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:
These consensus level changes undoubtedly mask variants present within each virus preparation.
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:
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.
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)|
|CGC||Polymorphism||15||17||3||55 -> 59||69.5% -> 72.7%||1.20E-99|
|CGC||Polymorphism||35||37||3||392 -> 396||19.4% -> 19.6%||4.00E-133|
|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|
|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|
|CA||Polymorphism||10,637||10,638||2||4145 -> 4148||47.00%||0|
|CT||Polymorphism||10,642||10,643||2||3972 -> 3978||49.0% -> 49.1%||0|
|TTC||Polymorphism||10,653||10,655||3||3579 -> 3632||49.4% -> 50.0%||0|
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.
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.
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|Prolonged shedding in urine|
|Lower viral laods|
|ZIKV-002 challenge virus sequence information|
|First viral loads later today or tomorrow morning|
|ZIKV-002 begins Monday|