Laboratoire de Virologie moléculaire, transfusionnelle et tropicale, Faculté de Médecine, 13385 Marseille, France1
Dept of Pathology, Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555, USA2
Author for correspondence: Rémi Charrel (at the Laboratoire de Virologie moléculaire, transfusionelle et tropicale).Fax +33 4 91 18 95 98. e-mail virophdm{at}lac2.gulliver.fr
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Abstract |
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GBV-Alab (Leary et al., 1997 ), GBV-Acal (Erker et al., 1998
) and GBV-Atri (Erker et al., 1998
) were isolated from Saguinus labiatus (tamarin), a Callithrix jacchusxpenicillata hybrid (marmoset) and Aotus trivirgatus (owl monkey), respectively. The strain isolated from the Callithrix hybrid, originally named GBV-Amx (Erker et al., 1998
), was renamed GBV-Acal in this study to avoid any confusion with viruses isolated from Saguinus mystax monkeys. The original GBV-A strain (Simons et al., 1995b
) was recovered from an S. labiatus tamarin. However, on the basis of its high degree of genetic similarity to other virus strains isolated from Saguinus nigricollis tamarins (Fig. 2a
), it was probably transmitted from an S. nigricollis tamarin during serial passage in different Saguinus species, as previously reported (Bukh & Apgar, 1997
).
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Because sequencing of complete genomes is an expensive and time-consuming procedure, studies of virus phylogeny are frequently performed by using subgenomic sequences. In the cases of HCV and GBV-C, subgenomic regions have been identified that reflect virus relationships observed by complete polyprotein analysis (Simmonds et al., 1993 , 1994
;Smith et al., 1995
, 1997
). We carried out a similar analysis of GBV-A isolates. The four complete GBV-A amino acid sequences and two GBV-C sequences (U36380 and U44402, acting as an outgroup) were aligned with the CLUSTAL W 1.7 program (Thompson et al., 1994
). Distances were calculated by using the gamma distance algorithm (
=2) and branching patterns were determined by the neighbour-joining method implemented in the MEGA software package (Kumar et al., 1993
) (Fig. 1b
). Phylogenetic analyses were performed on the complete polyprotein, the different virus genes and sequences of 1600 down to 100 amino acids. The robustness of the resulting groupings were tested by 500 bootstrap replications. Discrepant groupings were considered to be invalid even when supported by high bootstrap values. The best results were provided by the complete E2, NS5a and NS5b genes and by partial sequences in the E2 and NS5 regions (Fig. 1b
; see sequences giving bootstrap values >90% at both nodes A and B).
A similar analysis was performed in the 5' non-coding (5'NC) region, using progressively smaller nucleotide fragments, and distances were calculated by the JukesCantor algorithm (Fig. 2c). Only analyses performed with a 345 nucleotide fragment covering positions -190 and -535 provided the appropriate branching pattern with bootstrap support above 90% (Fig. 2c
). These results were confirmed when other distance algorithms (Kimura-2, Tamura, TajimaNei) and another phylogenetic method (unweighted pair group method with averages; UPGMA) were used.
In previous studies, 45 sequences of the 5'NC region of GBV-A were reported (Bukh & Apgar, 1997 ; Leary et al., 1996
). Although phylogenetic analysis of these sequences did not conform completely to the branching pattern observed by complete polyprotein analysis (Fig. 2c
), the distribution of distances between these 45 sequences is interesting. It shows that viruses exhibiting genetic distances greater than 14% originated from different monkey species (Fig. 2b
). The phylogenetic analysis (Fig. 2a
) allows eight clusters (A to H) to be distinguished, each corresponding to sequences recovered from the same monkey species. Analyses performed with JukesCantor or Kimura-2 algorithms and with UPGMA or maximum-parsimony methods showed similar results.
GBV-A sequences from S. mystax and S. labiatus are the most closely related. In nature, these two monkey species have contiguous distributions, but do not form mixed populations (C. Padua, personal communication). Although the possibility of cross-transmission of virus isolates cannot be completely excluded, it must be noted that these two species are genetically closer than any others in the genus Saguinus (Jacobs et al., 1995 ). Thus, the low genetic distances observed between GBV-A viruses isolated from S. mystax and S. labiatus may reflect the recent split between these two species during the evolution of New World monkeys. Only isolate SL-119 did not group with the other virus isolates recovered from the same species (in this case, S. labiatus). This may constitute strong evidence against cospeciation. Nevertheless, SL-119 did not group with any of the other isolates, meaning that this isolate may have been acquired from a monkey belonging to another species with which S. labiatus populations could share a contiguous distribution in nature (C. Padua, personal communication) or from contacts during transportation or captivity.
