Department of Medical Microbiology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK1
Department of Microbiology, School of Medicine, Universidad del Pais Vasco, 48080 Bilbao, Spain2
Centre for HIV Research, University of Edinburgh, Waddington Building, Kings Buildings, West Mains Road, Edinburgh EH9 3JN, UK3
Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin EH25 9RG, UK4
Department of Medicine, University of Natal, Congella, South Africa 40135
Author for correspondence: Donald Smith. Present address: Garden Cottage, Clerkington, Haddington, East Lothian EH41 4NJ, UK. e-mail Donald.B.Smith{at}gardencottage.screaming.net
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Abstract |
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Introduction |
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At present 33 epidemiologically unrelated GBV-C HGV complete virus genome sequences are available, including three of group 1 and several isolates of uncertain relationship to previously defined groupings. In addition, the recent discovery of closely related chimpanzee viruses (Adams et al., 1998 ) and the availability of a complete genome sequence (GBV-Ctro) (Birkenmeyer et al., 1998
) allows phylogenetic trees to be constructed using a more appropriate outgroup. We have therefore undertaken a re-analysis of the phylogenetic relationships of GBV-C/HGV complete genome sequences, the extent to which these can be reproduced by analysis of subgenomic regions and the implications of virus geographical variation for theories about its evolutionary history.
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Methods |
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Serum samples.
Sera were collected from individuals from Papua New Guinea, Sudan and The Gambia as part of population-based hookworm or malaria surveys, from pregnant women in the Democratic Republic of Congo (Mokili et al., 1999 ) and from blood donors from Saudi Arabia. Sera from South Africa were as described previously (Sathar et al., 1999
).
PCR amplification of GBV-C/HGV genomes.
RNA was extracted from plasma using a proteinase KSDS lysis buffer (Jarvis et al., 1994 ). The 5'-NCR was detected by nested RTPCR amplification using the outer primers 5' TGCCACCCGCCCTCACCCGAA 3' (positions -21 to -41) and 5' AGGTGGTGGATGGGTGAT 3' (-443 to -426), and the inner primers 5' GGRGCTGGGTGGCCYCATGCWT 3' (-76 to -97) and 5' TGGTAGGTCGTAAATCCCGGT 3' (-415 to -397). Amplification reactions were 30 cycles for each round and consisted of 94 °C for 36 s, 55 °C for 42 s and 72 °C for 90 s.
For samples that were PCR positive for the 5'-NCR [7/74 (9·4%) Papua New Guinea, 2/66 (3%) Sudan, 3/74 (4%) The Gambia and 1/48 (2%) Saudi Arabia], the E2 region was reverse transcribed and amplified from RNA purified in a combined reaction using a standard buffer system (Access PCR, Promega) according to the manufacturers instructions. Two sets of primers were used to amplify adjoining regions of the E2 gene. Set 1 consisted of outer primers 5' GCCTCHGCCAGCTTCATCAGRTA 3' (16821660) and 5' GGYAAYCCGGTGCGGTCVCCCYTGC 3' (12551279), and inner primers 5' AAAYACAAARTCCARVAGCARCCA 3' (16501627) and 5' TCCTACRCCATGACCAARATCCG 3' (12881310). Amplification conditions were 30 cycles of 94 °C for 18 s, 55 °C for 21 s and 72 °C for 90 s. Set 2 consisted of outer primers 5' ARCTYYGAACACCRSCGVACCAG 3' (14991477) and 5' GCCASYTGYACCATAGCYGC 3' (979998), and inner primers 5' ACCCRAACGTYCCRGTBGGAGGC 3' (13751353) and 5' GTNGYBGAGCTSTYCGAGTGGGG 3' (10271046).
