African origin of GB virus C determined by phylogenetic analysis of a complete genotype 5 genome from South Africa

A. Scott Muerhoff1, Thomas P. Leary1, Mahomed A. Sathar2, George J. Dawson1 and Suresh M. Desai1

1 Infectious Diseases Research and Development, Abbott Diagnostics Division, Abbott Laboratories, Dept 9NB, Bldg AP20-4, 100 Abbott Park Road, Abbott Park, IL 60064-6015, USA
2 Infectious Disease Unit, Nelson R. Mandela School of Medicine, Doris Duke Research Institute, University of KwaZulu-Natal, South Africa

Correspondence
A. Scott Muerhoff
scott.muerhoff{at}abbott.com


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
GB virus C (GBV-C), a positive-strand RNA virus, currently infects approximately one-sixth of the world's population. This virus exists as a collection of genotypes whose global distribution correlates with geographical origin. Genotyping of GBV-C isolates by phylogenetic analysis has relied upon the use of 5'-untranslated region (5'-UTR) sequences, however, complete genome sequences are used to demonstrate definitively their existence and geographical correlation. Initial identification of the fifth genotype from South Africa was based upon phylogenetic analysis of the 5'-UTR. It was sought to confirm this classification by analysis of full-length E2 genes from South African isolates and by analysis of a complete genotype 5 genome. Analysis of full-length E2 genes from 28 GBV-C-infected South African individuals revealed the existence of a unique group of 18 isolates, distinct from the other four genotypes. Bootstrap analysis provided strong support (95 %) for this fifth group. The remaining isolates were either genotype 1 (n=8) or 2 (n=2). Analysis of human E2 gene sequences, with the E2 gene from the chimpanzee variant GBV-Ctro included as the outgroup, produced a tree rooted on the genotype 1 branch. The complete genome nucleotide sequence of South African genotype 5 isolate D50 was determined. Phylogenetic analysis of the 5'-UTR and open reading frame produced congruent trees that grouped the sequences into five major genotypes. Inclusion of the corresponding region of the chimpanzee isolate GBV-Ctro in the analysis produced trees rooted on the branch leading to the genotype 5 isolate D50, suggesting an ancient African origin of GBV-C.

The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AY951953–AY951980 and AY949771.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
GB virus C (GBV-C) is a positive-strand RNA virus that was discovered in 1995 (Leary et al., 1996; Linnen et al., 1996; Simons et al., 1995a) shortly after the discovery of two related viruses, GB viruses A and B in tamarins (Muerhoff et al., 1995; Simons et al., 1995b). GBV-C and GBV-A are more closely related to each other than either is to GBV-B, with hepatitis C virus (HCV) being their next closest relative (Muerhoff et al., 1998; Ohba et al., 1996; Zuckerman, 1995). Since its first description, many groups have investigated the genetic diversity of GBV-C isolates from globally distributed human populations. These studies clearly demonstrated the existence of groups or genotypes of GBV-C viruses that correlated with geographical or ethnic origin (Muerhoff et al., 1997; Okamoto et al., 1997; Saito et al., 1998; Takahashi et al., 1997). Initially, three GBV-C genotypes were described originating from Africa (genotype 1), North America and Europe (genotype 2), and Japan (genotype 3). The number of GBV-C genotypes then expanded to include novel isolates discovered in Myanmar and Vietnam (Naito et al., 1999), and South Africa (Sathar et al., 1999b; Tucker et al., 1999), with both groups claiming identification of the fourth genotype. Analysis of the 5'-untranslated region (5'-UTR) from these novel isolates by Tucker and Smuts (2000), and subsequently by Sathar and York (2001), along with representatives of the three previously described genotypes, demonstrated the existence of five genotypes. It was thus proposed that the novel isolates from South-East Asia be known as genotype 4 and those from South Africa as genotype 5. This proposed classification was validated by Smith et al. (2000) who demonstrated the correlation of five genotypes with geographical origin by analysis of a small segment of the E2 gene. Furthermore, these investigators demonstrated that the evolutionary tree produced by analysis of the small E2 gene segment was congruent to that produced by analysis of complete genomes, at which time only representatives of genotypes 1 through 4 were available.

