Emergence of Zaire Ebola Virus Disease in Guinea

Patients

Blood samples were obtained from 20 patients who were hospitalized in Guéckédou, Macenta, and Kissidougou because of fever, diarrhea, vomiting, or hemorrhage. Demographic and clinical data for the patients were provided on the laboratory request forms. Clinical data were not collected in a systematic fashion. This work was performed as part of the public health response to contain the outbreak in Guinea; informed consent was not obtained.

Diagnostic Assays

Viral RNA was extracted from 50 to 100 μl of undiluted plasma and 1:10 diluted plasma with the use of the QIAmp viral RNA kit (Qiagen). Nucleic acid amplification tests for detection of filoviruses and arenaviruses were performed with the use of commercially available kits and published primers and probes^5-11 (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).

Viral Sequencing

Fragments amplified by filovirus L gene–specific primers were sequenced with the use of polymerase-chain-reaction (PCR) primers. Complete EBOV genomes were sequenced directly with the use of RNA extracted from serum obtained from three patients with high levels of viral RNA, as measured on real-time reverse-transcriptase–PCR (RT-PCR) analysis. The genome was amplified in overlapping fragments with the use of EBOV-specific primers. The fragments were sequenced from both ends with the use of conventional Sanger techniques. The sequence of the contigs was verified by visual inspection of the electropherograms.

Viral Isolation

About 100 μl of all serum samples was used to inoculate Vero E6 cells maintained in 25-cm² flasks in Dulbecco's modified Eagle's medium containing 2 to 5% fetal-calf serum and penicillin–streptomycin. Cells and supernatant were passaged several times. Virus growth in the cells was verified on immunofluorescence with the use of polyclonal mouse anti-EBOV–specific antibodies in the serum of mice challenged with EBOV or on the basis of an increase in viral levels in the cell-culture supernatant over several orders of magnitude, as measured on real-time RT-PCR.

Electron Microscopy

Specimens from two patients were prepared for electron microscopy with the use of a conventional negative-staining procedure. In brief, a drop of 1:10 diluted serum was adsorbed to a glow-discharged carbon-coated copper grid and stained with freshly prepared 1% phosphotungstic acid (Agar Scientific). Images were taken at room temperature with the use of a Tecnai Spirit electron microscope (FEI) equipped with a LaB6 filament and operated at an acceleration voltage of 80 kV.

Phylogenetic Analysis

We obtained all 48 complete genome sequences of filoviruses that are currently available from GenBank and aligned them with the new EBOV Guinea sequences (18,959 nucleotides). We used software designed to perform statistical selection of best-fit models of nucleotide substitution (jModelTest¹²) to identify the general time-reversible model of sequence evolution with gamma-distributed rate variation among sites (GTR+gamma) as the model that best describes the phylogenetic data. We used the Bayesian Markov Chain Monte Carlo method, as implemented in MrBayes 3.1.2 software,¹³ to infer the composition of one phylogenetic tree, using two runs of four chains with 1 million steps with a burn-in rate of 25% and the GTR+gamma model. A second tree was inferred for the same alignment with a maximum-likelihood method implemented in PhyML software¹⁴ under the GTR+gamma model with 1000 bootstrap replications. A reconstruction of the EBOV phylogenetic tree with the use of molecular clock models is provided in Fig. S1 in the Supplementary Appendix.

Epidemiologic Investigations

We gathered data on possible transmission chains from hospital records and through interviews with patients in whom EBOV infection was suspected and their contacts, affected families, inhabitants of villages in which deaths occurred, attendants of funerals, public health authorities, and hospital staff members.

Identification of the EBOV Strain

Table 1. Table 1. Demographic, Clinical, and Virologic Characteristics of 15 Patients with Confirmed Ebola Virus Disease during the 2014 Outbreak in Guinea.

Figure 2. Figure 2. Transmission Chains in the Outbreak of Ebola Virus Disease in Guinea.

Shown are transmission chains in the Ebola virus disease outbreak involving laboratory-confirmed cases. The presumed means of transmission of Zaire ebolavirus (EBOV), as revealed by epidemiologic investigation, are indicated by solid arrows. Dashed arrows indicate that the epidemiologic links are not well established. Laboratory-confirmed cases (C) are indicated with red circles, and suspected cases (S) are indicated with the case number. The inset image is an electron microscopic scan of the Guinean strain of EBOV in blood obtained from a patient. A typical complete virus particle, with the ends marked by arrows, and two degraded particles (arrowheads) are shown.

