Centre International de Recherches Médicales de Franceville, BP 769 Franceville, Gabon1
Institut für Tropenmedizin, Tübingen-Universität, Wilhelmstrasse 27, 72074 Tübingen, Germany2
Author for correspondence: Eric Leroy. Fax +241 67 72 95. e-mail leroy{at}cirmf.sci.ga
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Blood samples were obtained from nine individuals selected randomly during the EBOV outbreak in Booué, Gabon. These nine individuals comprised three symptomatic patients who died, three symptomatic patients who survived (Baize et al., 1999 ) and three asymptomatic infected individuals (Leroy et al., 2000
). In each group, one viral gene sequence was determined in a sample obtained at the beginning of the epidemic, while the other two were determined from samples obtained later during the epidemic (Georges-Courbot et al., 1997
). Samples were obtained with the patients' verbal, informed consent and all specimens were drawn and manipulated according to the WHO guidelines on virus haemorrhagic fever agents in Africa (WHO, 1985
). Blood samples, collected on EDTA, were transported on ice to CIRMF (Centre International de Recherches Médicales de Franceville, Gabon) for analysis. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficolldiatrizoate density-gradient centrifugation. Total RNA was extracted from PBMCs and first-strand cDNA was synthesized as described previously (Leroy et al., 2000
). Amplifications of cDNA were carried out as described previously (Leroy et al., 2000
) using the primers listed in Table 1
. PCR products were then excised from the gel and extracted using the QIAquick Gel Extraction kit (Qiagen). VP24 fragments were sequenced on an ALF Express DNA sequencer (Pharmacia Biotech) using the Autocycle 200 Sequencing kit (Pharmacia Biotech). GP, NP and VP40 fragments were sequenced by ACTgene Laboratories (France) using the ABI 373A or 377 Automated Sequencer (Applied Biosystems). The CLUSTAL W (Thompson et al., 1994
) profile alignment option was used to align the newly sequenced GP and NP genes of EBOV Gabon-96 to the filovirus nucleotide sequences of GP and NP that are available in GenBank. Gap-containing sites were removed prior to all analysis. Pairwise distances were calculated using the DNADIST program of the PHYLIP package (Felsenstein, 1993
) using the Kimura two-parameter model of nucleotide substitution. Phylogenetic trees were constructed using both the neighbour-joining (NJ) and the maximum-likelihood (ML) methods. All phylogenetic trees were inferred using PAUP (Swofford, 1998
). The sequences of the GP, NP, VP40 and VP24 genes of the EBOV strain isolated during the 1996 epidemic in Booué, Gabon are available from GenBank under accession numbers AY058898, AY058895, AY058896 and AY058897, respectively.
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The deduced GP amino acid sequences were compared to those of all known EBOV sequences. The mean genetic diversity between Booué-96 and the other Zaire subtype strains was much lower (12%) than that between Booué-96 and the other three subtypes (4271%), at both the nucleotide and amino acid levels, suggesting that Booué-96 belongs to the Zaire subtype (Fig. 2). Analysis of multialignment sequences in the sequenced region of the GP gene (2174 bp) showed only 36 nucleotide substitutions and 15 amino acid changes between Booué-96 and Mayinga-76 (Fig. 1a). Most of these mutations were located in the middle of the GP gene, which encodes a variable region that seems to be subtype-specific. In this hypervariable region of 180 nucleotides (60 amino acids), we have observed 14 nucleotide substitutions (including 10 nonsynonymous) between Booué-96 and Mayinga-76 and three nucleotide substitutions (all nonsynonymous) between the two Gabon strains. Almost 40% of substitutions are located in less than 9% of the GP gene sequence. The two Gabon strains form a subcluster within the Zaire subtype (Fig. 1
). The nucleotide distance in the GP gene is only 0·39% (eight substitutions) between Booué-96 and Gabon-94, while the nucleotide distance with Mayinga-76 and Kikwit-95 is 1·79% (36 substitutions) and 1·04% (21 substitutions), respectively. At the amino acid level, the genetic distance between Booué-96 and Gabon-94 is only 0·99%, while the genetic distance with Mayinga-76 and Kikwit-95 is 2·14% and 0·99%, respectively. The sequenced region of the GP gene presented in Fig. 1(a)
contains 18 N-glycosylation sites. Only one site, located in the hypervariable region, had changed from NDS to NAS between the Zaire and Gabon strains.
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Phylogenetic analysis of the VP24 gene showed only one and eight substitutions in the Booué-96 sequences from the three deceased patients, the three survivors and the four asymptomatic individuals relative to Gabon-94 and Zaire-76, respectively, indicating a low genetic variability in this gene. Interestingly, another asymptomatic individual had exactly the same VP24 sequence as the Gabon-94 strain. This indicates that several virus variants can cocirculate during the same Ebola outbreak and that the variant corresponding to this asymptomatic individual is minor. The low genetic variability of EBOV was shown further by analysis of the NP and GP genes. The high mutation rate between Booué-96 and the Côte d'Ivoire, Sudan and Reston subtypes, and the low mutation rate between Booué-96 and the other Zaire subtype strains demonstrates that Booué-96 belongs to the Zaire subtype.
One feature of members of the family Filoviridae is the contrast between the high genetic diversity between subtypes and the low intrasubtype variability. Indeed, the Booué strain diverges from other strains of the Zaire subtype by only 12%, despite the fact that Booué-96 and Zaire-76/95 were isolated 20 years apart and more than 3000 km apart. This is consistent with another study of the most variable 249 nucleotide region of the GP gene (Rodriguez et al., 1999 ). In contrast to most RNA viruses, EBOV is characterized by high genetic stability, which may be due to four main factors: low error rate of RNA polymerase, slow replication in the natural host, small number of natural hosts and weak immunological pressure. In contrast with other genes, the rate of nonsynonymous substitutions between the two Gabon strains in the NP gene is significantly higher than that between Booué-96 and Mayinga-76 and between Booué-96 and Kikwit-95 (1·27 versus 0·95 and 0·63, respectively). This comparatively high level of nonsynonymous substitutions between the two Gabon strains in the NP gene relative to other genes points to selective constraints on NP. It has been shown that when nonsynonymous substitutions predominate over synonymous substitutions in a viral gene, this gene is subjected to positive selection (Hughes & Nei, 1988
). This positive Darwinian selection is driven generally by host immune pressure (Fitch et al., 1991
; Hughes & Nei, 1989
). This particular NP pattern observed in the two Gabon strains of EBOV differs from the evolutionary patterns of most RNA virus genes, in which synonymous substitutions predominate strongly over nonsynonymous substitutions (the neutral theory of molecular evolution) (Gojobori et al., 1990
). Two main hypotheses may be proposed to explain this particular NP pattern: (i) the two Gabon strains may have diverged much earlier than suggested by GP analysis (Suzuki & Gojobori, 1997
) and (ii) the two Gabon strains diverged recently, but the separation was accompanied by a change in the natural host (species or genus), leading to different immunological pressures on the NP gene. Nevertheless, the coexistence of two different strains, without apparent genetic recombination, in similar geographical and temporal conditions may be possible if the viruses have their own ecological niche. These features of EBOV genetic structure may have implications for the development of treatments and vaccines against EBOV infection. The NP gene pattern of nonsynonymous nucleotide substitutions suggest that NP is subject to functional constraints that induce relatively rapid molecular evolution under immunological pressure. For this reason, the NP gene is a potential candidate target for antiviral drugs. Likewise, the extremely high genetic stability in each EBOV subtype may have implications for the development of vaccines.
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References |
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Received 5 July 2001;
accepted 31 August 2001.