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In March 2014, the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea. Virologic investigation identified Zaire ebolavirus (EBOV) as the causative agent. Full-length genome sequencing and phylogenetic analysis showed that EBOV from Guinea forms a separate clade in relationship to the known EBOV strains from the Democratic Republic of Congo and Gabon. Epidemiologic investigation linked the laboratory-confirmed cases with the presumed first fatality of the outbreak in December 2013. This study demonstrates the emergence of a new EBOV strain in Guinea.

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. 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-cm 2 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. 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 (jModelTest ) 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, 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 software 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. Identification of the EBOV Strain 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.

In addition, we performed EBOV-specific real-time RT-PCR assays targeting the glycoprotein (GP) or nucleoprotein (NP) gene. Samples from 15 of 20 patients tested positive on the conventional L gene PCR assay and the real-time assays ( Table 1 Demographic, Clinical, and Virologic Characteristics of 15 Patients with Confirmed Ebola Virus Disease during the 2014 Outbreak in Guinea.

EBOV was identified in the serum of one patient on electron microscopy ( 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., 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. 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. Festo Fluidsim Download Kostenlos Deutschkurs. Sequencing of Samples from Patients 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 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. Black Shades 4 8 Crackers there. 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 ().