To detect the causative agent, we used conventional Filoviridae-specific RT-PCR assays targeting a conserved region in the L gene to test samples obtained from 20 hospitalized patients who were suspected of being infected with a hemorrhagic fever virus.^5,6,9 In addition, we performed EBOV-specific real-time RT-PCR assays targeting the glycoprotein (GP) or nucleoprotein (NP) gene.^7,10 Samples from 15 of 20 patients tested positive on the conventional L gene PCR assay and the real-time assays (Table 1). EBOV was identified in the serum of one patient on electron microscopy (Figure 2, inset) and was isolated in cell culture from 5 patients. None of the samples were positive for Lassa virus on Lassa virus–specific RT-PCR assays.^8,11 Sequencing of the fragments amplified by the L gene RT-PCR assays revealed EBOV sequences. The partial L gene sequences were identical for all confirmed cases, except for a synonymous T-to-C polymorphism at position 13560, which was found in Patients C12 and C14.

Sequencing of Samples from Patients

Figure 3. Figure 3. Phylogenetic Analysis of the Ebolavirus Genus, Including the EBOV Strains from Guinea.

The phylogenetic tree was inferred with the use of the Bayesian Markov Chain Monte Carlo method. A second tree that was inferred for the same set of sequences with a maximum-likelihood method confirmed the Bayesian tree (data not shown). Bayesian posterior probabilities and bootstrap percentages (1000 replicates of the maximum-likelihood tree) are shown on the branches. For clarity of presentation, the branches for the non-EBOV species were shortened and condensed (dashed branches). The GenBank accession number, strain designation, country of origin, and year of isolation are indicated on the EBOV branches. The EBOV Guinea strain is available from the European Virus Archive (www.european-virus-archive.com).

The EBOV in samples obtained from three patients was completely sequenced with the use of conventional Sanger techniques (GenBank accession numbers, KJ660346, KJ660347, and KJ660348; the sequences in the preliminary report have been updated). The three sequences, each 18,959 nucleotides in length, were identical, with the exception of a few polymorphisms at positions 2124 (G→A, NP552 glycine→glutamic acid), 2185 (A→G, synonymous), 6909 (A→T, sGP291 arginine→tryptophan), 9923 (T→C, synonymous), 13856 (A→G, L759 aspartic acid→glycine), and 15660 (T→C, synonymous). The Guinean EBOV strain showed 97% identity to EBOV strains from the Democratic Republic of Congo and Gabon. Phylogenetic analysis of the full-length sequences by means of Bayesian and maximum-likelihood methods revealed a separate, basal position of the Guinean EBOV within the EBOV clade (Figure 3).

Clinical and Epidemiologic Analysis

The prominent clinical features of the EBOV infection in the confirmed cases were fever, severe diarrhea, and vomiting; hemorrhage was less frequent. The case fatality rate in the initial cases was 86% (12 of 14 patients with a known outcome died). Confirmed cases originated from hospitals in Guéckédou, Macenta, Nzérékoré, and Kissidougou prefectures (Figure 1). We performed an epidemiologic look-back investigation of the transmission chains by reviewing hospital documentations and interviews with affected families, patients with suspected disease, and inhabitants of villages in which cases occurred.

According to the initial epidemiologic investigation, the suspected first case of the outbreak was a 2-year-old child who died in Meliandou in Guéckédou prefecture on December 6, 2013 (Figure 2). A second investigation confirmed the origin of the outbreak in Meliandou but revealed a somewhat different timing of the early events (including the death of Patient S1 at the end of December and the deaths of Patients S2, S3, and S4 in January). Patient S14, a health care worker from Guéckédou with suspected disease, seems to have triggered the spread of the virus to Macenta, Nzérékoré, and Kissidougou in February 2014. As the virus spread, 13 of the confirmed cases could be linked to four clusters: the Baladou district of Guéckédou, the Farako district of Guéckédou, Macenta, and Kissidougou. Eventually, all clusters were linked with several deaths in the villages of Meliandou and Dawa between December 2013 and March 2014.

Initial Phase of the Outbreak

Figure 4. Figure 4. Number of Suspected Cases of Ebola Virus Disease, According to Prefecture and Week.

The cases in Guéckédou, Macenta, and Kissidougou prefectures were recorded by the local public health authorities in collaboration with the World Health Organization and Médecins sans Frontières.