To extend the work of Smith et al. (2000), we sequenced the complete E2 gene from a cohort of GBV-C RNA-positive individuals from the province of KwaZulu-Natal, South Africa and confirmed the existence of genotype 5 isolates in this population, possibly existing as two genotype 5 subgroups. One of these isolates was chosen for complete genome sequencing and subsequent phylogenetic comparison, to all currently known GBV-C full-length genome sequences, to substantiate the existence of genotype 5. Additional analyses were performed using the GBV-C isolate from chimpanzees (Birkenmeyer et al., 1998) to determine the ancestral origin of GBV-C.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Patients.
Sera were from individuals previously shown to be infected with GBV-C (Sathar et al., 1999a). They included patients with chronic renal failure undergoing maintenance haemodialysis (D), patients with chronic liver disease (L) and adult volunteer blood donors (B). Patients were identified only by code number and their corresponding medical classification (D, L or B).

Amplification of GBV-C sequences.
Serum from a total of 30 GBV-C-infected individuals was available for testing. RNA was isolated from 0·05 to 0·10 ml of serum using the Trizol-LS Reagent (Invitrogen/Gibco-BRL) according to the manufacturer's instructions. The RNA was then reverse transcribed using random hexamers and MMLV reverse transcriptase. The cDNA was used as the template in PCRs designed to amplify the entire E2 gene of GBV-C using the methods previously described (Muerhoff et al., 2003). cDNAs prepared from the South African genotype 5 isolate D50 were used to generate a series of overlapping amplicons spanning the entire genome. Amplification of the 5'-UTR was done as previously described (Muerhoff et al., 1996a). The region of the genome spanning the 3' terminus of the 5'-UTR and the 5' terminus of the E2 gene was amplified as overlapping amplicons using primer pairs ntrCS1/E1Fcon, ntrCS3/E2SeqAR (Muerhoff et al., 1996a, 2003) and E1FconR/C2200R; regions downstream of E2 were produced using gene-specific primers (primer sequences are available from the authors upon request). Amplicons were approximately 700–1500 bp in length with an average overlap of approximately 50 % and covered the entire genome except for a small portion of the 3'-UTR. PCR products were analysed by agarose gel electrophoresis, purified using the Qiagen PCR Purification kit, and then sequenced directly using the amplification primers and the ABI Big Dye Terminator v1.1 Cycle Sequencing kit. Sequences were determined by using the ABI model 3100 Genetic Analyser (Applied Biosystems) and contigs assembled using Sequencher v4.1.4 for Windows (GeneCodes).

Phylogenetic analysis.
Phylogenetic analysis was performed using the program MEGA2 for Windows (Kumar et al., 2001) on nucleotide sequence alignments made using CLUSTALW (Thompson et al., 1994). Sequence alignments were edited using the program GeneDoc (Nicholas et al., 1997). Genotyping was performed by phylogenetic analysis of E2 gene sequences from the patients and the corresponding region from the available full-length GBV-C genome sequences: AX338086, AB003288–AB003293, AB008335, AB008342, AB013500, AB013501, AB018667, AB021287, AF006500, AF031827–AF031829, AF070476, AF081782, AF104403, AF121950, AF309966, AY196904, D87255, D87262, D87263, D87708–D87715, D90600, D90601, U36380, U44402, U45966, U63715, U75356 and U94695. Additional E2 sequences from GBV-C genotype 1 and 2 isolates from Germany were included in the genotyping dataset (Muerhoff et al., 2003). The sequence dataset used for 5'-UTR analysis comprised the corresponding region from the South African isolate D50 and the full-length isolates listed above. Distances between nucleotide sequences were determined using the Jukes–Cantor method (Jukes & Cantor, 1969). Neighbour-joining trees were constructed by the method of Saitou & Nei (1987). Gamma distances were calculated by using the gamma parameter (alpha) estimated from the alignments using PAUP* version 4.0 for Windows (Sinauer Associates). Complete open reading frame (ORF) nucleotide sequences were analysed at synonymous sites by the method of Nei & Gojobori (1986) using Jukes–Cantor correction with neighbour-joining trees constructed as described above. Bootstrapping was performed on 1000 resamplings of the alignments.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Genotyping by E2 gene analysis
Serum from a cohort of 30 South African individuals, previously shown to be GBV-C RNA positive (Sathar et al., 1999b), was extracted for RNA and the GBV-C E2 gene was amplified by RT-PCR. Twenty-eight full-length E2 gene sequences (1004 nt, positions 610–1613 of the U36380 ORF) were obtained and aligned with the corresponding region from all full-length GBV-C genome sequences available in the public domain. Also included were additional E2 genotype 1 and 2 sequences from Germany (Muerhoff et al., 2003). Phylogenetic analysis using gamma distances resulted in the clustering of the GBV-C reference sequences according to their previously determined genotypes (Muerhoff et al., 2003; Smith et al., 2000). Two South African isolates grouped with genotype 2 and eight were subsumed by the genotype 1 reference isolates. However, 18 of 28 South African E2 gene sequences formed a separate, distinct group (Fig. 1) thereby defining the fifth genotype. Bootstrap analysis of the E2 sequence alignment provided strong support (>98 %) for the existence genotypes 1–5 in agreement with previous analysis of 5'-UTR (Sathar & York, 2001; Tucker & Smuts, 2000) or the E2 gene (Smith et al., 2000). There also appeared to be two distinct type 5 subgroups, supported by bootstrap values 94 and 98 %.



