The authors have the following interests: Jens H. Kuhn is employed by Tunnell Government Services, Inc. There are no patents, products in development or marketed products to declare. This employment does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Conceived and designed the experiments: GP. Performed the experiments: JRK CAR MRW JTL ERN BPP KG KP. Analyzed the data: JRK JTL JW JHK GP. Wrote the paper: JRK CAR JHK GP.
The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit “R4368” and its 2014 replacement “R4415.” We also provide characterization of the master stock used to create “R4415.” The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results.
Ebola virus disease (EVD) is a frequently lethal human viral hemorrhagic fever caused by four distinct ebolaviruses (Bundibugyo virus, Ebola virus, Sudan virus, and Taï Forest virus). EVD occurs sporadically and usually affects no more than several hundred people during an outbreak (reviewed in [
Taxonomically, EBOV is the only member of the species
The development, evaluation, and final licensure of medical countermeasures (MCMs) against EVD in the US is critically dependent on standardized animal models of filovirus infection and standardized assays and reagents [
All ebolaviruses make use of co-transcriptional editing of their glycoprotein-encoding
EBOV, Sudan virus (SUDV), and possibly other ebolaviruses adapt to different
Here, we report the coding-complete genome sequence (see [
A clinical specimen (designated Centers for Disease Control and Prevention Special Pathogens Branch Log [CDC SPBLOG] 9510621) was obtained from an EBOV-infected 65-year old female patient during an EVD outbreak that occurred in 1995 around Kikwit, Zaire (today Democratic Republic of the Congo, COD). The patient’s disease onset was recorded as April 29, 1995. She was hospitalized on May 1, 1995, and died on May 5, 1995. The clinical specimen, most likely plasma or serum, was obtained from the patient on May 4, 1995. How the patient became infected and what medical care she may have received is unclear. Unfortunately, chain-of-custody records that further detail the origin of 9510621 or its shipment to the CDC are not available anymore, and EBOV titration was not attempted from this specimen prior to cell-culture passage.
The first passage of EBOV/Kik-9510621, designated “virus seed pool (VSP) 807223” (
At USAMRIID, “VSP 807224” was stored frozen at -70°C and designated there as “135” (passage 2). After an additional passage in Vero E6 cells, the virus, now designated “134” (passage 3), was sequenced by Chain
Independently at UTMB, “VSP 807223” was passaged one time on Vero E6 cells, harvested after a 10-day incubation period on February 21, 2012, and stored in 1.0-ml aliquots as “WRC000121” (passage 2; MOI unknown). A titer of 1.8E+07 pfu/ml was determined (titration method unknown). On May 1, 2012, UTMB transferred “WRC000121” to USAMRIID, where the virus was stored at -70°C and designated as “R4414” (passage 2). This material was passaged at an MOI of 0.001 on Vero E6 cells to prepare NHP challenge stock “R4415” (passage 3), which was harvested on June 11, 2013, after a 12-day incubation period (time to develop 2–3+ CPE) with a titer of 1.31E+06 pfu/ml determined by agarose-based plaque assay [
Vials (1 ml) of EBOV/Kik-9510621 challenge stocks were stored and maintained at USAMRIID at -70°C. “R4368” (passage 4) was stored since July 24, 2011, and thawed on March 2, 2012; “R4414” (passage 2) and “R4415” (passage 3) were stored since June 11, 2013, and were thawed in January 2014. An aliquot of 100 μl of each thawed virus stock was placed in 3:1 TRIzol (Life Technologies, Carlsbad, CA). Nucleic acids were isolated from TRIzol-treated material, and genome sequence was determined on Illumina technology (MiSeq or HiSeq) with EBOV-specific oligonucleotides following sample preparation performed as described in [
For rapid amplification of cDNA ends (RACE), “R4415” (passage 3) RNAs were extracted with Zymo Direct-Zol (Zymo Research Corporation, Irvine, CA) from cell-culture supernatant in TRIzol according to the manufacturers' instructions. SMARter RACE 5’/3’ kit (Clontech Laboratories, Inc., Mountain View, CA) was used to amplify both 5' and 3' untranslated regions (UTRs) of the virus genome from the extracted RNAs. The kit's two-stage nested PCR protocol was found to be optimal. The gene-specific primers for each RACE experiment are as follows 5’ RACE (outer primer):
Research has been reviewed for compliance with dual-use guidelines and approved for publication by the USAMRIID Institute Biosafety Committee (IBC) and the Operational Security office.
Compared to EBOV/Kik-9510621 “134” (passage 3), “R4368” (passage 4) acquired only one consensus-level substitution (nucleotide position 7,327). Six intrahost single nucleotide variants (iSNVs) (≥2% of the population) were detected and are reported in
Reference position (“134” (passage 3)) | Reference base | SNP base | SNP % | Codon | Gene | Depth |
---|---|---|---|---|---|---|
5,878 | T | g | 2.84 | 1374 | ||
6,139 | C | t | 2.07 | P (CCA) @34 L (CtA) | 1595 | |
6,179 | G | t | 8.57 | E (GAG) @47 D (GAt) | 1541 | |
7,298 | T | c | 3.98 | Synonymous | 804 | |
7,327 | C | T | 99.61 | P (CCA) @430 L (CTA) | 507 | |
10,833 | G | a | 2.35 | R (AGA) @163 K (AaA) | 1533 | |
16,365 | A | g | 2.76 | Q (CAG) @1595 R (CgG) | 1560 |
SNP, single nucleotide polymorphism.