The region of NS5A where duplications are observed in some isolates (Tanaka et al., 1998 ) was amplified for 5'-NCR-PCR positive samples from the Democratic Republic of Congo (n=7), The Gambia (n=4), Sudan (n=2) and Papua New Guinea (n=8) using outer primers 5' CACAATAGGCTGTATGGTTCTGG 3' (positions 67366714) and 5' CCATCGCCWGCACTWATCTCGG 3' (positions 64096430), and inner primers 5' TACRGARAGRGCCACRTTGAAGAC 3' (positions 65736550) and 5' ACNGAGAGCAGCTCAGATGAG 3' (positions 64336453). Amplifications were started at 80 °C followed by 30 cycles of 94 °C 18 s, 52 °C 21 s and 72 °C 90 s. The size of amplified DNA fragments was assessed by electrophoresis through 4% Metaphor agarose gels, with expected sizes of 140 or 180 bp for fragments with and without a duplication.
Nucleotide sequencing and phylogenetic analysis.
Nucleotide sequences of amplified fragments were obtained by direct sequencing of amplified genome regions using Thermosequenase (Amersham) in reactions containing 33P-labelled dideoxynucleotides. Potential RNA secondary structures were investigated using RNADraw version 1.0 (Matzura, 1995 ).
Complete coding region sequences were analysed by three different methods. (1) Distances were estimated using an F84 model of substitution (Felsenstein, 1984 ), which allows for unequal transition/transversion rates and unequal base frequencies. 100 bootstrapped datasets, distance matrices, neighbour joining trees and a consensus tree were produced using SEQBOOT, DNADIST, NEIGHBOR and CONSENSE, all part of the PHYLIP package (Felsenstein, 1993
). (2) Synonymous and nonsynonymous distances were estimated using Method I of Nei & Gojobori (1986)
for 200 datasets produced by bootstrapping codons (D. Haydon, unpublished). Neighbour joining trees and a consensus tree were produced as above. (3) Distances were estimated using a Tamura & Nei (1993)
model of substitution, a more general form of the F84 model that also allows for unequal transition rates between purines and pyrimidines, together with rate heterogeneity modelled as a proportion of invariable sites plus eight rates taken from a discrete gamma distribution using parameters estimated from the data. 100 bootstrapped datasets were produced using SEQBOOT, and distance matrices calculated with the programs PUZZLE version 4.0.2 (Strimmer & von Haeseler, 1996
) and PUZZLEBOOT version 1.01 (Holder & Roger, 1999
) using parameters estimated from the dataset. Trees with the lowest least-squares deviation were produced using the program FITCH applying global search, and a consensus tree produced using CONSENSE. Phylogenetic trees of subgenomic regions were produced with MEGA (Kumar et al., 1993
) using the Kimura 2-parameter model of substitution on datasets of 100 bootstrap replicates.
In order to determine which GBV-C/HGV group was closest to the outgroup GBV-Ctro (AF070476), we used a reduced dataset consisting of three sequences from each group (Group 1, HGU36380, AB003291, AB013500; Group 2, AB013501, U44402, U63715; Group 3, AB003288, D90601, U94695; Group 4, AB003292, AB018667, AB021287). We estimated the likelihood of the outgroup clustering with each of the four groups under four different models of nucleotide substitution: (i) an F84 model; (ii) an F84 model allowing for rate variation modelled using a discrete gamma distribution with eight categories; (iii) as for (ii), but allowing for different transition/transversion ratios, different base frequencies, different rates and different levels of rate heterogeneity for each gene; and (iv), as for (ii) but allowing parameters to differ for each codon position. These models were fitted and the bootstrap support for each cluster compared using the fast approximate bootstrap procedure of Kishino & Hasegawa (1989) as implemented in the PAML version 2.0 package (Yang, 1999
). Similar analyses of amino acid sequences were carried out using Phylip 3.57c (Kimura substitution matrix), or RELL [Jones et al. (1992)
substitution matrix)] assuming gamma rate heterogeneity.
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Results |
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Subgenomic regions
Next, we investigated the extent to which subgenomic regions of GBV-C/HGV bear the same phylogenetic relationships as do entire virus genomes. Analysis of individual virus genes failed to produce congruent phylogenetic trees with the sole exception of the E2 gene (Fig. 3). Similar analysis of subgenomic fragments of 2000, 1000 or 500 nt produced congruent trees only for fragments including the COOH-terminal region of the E2 gene. Analysis of the entire 3'-terminal half of the virus genome or any of its subfragments failed to produce congruent trees.