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Fig. 1. Unrooted phylogenetic tree produced from alignment of complete GBV-C E2 gene sequences (1004 nt, residues 610–1613 of U36380 ORF, numbering according to Muerhoff et al., 2003). Jukes–Cantor gamma distances (a=0·31) were calculated followed by tree construction by the neighbour-joining method. Reference E2 sequences from complete GBV-C genomes are identified by their GenBank accession numbers; E2 sequences from Germany (E2 suffix, e.g. 1283E2) were reported previously (Muerhoff et al., 2003). Sequences from South Africa are designated by the ‘L’, ‘D’ or ‘B’ prefixes. The line lengths are proportional to the distances between sequences. Bootstrap values from 1000 replicates are shown as percentages.

 
Analysis of full-length genome sequences
One of the South African isolates, D50, was chosen for sequencing of the complete genome. Alignment of the D50 ORF nucleotide sequence with 41 GBV-C complete ORF nucleotide sequences was performed using CLUSTALW. The alignment was then analysed at synonymous sites only because our previous study had shown the strong bias among GBV-C isolates against non-synonymous substitutions (Muerhoff et al., 1997). The representative genotype 5 isolate, D50, resided on a branch near type 1 sequences with its node very close to the tree centre, suggesting it represents a unique non-type 1 isolate. Bootstrap support for group 1 was 67 % and there was 88 % support for the node leading to D50. Bootstrap values of 100 % were obtained for genotypes 2, 3 and 4 (data not shown). Thus, unrooted phylogenetic trees produced by E2 gene and ORF analysis were in agreement.

When the nucleotide sequence of the GBV-C chimpanzee variant, GBV-Ctro, (AF070476) was included as the outgroup isolate in the analysis of complete ORFs at synonymous sites, the root of the tree was placed on the genotype 5 isolate D50 (Fig. 2). Bootstrap support was 77 % for group 1, 83 % for group 4, 80 % for group 3 and 97 % for group 2; however, support for the D50 node was only 53 %. The Egyptian sequence U63715 (Erker et al., 1996) appeared isolated on its own branch between genotypes 2 and type 4 (97 % bootstrap support) although it occupied a position closer to the genotype 2 sequences. The analysis of E2 sequences placed this isolate firmly into the genotype 2 clade. As with Egyptian isolate U63715, the Bolivian isolate AB013501 (Konomi et al., 1999) appears to occupy an isolated position within the tree (90 % bootstrap support). Analysis of E2 sequences identified this isolate as genotype 2. Removal of the putative recombinant forms of GBV-C [i.e. AB013501, AB021287, U75356; (Worobey & Holmes, 2001)] did not significantly alter the topologies of the tree. Analysis of ORF nucleotide sequences using Jukes–Cantor gamma distances produced a tree with topology similar to that produced by other methods with strong bootstrap support for genotype 2, 3 and 4 (70–96 %) but only 50 % support for groups 1 and 5 (i.e. D50). Removal of putative recombinant forms from the analysis increased bootstrap support for genotype 1 to 65 % though support for genotype 5 remained <50 %; the root of the tree was on the branch leading to genotype 4 (data not shown).



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Fig. 2. Phylogenetic tree produced from alignment of complete ORF nucleotide sequences from 41 full-length GBV-C genomes and GBV-Ctro. The neighbour-joining tree was produced from synonymous distances calculated by Nei & Gojobori method. Bootstrap values from 1000 replicates are shown as percentages at the nodes of each of the four major groups and the nodes leading to AB013501 and U63715. The line lengths are proportional to the distances between sequences except for the line leading to GBV-Ctro. Isolates are identified by their GenBank accession numbers. Putative recombinant forms are boxed.

