Characterisation of infectious Ebola virus from the ongoing outbreak to guide response activities in the Democratic Republic of the Congo: a phylogenetic and in vitro analysis

Abstract

Background: The ongoing Ebola virus outbreak in the Ituri and North Kivu Provinces of the Democratic Republic of the Congo, which began in July, 2018, is the second largest ever recorded. Despite civil unrest, outbreak control measures and the administration of experimental therapies and a vaccine have been initiated. The aim of this study was to test the efficacy of candidate therapies and diagnostic tests with the outbreak strain Ituri Ebola virus. Lacking a virus isolate from this outbreak, a recombinant Ituri Ebola virus was compared with a similarly engineered Makona virus from the 2013-16 outbreak.

Methods: Using Ebola virus sequences provided by organisations in DR Congo and a reverse genetics system, we generated an authentic Ebola virus from the ongoing outbreak in Ituri and North Kivu provinces. To relate this virus to other Ebola viruses in DR Congo, we did a phylogenetic analysis of representative complete Ebola virus genome sequences from previous outbreaks. We evaluated experimental therapies being tested in clinical trials in DR Congo, including remdesivir and ZMapp monoclonal antibodies, for their ability to inhibit the growth of infectious Ituri Ebola virus in cell culture. We also tested diagnostic assays for detection of the Ituri Ebola virus sequence.

Findings: The phylogenetic analysis of whole-genome sequences from each Ebola virus outbreak suggests there are at least two Ebola virus strains in DR Congo, which have independently crossed into the human population. The Ituri Ebola strain initially grew slower than the Makona strain, yet reached similar mean yields of 3 × 10⁷ 50% tissue culture infectious dose by 72 h infection in Huh-7 cells. Ituri Ebola virus was similar to Makona in its susceptibility to inhibition by remdesivir and to neutralisation by monoclonal antibodies from ZMapp and other monoclonal antibodies. Remdesivir inhibited Ituri Ebola virus at a 50% effective concentration (EC₅₀) of 12nM (with a selectivity index of 303) and Makona Ebola virus at 13nM (with a selectivity index of 279). The Zmapp monoclonal antibodies 2G4 and 4G7 neutralised Ituri Ebola virus with a mean EC₅₀ of 0·24 μg/mL and 0·48 μg/mL, and Makona Ebola virus with a mean EC₅₀ of 0·45 μg/mL and 0·2 μg/mL. The Xpert Ebola and US Centers for Disease Control and Prevention real-time RT-qPCR diagnostic assays detected Ituri and Makona Ebola virus sequences with similar sensitivities and efficiencies, despite primer site binding mismatches in the Ituri Ebola virus.

Interpretation: Our findings provide a rationale for the continued testing of investigational therapies, confirm the effectiveness of the diagnostic assays used in the region, and establish a paradigm for the use of reverse genetics to inform response activities in an outbreak.

Funding: US Centers for Disease Control and Prevention.

Figure 1:. Phylogenetic analysis of representative complete Ebola virus genomic sequences

Bayesian coalescent phylogenetic analysis with Markov chain Monte Carlo sampling was used to infer a molecular clock to estimate the dates of origin. (A) Maximum-likelihood analysis of full-length genomes showing Ebola virus sequences cluster separately from Bundibugyo virus. Scale bar is substitutions per site. (B) Maximum clade credibility tree of the Bayesian analysis of the Ebola-Ituri sequence with those from other Ebola virus outbreaks. The most recent common ancestor, with years since 2018, and posterior probability values are shown at the nodes, and the 95% highest posterior density interval is shown in parentheses. The evolutionary rate was estimated to be 4·6 × 10⁻⁴ nucleotide substitutions per site per year (2·8 × 10⁻⁴ – 6·3 × 10⁻⁴ highest posterior density), typical for an RNA virus with a 19 kb genome. The Ebola-Ituri genome used in this study is marked with a star. (C) Locations of Ebola virus outbreaks within the Democratic Republic of the Congo.

Figure 2:. Recombinant EBOV-Ituri genome and replication kinetics of EBOV-Ituri and EBOV-Makona in cell culture

(A) Schematic of the recombinant EBOV-Ituri genome in viral complementary sense with reporter ZsG expressed with the gene for virus membrane protein VP40 and separated by the gene for the self-cleaving 2A peptide. (B) Time course of viral growth. Huh-7 cells were infected at a multiplicity of infection of 0·2. Samples of media were harvested at varying time points and virus titres were measured. Data represent the mean of three biological replicates and the error bars represent the SD. EBOV=Ebola virus. TCID₅₀=50% tissue culture infectious dose.

Figure 3:. Performance of diagnostic assays on the Ebola-Ituri sequence

(A) Results from the CDC NP2 real-time RT-qPCR assay, using dilution series of Ituri or Makona Ebola virus RNA. (B) Results from the CDC VP40 real-time RT-qPCR assay and the modified VP40 assay that accounts for the mismatch in the NP2 reverse primer (654G), using dilution series of Ituri or Makona Ebola virus RNA. For each assay, a representative of at least three independent experiments is shown. CDC=US Centers for Disease Control and Prevention.