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Since a 500 nt fragment of the E2 gene could reproduce the phylogenetic relationships of the complete genome sequences, we next analysed this region in more detail (Figs 4 and 5
). Congruent phylogenetic trees were produced using a region as small as 200 nt (positions 13441543). The shortest region that gave a congruent tree with more than 98% bootstrap support for each group was the 600 nt region from positions 9941594. This region also produced a congruent tree when analysed at synonymous sites (>85% support) but not at nonsynonymous sites.
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Similar analysis of 5'-NCR sequences is complicated by the large number of sequences available within this region (1409 accessions) and the inconsistent phylogenetic relationships of all but the largest fragments (Fig. 6). Visual examination for motifs distinct from those typical of groups 1, 2, 3 and 4 revealed a small number of unusual sequences, but of these only the variants from South Africa (Sathar et al., 1999
) described above, and isolates from Spain (Lopez-Alcorocho et al., 1999
) grouped separately from the 5'-NCR sequences of complete genome sequences for the region -388 to -1. These later isolates could represent recombinants since they have motifs typical of group 3 sequences between positions -489 and -459 but the remainder of the 5'-NCR is similar to, although distinct from, that of group 2 isolates.
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Discussion |
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An unexpected finding of this study is that the phylogenetic relationships of complete GBV-C/HGV sequences (but not subgroupings) can be reproduced by short COOH-terminal fragments of the E2 gene (Fig. 4). Previous studies of smaller numbers of sequences have shown that phylogenetic relationships are inconsistent when comparisons are made for individual genes (Takahashi et al., 1997b
) or subgenomic fragments (Smith et al., 1997b
). Although three (Cong et al., 1999
) or four (Lim et al., 1997
) phylogenetic groupings are observed when fragments larger than 1500 nucleotides including the E2 gene are compared, interpretation of these studies is complicated by idiosyncratic labelling, the absence of group 4 isolates and the failure to assess the robustness of groupings. We show here that analysis of a 200 nt fragment from the centre of the E2 gene (positions 13441543) provides >75% bootstrap support for all four phylogenetic groupings, while a 600 nt region (positions 9941594) provided >98% support. This is in stark contrast to other coding regions: all subgenomic fragments of 2000 nt or less that did not contain this region of E2 failed to produce congruent trees with the sole exception of a 2000 nt fragment encompassing the remainder of the E2 gene, NS2 and the NH2-terminal half of NS3 (Fig. 3
). An important unresolved question is the extent to which some complete genome sequences represent recombinants between different groups. Inconsistent relationships were observed for some sequences between the 5'-NCR and coding regions or between different coding regions. However, the aberrant groupings were typically weak and inconsistent suggesting that these do not represent simple recombinants.
Our analysis helps to clarify previous conflicting studies on the extent to which subgenomic regions can be used to identify GBV-C/HGV phylogenetic groupings. Most early studies of virus diversity concentrated on a 118135 nt fragment within NS3 that was the first part of the genome to be sequenced (Simons et al., 1995 ; Masuko et al., 1996
; Heringlake et al., 1996
; Kao et al., 1996
; Berg et al., 1996
; Schreier et al., 1996
; Tsuda et al., 1996
; Schmidt et al., 1996
; Muerhoff et al., 1997
; Pickering et al., 1997
; Ibanez et al., 1998
). However, the phylogenetic conclusions of these studies appear to be unreliable since analysis of even the entire NS3 gene fails to produce congruent groupings. Inconsistent phylogenetic groupings have also been observed for the NH2 terminus of E2 (Lim et al., 1997
; Muerhoff et al., 1997
), 354 nt of NS5A/NS5B (Viazov et al., 1997
) or 279 nt of NS5B (Muerhoff et al., 1997
). Finally, although analysis of a 2600 nt fragment containing both NS5A and NS5B produced a congruent tree for 12 isolates from groups 1, 2 and 3 (Khudyakov et al., 1997
), comparison of this region from the 33 complete genome sequences gave aberrant groupings of some isolates with only marginal support for groups 1 and 4 (data not shown).