 
Analysis of 5'-UTR sequences
Previous phylogenetic studies utilizing GBV-C 5'-UTR sequences have generally shown good correlation between groupings and geographical origin (Hsieh et al., 1997; Muerhoff et al., 1996b; Smith et al., 1997, 2000). To establish the validity of this method and to attempt to identify the root of the GBV-C phylogenetic tree, analysis of the 5'-UTR was performed using the corresponding region from the available full-length isolates and the chimpanzee variant, GBV-Ctro. Trees were examined for congruence to the ORF and E2-based trees and bootstrap support for each group. Analysis of the 5'-UTR from 37 of the available 41 complete human GBV-C genomes allowed for the examination of the longest possible region (388 nt, corresponding to bases 147–535 of U36380 where base 1 is at the 5'-end of the genome). The resulting neighbour-joining tree is shown in Fig. 3. The topology of this tree is nearly identical to that produced by analysis of the coding region or E2 gene. The exception is the putative recombinant AB013501 which grouped with type 3 sequences with 93 % bootstrap support, though it grouped with genotype 2 based on E2 sequence analysis. This agrees with the findings of Worobey & Holmes (2001) that this isolate represents a recombinant form of GBV-C. Genotypes 2 and 4 were supported by 83 and 88 % bootstrap values, respectively, in the 5'-UTR tree while type 1 sequences were supported at 65 %. The branch leading to genotype 5 isolate D50 was located nearest the genotype 1 group and its node was not as near the centre of the tree as observed upon ORF analysis. Inclusion of the 5'-UTR of GBV-Ctro as the outgroup resulted in a tree with the root closest to D50 as was observed for the coding region tree.



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Fig. 3. Rooted phylogenetic tree produced from alignment of GBV-C 5'-UTR (nt 147–535 based on U36380 genome) from 37 GBV-C genome sequences. The 5'-UTR from GBV-Ctro (AF070476) was included as the outgroup. The neighbour-joining tree was produced from Jukes–Cantor distances. Bootstrap values from 1000 replicates are shown as percentages. The line lengths are proportional to the distances between sequences except for the line leading to the outgroup, GBV-Ctro (chimpanzee variant). Symbols used to designate genotypes: type 1, open triangles; type 2, closed squares; type 3, closed circles; type 4, closed triangles; type 5, open square. Putative recombinant isolate AB013501 is shown by the semi-solid circle.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
GBV-C exists as a collection of closely related variants or genotypes. Historically, the 5'-UTR was first used for the delineation of genotypes via phylogenetic analysis since analysis of small portions of the coding region resulted in an unresolved, or star, phylogeny (Muerhoff et al., 1997). Subsequently, the availability of complete genome sequences allowed comparison of groupings obtained by complete coding region analysis with that obtained from smaller portions of non-coding or coding regions (Smith et al., 2000). Validation of tree topology produced by subgenomic regions was obtained when groupings agreed with that produced from examination of the complete coding region. Smith and colleagues demonstrated this for a small segment of the E2 gene. The recent identification of five GBV-C genotypes was based upon comparison of the 5'-UTR from South African isolates and representatives of the other four major groups (Tucker & Smuts, 2000; Sathar & York, 2001). We sought to confirm the existence of this fifth genotype by analysis of E2 sequences from South African isolates and the analysis of the complete genome of a genotype 5 isolate.

Sequencing and phylogenetic analysis of complete E2 genes from 28 infected individuals from the KwaZulu-Natal province of South Africa demonstrated that 18 sequences represented a variant distinct from the other four previously described genotypes (Fig. 1), thereby defining a fifth genotype. Among the remaining 10, two were infected with genotype 2 and eight with genotype 1. Bootstrap support was >98 % for each of the five groups with genotype 5 consisting of two subtypes (5a and 5b). Calculation of Jukes–Cantor gamma distances from an alignment that included GBV-Ctro as the outgroup produced a tree with the root placed on the branch leading to isolate D69 (genotype 1 from South Africa) with bootstrap support of 64–72 % for the five groups (data not shown). Based on this analysis, GBV-C genotype 1 isolates, which predominate in Africa, appear to be the most divergent of the human isolates.

