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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AY951953AY951980 and AY949771.
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INTRODUCTION |
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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.
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METHODS |
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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 7001500 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, AB003288AB003293, AB008335, AB008342, AB013500, AB013501, AB018667, AB021287, AF006500, AF031827AF031829, AF070476, AF081782, AF104403, AF121950, AF309966, AY196904, D87255, D87262, D87263, D87708D87715, 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 JukesCantor 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 JukesCantor correction with neighbour-joining trees constructed as described above. Bootstrapping was performed on 1000 resamplings of the alignments.
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RESULTS |
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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 JukesCantor gamma distances produced a tree with topology similar to that produced by other methods with strong bootstrap support for genotype 2, 3 and 4 (7096 %) 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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DISCUSSION |
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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 JukesCantor 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 6472 % 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 88100 % for genotypes 25 (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 7173 % 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 JukesCantor 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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Received 23 December 2004;
accepted 28 February 2005.
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