431||1,431||1||T -> I||1,325||18,049||Substitution||18.80%||0
|C||Polymorphism||1,443||1,443||1||I -> T||1,337||18,126||Substitution||16.50%||0
|CT||Polymorphism||10,611||10,612||2|| || ||5489 -> 5551||34.7% -> 34.9%||0
|CCA||Polymorphism||10,615||10,615||1|| || ||5,416|| ||35.10%||0
|A||Polymorphism||10,618||10,618||1|| || ||5,111|| ||37.40%||0
|TC||Polymorphism||10,620||10,621||2|| || ||5066 -> 5074||37.6% -> 37.7%||0
|CTG||Polymorphism||10,625||10,627||3|| || ||4877 -> 4905||39.4% -> 39.6%||0
|T||Polymorphism||10,635||10,635||1|| || ||4,210|| ||46.40%||0
|CA||Polymorphism||10,637||10,638||2|| || ||4145 -> 4148||47.00%||0
|A||Polymorphism||10,640||10,640||1|| || ||4,051|| ||48.20%||0
|CT||Polymorphism||10,642||10,643||2|| || ||3972 -> 3978||49.0% -> 49.1%||0
|G||Polymorphism||10,645||10,645||1|| || ||3,951|| ||49.40%||0
|TTC||Polymorphism||10,653||10,655||3|| || ||3579 -> 3632||49.4% -> 50.0%||0
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