The presence of different host species and the low genetic distance reported among viruses isolated from a unique monkey species suggested that these viruses shared a long-term evolutionary relationship with their respective primate hosts. Based on present-day genetic evidence, a cospeciation mechanism was evoked. To investigate this hypothesis, we compared the phylogenetic relationships observed among GBV-A and GBV-C isolates with the phylogeny of their primate hosts.
Phylogenetic analysis of the primate hosts was achieved by using the complete nucleotide sequences of the -globin gene (Fig. 3a
). Because sequences of
-globin have not been determined for S. labiatus and A. trivirgatus, sequences from surrogate species (Saguinus midas and Aotus azarai, respectively) were included for analysis. S. labiatus and S. midas belong to the same clade within the genus Saguinus (Jacobs et al., 1995
). A. azarai is currently the only species in the genus Aotus for which the
-globin gene sequence is available from nucleotide databases.
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The rate of evolution of GB viruses in their respective primate hosts was estimated (Fig. 3b, c
). The GBV-A and GBV-C lineages were analysed separately. The different points on each curve reflect the correlation between the time of the splitting event between two given primate species during their evolution (x-axis) and the present-day genetic distances between virus strains that infect these two host species (y-axis). The genetic distance corresponding to the split between New and Old World primates, estimated at 35 million years before the present (MYBP) (Fleagle, 1988
), has been determined by using the average genetic distance (gamma distance with
values of 2 and 0·5) observed between the complete GBV-C (human and chimpanzee isolates) and GBV-A sequences. Therefore, the GBV-A and GBV-C curves share the same origin. The evolutionary split between callitrichines and Aotus has been estimated at 18 MYBP (Porter et al., 1997
) and that between Saguinus (tamarins) and Callithrix (marmosets) at 9 MYBP (Porter et al., 1997
; Schneider et al., 1993
). On the basis of fossil records and molecular evidence, the split between Pan and Homo has been estimated at around 6 MYBP (Hill & Ward, 1988
; Horai et al., 1995
) and the emergence of Homo sapiens occurred around 0·15 MYBP (Cann et al., 1987
). The calculation of the genetic distances between virus isolates was performed by using the gamma distance with
values of 2 (Fig. 3b
) and 0·5 (Fig. 3c
).
The resulting curves show that the rate of evolution in both virus lineages is globally a linear function of time. This suggests that the accumulation of genetic diversity has occurred at a constant rate and demonstrates that virus evolution has followed a similar model in the two lineages. This might not be expected in a case of cross-species virus transmission. By contrast, these data are consistent with the hypothesis of a cospeciation mechanism between the viruses and their respective primate hosts. This mechanism of evolution has been described for other viruses, such as arenaviruses (Bowen et al., 1997 ) and hantaviruses (Morzunov et al., 1998
).
The curves illustrating the evolution of GBV-A and GBV-C lineages present similar slopes, suggesting that both viruses evolved at the same rate in their respective hosts. Between 35 and 6 MYBP, this rate is 3x10-8 amino acid substitutions per site per year. In the last 150000 years, the rate of evolution of human GBV-C increased to 3x10-7 amino acid substitutions per site per year. This phenomenon could be related to the important growth in the population of Homo sapiens. Numerous epidemiological studies have demonstrated that of the 6 billion individuals currently living on earth, more than 1 billion have been infected with GBV-C. This implies a formidable increase in the virus population of human GBV-C. According to the data obtained using a gamma distribution with =2 for the calculation of genetic distances, the increase in the rate of evolution may have begun with the emergence of the very first human ancestors of Homo sapiens, 6 MYBP.
Globally, these data support the hypothesis of a slow genetic evolution of the GBV-C lineage. This result is consistent with the findings of Adams et al. (1998) , who studied the evolution of GBV-C in humans and chimpanzees. Nakao et al. (1997)
reported a higher rate of evolution (2x10-4 amino acid substitutions per site per year) based on analysis of virus sequences obtained from a haemodialysed patient over an 8·4 year follow-up. However, it has been demonstrated that extrapolation of the rate of non-synonymous substitution over a short period of time to reconstruct the history of viruses may tend to underestimate the actual time of divergence (Smith et al., 1997
), probably because this kind of study does not take into account inter-individual virus transmission, which is a crucial parameter of virus dynamics.
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References |
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Received 13 January 1999;
accepted 6 May 1999.