Ebola Virus Transmission Initiated by Systemic Ebola Virus Disease Relapse

Summary During the 2018-2020 Nord Kivu Ebola virus disease (EVD) outbreak in the Democratic Republic of the Congo, an individual who had received the Merck rVSV-ZEBOV vaccine was diagnosed with EVD. His treatment included an Ebola virus-specific monoclonal antibody (mAb114), and he recovered within 14 days but re-presented six months later with severe EVD-like illness and Ebola virus viremia and died. We initiated an epidemiological and genomic investigation that showed the patient had a relapse of acute EVD, which led to a transmission chain that resulted in 91 cases spanning six health zones over four-months.


Ethics statement
The use of mAb114 during the June 2019 treatment was conducted under the Monitored Emergency Use of Unregistered and Investigational Interventions (MEURI) protocol which was review by Institut National de Recherche Biomédicale internal review board (IRB). The Ministry of Health (MoH) of the Democratic Republic of the Congo (DRC) approved this study. Oral consent was obtained at the homes of patients or in the Ebola Treatment Units prior to any sample collection by a team, including staff members of the MoH.

Viral sequencing
RNA was extracted from serum samples using the Qiagen Viral RNA Mini kit. cDNA was prepared using the ThermoFisher 1 st strand synthesis system, and amplicons prepared using Q5 mastermix (New England Biolabs) and EBOV-specific amplicons generated using PrimalSeq. 1 Amplicons were quantified using a Qubit fluorometric quantification device (ThermoFisher) with dsDNA broad range kit and diluted to <500 ng for input into library preparation. Libraries were prepared using the Illumina Nextera DNA Flex kit with IDT for Illumina Unique Dual indexes, quantified, and loaded on the Illumina iSeq 100 for 2 x 150 cycles or Illumina MiSeq for 2 x 150 cycles.

Viral sequencing analysis
Short read data were analyzed with the iVar (v1.0.1) 2 using reference sequence MK007330. The reads were trimmed using the ivar trim command with a quality threshold of 20 and with optione, to prevent the removal of reads that are not directly attached to amplicon primers following Nextera Flex library prep. Consensus sequences were called using the ivar consensus command, and a minimum coverage depth threshold of 50x, to prevent inadvertent contamination. Negative controls were included in each run to monitor potential contamination.
Multiple sequence alignment was performed with MAFFT 3 after retaining only genomes that were >95% complete. The maximum likelihood analysis was performed using IQ-TREE using ModelFinder to select the best-fit model. 4 The Nextstrain phylogenetics platform was used as part of the analysis. 5 Bayesian phylogenetic analysis was performed using BEAST v1.10.5 to infer time-resolved phylogenies. 6 We used an SDR06 nucleotide substitution model with a local clock model and a non-informative continuous time Markov chain reference prior (CTMC) on the molecular clock rate and a Skygrid coalescent prior. All the Bayesian analyses were run for 80 million Markov chain Monte Carlo steps, sampling parameters and trees every 10,000 generations. 7,8 Tracer v1.7 was used to ensure run convergence (effective sample size > 200). 9 The BEAST XML and log files are available at https://github.com/andersen-lab/paper_2020_drcebola. The final figures were created using baltic (https://github.com/evogytis/baltic) and Phylo (https://biopython.org/wiki/Phylo).

EBOV IgG ELISAs
Anti-human Ebola GP IgG ELISA data was generated using the respective Alpha Diagnostic International kits, according to manufacturer's instructions. In brief, patient serum was diluted as indicated. Final incubation with TMB substrate was carried out for 15 minutes, before stopping solution was added. ELISA Plates were read at 450nm, and optical density at 630nm was subtracted to normalize well background. To analyze the data, blank background signal was subtracted from all data. EC50 binding titers were determined using Graphpad Prism 8.

Human exome sequencing and analysis
Human genomic DNA was isolated from blood using the Qiagen DNA Blood and Tissue kit. Exome sequencing libraries were prepared using the Illumina Nextera Flex for Enrichment kit, utilizing the Illumina Exome Panel as enrichment oligos and IDT for Illumina Unique Dual indexes according to manufacturer's instructions. Sequencing libraries were analyzed on an Agilent TapeStation using the high-sensitivity DNA reagents before dilution and loading. Two exome libraries were prepared from two independent DNA extractions of the same sample, to maximize exome coverage. Both libraries were loaded on a single Illumina MiSeq run, using 2 x 300 cycle V3 chemistry to maximize the coverage depth. Close to 20 million unique reads were obtained, resulting in an average depth of 69X across all variants found. Sequence quality control was done using the FastQC software package (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and sequences were mapped to hg19 using the BWA aligner. 10 SNP and INDEL calling, annotation, classification and in-depth analyses were carried out with the Genoox platform (https://www.genoox.com/). The analysis focused on genes causing immunodeficiency disorders. Upon analysis of likely pathogenic mutations using a built-in tool in the Genoox platform we identified a single likely pathogenic variant, c.356_357insC (p.Glu119fs), in the SH2D1A gene (Supplementary Table 2). This variant has been reported to cause Lymphoproliferative Syndrome, OMIM # 308240. 11 However, Sanger sequencing confirmation revealed that this variant arose from a sequencing error, and was in fact not present in the patient's genome.
Neutralization assay mAb114 recognition of patient EBOV GP mutants was evaluated using a single-round infection and neutralization assay. Lentiviruses were produced bearing at their surface EBOV GP from either Ituri wildtype GP (first sequenced virus 18FHV089 12 ), patient's first infection sequenced GP (d1, MAN4194) and patient's second infection sequenced GP (d171, MAN12309). Neutralization was performed as previously described. 13 Briefly, HEK293T cells were exposed to pseudovirus mixed with serial dilutions of mAb114 at concentrations from 0.0001-10 μg/mL. Luciferase activity measured as relative luminescence unit (RLU) was obtained after lysis of target cells by using a Luciferase Assay System Bright Glo (Promega) and an Envision Plate Reader (PerkinElmer). Assays were performed three times, each with samples in triplicate.