This report is focused on the initial phase and geographic origin of the EBOV outbreak. Before the end of March 2014 (week 13), a total of 111 clinically suspected cases with 79 deaths (71% case fatality rate on the basis of clinical suspicion) had been recorded in the prefectures of Guéckédou, Macenta, and Kissidougou. According to the timeline of the transmission chains (Figure 2), the outbreak of confirmed disease started in the prefecture Guéckédou and then spread to Macenta and Kissidougou (Figure 4). The male-to-female ratio among patients who died was 41:59; the median age was 35 years (interquartile range, 25 to 51).

This study demonstrates the emergence of EBOV in Guinea. The high degree of similarity among the 15 partial L gene sequences, along with the three full-length sequences and the epidemiologic links between the cases, suggest a single introduction of the virus into the human population. This introduction seems to have happened in December 2013. Further epidemiologic investigation is ongoing to identify the presumed animal source of the outbreak. It is suspected that the virus was transmitted for months before the outbreak became apparent because of clusters of cases in the hospitals of Guéckédou and Macenta. This length of exposure appears to have allowed many transmission chains and thus increased the number of cases of Ebola virus disease.

The clinical picture of the initial cases was predominantly fever, vomiting, and severe diarrhea. Hemorrhage was not documented for most of the patients with confirmed disease at the time of sampling but may have developed later in the course of the disease. The term Ebola virus disease (rather than the earlier term Ebola hemorrhagic fever) takes into account that hemorrhage is not seen in all patients¹⁵ and may help clinicians and public health officials in the early recognition of the disease. The case fatality rate was 86% among the early confirmed cases and 71% among clinically suspected cases, which is consistent with the case fatality rates observed in previous EBOV outbreaks.^15-17

Phylogenetic analysis of the full-length sequences established a separate clade for the Guinean EBOV strain in a sister relationship with other known EBOV strains. This suggests that the EBOV strain from Guinea has evolved in parallel with the strains from the Democratic Republic of Congo and Gabon from a recent ancestor and has not been introduced from the latter countries into Guinea. However, the determination of both the timing of the introduction of the virus into Guinea and its phylogenetic origin also depend on our understanding of the evolutionary rate of EBOV in nature (Fig. S1 in the Supplementary Appendix). A reliable estimate for this rate, such as one derived from archaeological calibration,¹⁸ may be required to answer these questions.

Potential reservoirs of EBOV, fruit bats of the species Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata, are present in large parts of West Africa.¹⁹ Therefore, it is possible that EBOV has circulated undetected in this region for some time. The emergence of the virus in Guinea highlights the risk of EBOV outbreaks in the whole West African subregion.

The authors' affiliations are as follows: the National Reference Center for Viral Hemorrhagic Fevers (S.B., D.P., A.B., S.M.), Unité de Biologie des Infections Virales Emergentes, Institut Pasteur (S.B.), Centre International de Recherche en Infectiologie (CIRI), Université de Lyon, INSERM Unité 1111, Ecole Normale Supérieure de Lyon, Université Lyon 1 (S.B.), and Laboratoire P4 INSERM–Jean Mérieux (D.P., A.B., S.M., H.R.), Lyon, and Epicentre (A. Tiffany) and Pole de Génotypage des Pathogènes, Unité de Recherche et d'Expertise Environnement et Risques Infectieux, Institut Pasteur (V.C.), Paris — all in France; Bernhard Nocht Institute for Tropical Medicine, World Health Organization (WHO) Collaborating Center for Arbovirus and Hemorrhagic Fever Reference and Research, and the German Center for Infection Research (DZIF), Partner Site Hamburg — both in Hamburg, Germany (L.O., T.R., D.C., M.G., M.P., D.T., J.S.-C., S.G.); Institut National de Santé Publique (L.K.), Université Gamal Abdel Nasser de Conakry, Laboratoire des Fièvres Hémorragiques en Guinée (N.M., B.S.), Hôpital National Donka, Service des Maladies Infectieuses et Tropicales (M.S.S.), Ministry of Health Guinea, Prevention and Disease Control (S.K.), and WHO (E.R.M., E.H., A.K.D.), Conakry, Section Prévention et Lutte contre la Maladie à la Direction Régionale de la Santé de Nzérékoré, Nzérékoré (M.L.), and Section Prévention et Lutte contre la Maladie à la Direction Préfectorale de la Santé de Guéckédou (A. Traoré) and Hôpital Préfectoral de Guéckédou (M.K.), Guéckédou — all in Guinea; Médecins sans Frontières, Brussels (H.D.C., M.V.H.); Médecins sans Frontières (A.T., G.D.) and WHO (P.F.) — both in Geneva; and WHO, African Regional Office, Brazzaville, Republic of Congo (B.I.).