Reference position (“134” (passage 3)) | Reference base | SNP base | SNP % | Codon | Gene | Depth |
---|---|---|---|---|---|---|
1,401 | G | a | 2.08 | G:GGT @ 311 → D:GaT | 1,008 | |
5,830 | T | a | 2.18 | 1,789 | ||
6,179 | G | t | 100.00 | E:GAG @ 47 → D:GAt | 2,887 | |
6,231 | T | c | 3.97 | S:TCA @ 65 → P:cCA | 3,956 | |
6,384 | C | a | 4.33 | P:CCT @ 116 → T:aCT | 5,264 | |
7,327 | C | t | 99.90 | P:CCA @ 430 → L:CtA | 995 | |
7,669 | C | t | 33.00 | T:ACA @ 544 → I:AtA | 912 | |
10,344 | C | a | 4.60 | 5,108 | ||
10,833 | G | a | 99.60 | R:AGA @ 163 → K:AaA | 3,422 | |
11,283 | A | c | 2.89 | 3,361 | ||
11,498 | G | a | 6.05 | 1,123 | ||
12,065 | G | a | 4.21 | G:GGT @ 162 → S:aGT | 4,798 | |
12,153 | G | t | 5.38 | W:TGG @ 191 → L:TtG | 5,246 | |
14,184 | C | t | 2.13 | S:TCG @ 868 → L:TtG | 1,880 |
SNP, single nucleotide polymorphism.
Reference position (“134” (passage 3)) | Reference base | SNP base | SNP % | Codon | Gene | Depth |
---|---|---|---|---|---|---|
520 | T | c | 10.60 | S:TCT @ 17 →→ S:TCc | 4,330 | |
530 | T | c | 10.15 | Y:TAC @ 21 → H:cAC | 4,403 | |
542 | T | c | 9.84 | L:TTG @ 25 → L:cTG | 4,483 | |
606 | T | c | 10.70 | V:GTA @ 46 → A:GcA | 4,831 | |
1,274 | A | g | 7.83 | R:AGG @ 269 → G:gGG | 5,62 | |
5,830 | T | c | 3.20 | 2,871 | ||
6,179 | G | t | 99.90 | E:GAG @ 47 → D:GAt | 8,337 | |
6,384 | C | a | 3.4 | P:CCT @ 116 → T:aCT | 11,383 | |
7,327 | C | t | 99.940 | P:CCA @ 430 → L:CtA | 1,048 | |
7,669 | C | t | 98.70 | T:ACA @ 544 → I:AtA | 1,486 | |
10,344 | C | a | 4.20 | 9,735 | ||
10,833 | G | a | 100.00 | R:AGA @ 163 → K:AaA | 4,642 | |
11,283 | A | c | 3.91 | 1,844 | ||
11,498 | G | a | 5.15 | 466 | ||
12153 | G | a | 4.95 | W:TGG @ 191 →.:TaG | 4,910 | |
13994 | C | a | 4.65 | Q:CAA @ 805 → K:aAA | 7,614 | |
16247 | T | c | 2.19 | S:TCA @ 1556 → P:cCA | 2,838 |
SNP, single nucleotide polymorphism.
6U/9U (ssGP phenotype) | 7U (sGP phenotype) | 8U (GP1,2 phenotype) | |
---|---|---|---|
3.8% (373) | 11.2% (1,090) | 85.0% (8,300) | |
0.6% (149) | 97.5% (23222) | 1.8% (439) | |
0.3% (40) | 88.8% (11231) | 10.9% (1378) |
GP, glycoprotein; sGP, soluble glycoprotein; ssGP, small secreted glycoprotein; 7–9U, 7–9 uridylyl glycoprotein (
We have provided here a concise report on the history and genomic characterization of the USAMRIID Ebola virus/H.sapiens-tc/COD/1995/Kikwit-9510621 NHP challenge stocks “R4368” (passage 4) and “R4415” (passage 3), as well as the “R4415” (passage 3) predecessor, “R4414 (passage 2).” “R4368” (passage 4) was used between July 2011 and December 2014 in both
The authors thank Dr. Pierre E. Rollin (CDC, Atlanta, GA) and Dr. Thomas W. Geisbert (UTMB) for providing the background information on EBOV/Kik-951062 stocks, the USAMRIID virus production team of Brian Kearney, Scott Olschner, Priscilla Williams, Dr. Anna N. Honko, and Joshua C. Johnson for experimental support, and Laura Bollinger (IRF-Frederick) for critically editing the manuscript.