The complete genome of genotype 5 isolate D50 was determined and no unique insertions or deletions were observed when the nucleotide sequence was compared to all other genome sequences available. Phylogenetic analysis, exclusive of the chimpanzee variant, at synonymous sites produced an evolutionary tree with five genotypes clearly defined with bootstrap support ranging from 67 % for genotype 1 to 88–100 % for genotypes 2–5 (data not shown). Isolate D50, the sole genotype 5 representative, branched near genotype 1 (88 % bootstrap value) but was not subsumed by this group; its node was very close to the root of the tree suggesting it represents a unique GBV-C clade. Removal of the putative recombinant forms of GBV-C (AB021287 from Myanmar; U75356 from China; AB013501 from Bolivia) resulted in a similar tree with bootstrap support of 100 % for genotypes 2, 3 and 4 and 71–73 % for groups 1 and 5. Phylogenetic analysis at synonymous sites with GBV-Ctro included as the outgroup, produced a tree with the four major genotypes supported by bootstrap values >77 % and with the root placed on the branch leading to D50, though bootstrap was rather low (only 53 %) (Fig. 2). Analysis at non-synonymous sites yielded a tree with nearly identical groupings but with the isolate AB003291 (genotype 1) as ancestral. This isolate was shown by Pavesi (2001) to be ‘ancestral’ upon analysis of highly conserved genomic regions but not by analysis of the entire ORF. The placement of the tree root at the D50 branch does not agree with results obtained by analysis of E2 gene sequences using gamma distances where the root was placed at genotype 1 (isolate D69). Analysis of ORF sequences using Jukes–Cantor gamma distances rooted the tree at D50 but bootstrap support was low for groups 1 and 5, only 50 %, but >70 % for groups 2, 3 and 4. Hence, a different root is identified depending upon the genomic region examined and the method used to calculate the tree. However, all methods used herein defined either a genotype 1 or genotype 5 isolate as most divergent, suggesting that the origin of GBV-C is in Africa.

The discrepancy between the E2 and ORF trees with respect to root placement may be due to sampling bias in that too few genotype 1 or 4 sequences were included (only three genomes of each are currently available). Alternatively, the higher degree of nucleotide sequence variation within E2 as compared with the entire genome could be obscuring ancient relationships. Thus, the E2 gene, while being quite useful for genotyping present-day isolates, is insufficient for distant relationships. It may be necessary to obtain full-length genome sequences from additional genotype 1, 4 and 5 isolates before this discrepancy can be resolved. Artefacts due to the inclusion of recombinant forms of GBV-C (i.e. U75356, AB021287 and AB013501; Worobey & Holmes, 2001) in the analyses were eliminated since trees with identical roots were obtained when all recombinant forms were excluded from the datasets.

It was assumed that because the 5'-UTR of GBV-C shares sequence and secondary structure motifs with GBV-A and GBV-B, as well as HCV (Honda et al., 1999; Muerhoff et al., 1995, 1998; Simons et al., 1996), it may possess the proper concentration of invariant sites by which distant evolutionary relationships may be measured. Trees produced from analysis of 5'-UTR sequences were not rooted at genotype 1 as was observed for E2 gene analysis, instead the root was placed on the branch leading to genotype 5 (Fig. 3) – in agreement with the ORF tree. Correction for among-site variation using the gamma distances estimated from the alignments provided consistent bootstrap support (>70 %) for groups 1 and 4 but support for 2, 3 and 5 was generally less than 60 %. In addition, use of gamma distances resulted in trees rooted at genotype 4 isolates, unlike the E2 gene or ORF trees where genotype 1 or 5 appeared to be the most divergent. The reason for this discrepancy is unclear. Thus, while it is possible to use the 5'-UTR, the E2 gene or full-length ORF sequences to establish GBV-C groupings, it remains to be seen which subregion may be useful for conclusive demonstration of ancient origin.

Previous studies have attempted to establish the origin of GBV-C by comparative analysis with either GB virus A (GBV-A) (Tanaka et al., 1998) or with GBV-Ctro (Pavesi, 2001; Smith et al., 2000) or with all GB viruses (Charrel et al., 1999). While comparison to the chimpanzee variant GBV-Ctro is the most logical given the close evolutionary relatedness of humans and chimps – as opposed to humans and New World primates, the natural hosts for GBV-A – the ancestral GBV-C genotype has not been easy to discern. Smith and colleagues did not find statistical support for any genotype as being ancestral by using an F84 model of nucleotide substitution and Pavesi (2001) could not establish ancestry by analysis of complete genomes by using methods similar to those used here. These studies used different datasets of full-length GBV-C genomes. Charrel et al. (1999) provided the most compelling case for African origins of GBV-C by providing evidence for co-speciation between the GB viruses (A, B and C) and their primate hosts. Assuming the co-speciation hypothesis to be correct, the origin of GBV-C must lie within Africa. This is supported by the fact that the intragroup genetic distance of GBV-C type 1 isolates is larger that that for groups 2, 3 or 4 (Table 1), suggesting a long term pattern of human infection in Africa that is not apparent elsewhere.


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Table 1. GBV-C mean intragroup genetic distances calculated from an alignment of the entire coding region nucleotide sequences of 35 unique isolates

Distance values were calculated (a) at all sites using Jukes–Cantor gamma distances (a=0·22) or (b) at synonymous sites only using the Nei & Gojobori method. SE, Standard error.

 


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 23 December 2004; accepted 28 February 2005.



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