The inconsistent phylogenetic relationships of GBV-C/HGV subgenomic coding regions contrasts with that observed for HCV where analysis of a variety of subgenomic regions reproduces the phylogenetic relationships of complete genomes (Simmonds et al., 1994 ; Tokita et al., 1998
). This difference could arise if different regions of the GBV-C/HGV genome were subject to different evolutionary processes in which case combining these regions could produce a less reliable reconstruction than the analysis of individual regions (Bull et al., 1993
). However, the validity of the analysis based on complete genome sequences is supported by the correlation between their phylogenetic relationships and their geographical origin. An alternative explanation is that the amino acid sequence of the GBV-C/HGV polyprotein is well conserved (dN:dS 0·033, divergence <11%) relative to HCV subtypes (dN:dS 0·094, divergence <10%). Phylogenetic groupings of GBV-C/HGV therefore rely more on variation at synonymous sites, many of which are invariant or saturated, possibly because of the presence of extensive RNA secondary structures within the GBV-C/HGV genome (Simmonds & Smith, 1999
). Consequently, GBV-C/HGV isolates with a common evolutionary origin may share only a small number of polymorphisms and analysis of subgenomic regions could often fail to produce congruent phylogenetic trees.
There are several potential explanations for our observation that analysis of the E2 gene or specific subfragments produces phylogenetic trees congruent with those observed for complete genome sequences. First, since E2 is the most variable part of the GBV-C/HGV polyprotein (Katayama et al., 1998 ; Erker et al., 1996
), the phylogenetic relationships of complete genome sequences could depend entirely on substitutions in E2. However, identical groupings are observed if E2 is excluded and most amino acid substitutions occur at the NH2 terminus of E2 (Lim et al., 1997
; Katayama et al., 1998
; Cong et al., 1999
), whereas it is only the central and 3' regions that produce congruent phylogenetic trees, and then only for synonymous rather than nonsynonymous substitutions. A second potential explanation is that this region of the genome encodes an open reading frame on the anti-sense strand (nucleotides 8701226, Fig. 4
), possibly encoding a nucleocapsid protein (Kondo et al., 1998
). This would constrain the accumulation of substitutions and so retain evidence of phylogenetic relationships. However, although synonymous substitutions are suppressed in this part of the genome (Muerhoff et al., 1997
; Simmonds & Smith, 1999
), phylogenetic analysis of the anti-sense reading frame fails to produce a congruent tree (Fig. 4
). Another potential source of constraint in this region of the genome would be if translation of the anti-sense reading frame was dependent on an upstream internal ribosome entry site. Indeed, substitutions in this region of the genome are frequently covariant and associated with potential RNA secondary structures (Simmonds & Smith, 1999
) (Fig. 7
), while RNA folding predictions for representatives of groups 13 identify structures with free energies one standard deviation below those of random sequences of the same base composition. However, these RNA structures were not observed in sequences from group 4 isolates, and the anti-sense reading frame is 10 residues longer in some group 3 isolates and lacks the initial AUG codon in GBV-Ctro.
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Finally, since a virus related to GBV-C/HGV is present in chimpanzees (Adams et al., 1998 ; Birkenmeyer et al., 1998
), while New World monkeys harbour more distantly related but species-specific variants (Bukh & Apgar, 1997
; Leary et al., 1996a
) it is possible that GBV-C/HGV has been continuously present in human populations since speciation. In this case the virus appears to have evolved at an overall rate of less than 10-5 per site per year (Suzuki et al., 1999
; Simmonds & Smith, 1999
), although its rate of sequence evolution measured in longitudinal studies is similar to that of other RNA viruses. Further sequence analysis of the E2 region from GBV-C/HGV variants isolated from different geographical regions may help to clarify the origins of this unusual virus.
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Acknowledgments |
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Footnotes |
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Received 17 August 1999;
accepted 30 November 1999.