National Retrovirus Reference Center, Department of Hygiene and Epidemiology, Athens University Medical School, 75 Mikras Asias Street, GR-115 27 Athens (Goudi), Greece1
Red Cross Hospital, Athens, Greece2
Tzanio General Hospital, Piraeus, Greece3
Rega Institute for Medical Research and University Hospitals, Katholieke Universiteit Leuven, Belgium4
Author for correspondence: A. Hatzakis.Fax +30 1 748 6382. e-mail ahatzak{at}cc.uoa.gr
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Abstract |
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In the current study, we present the analysis of the complete full-length HIV-1 sequence (99GR303) isolated from a Greek sailor who was probably infected in Africa, a location to which he had travelled several times; it is thought that the HIV-1 infection was acquired in Sierra Leone. For this patient, HIV-1 seropositivity was first documented in 1996; plasma samples have been obtained on a regular basis since then for follow-up. For the purposes of the current study, RNA obtained from cryopreserved plasma samples from 1999 was utilized.
RNA was extracted from plasma using the Total RNA Isolation kit (Ambion). Catalysed by reverse transcriptase (RT), cDNA was synthesized with random hexamers and oligo(dT) primers from the GeneAmp RNA PCR kit (Roche), according to manufacturers recommendations.
All genomic regions were amplified by nested PCR using HIV-1-specific primers, as described previously (Paraskevis et al., 2000 ). More specifically, the complete genome of the 99GR303 isolate was obtained by nested PCR amplification of overlapping DNA fragments of approximately 500 bp in length. All second-round PCR products were cloned into the pT-Adv vector (Clontech) and, for each PCR fragment, an individual clone was sequenced directly on both complementary strands using a VGI automated DNA sequencer (Visible Genetics).
Phylogenetic analysis of two fragments of the partial RT region of pol, which was initially examined for monitoring genotypic resistance to anti-retroviral drugs, revealed that the phylogenetic position of the 99GR303 isolate differed in these fragments, suggesting that it may contain an intersubtype recombinant (data not shown). To further investigate the recombination pattern of this isolate, the full-length genome was sequenced and analysed in detail.
To examine for any potential relationships of the 99GR303 isolate with any of the HIV-1 subtypes AD, FH, J and K characterized previously, we performed a bootscanning plot using the SimPlot software (Fig. 1) (Ray, 1998
). More specifically, trees were constructed for a window of 400 bp moving in steps of 50 bp. Bootstrap values obtained supported the clustering of the 99GR303 isolate with the different subtypes and were plotted across the alignment (Gao et al., 1996
). The bootscanning plot suggested that the 99GR303 isolate comprised genomic regions belonging to subtypes A, G, K and J, as well as of regions that did not show any close relationship with any of the HIV-1 subtypes characterized previously (Fig. 1
). Subtype classification for each region was confirmed by phylogenetic analysis using the neighbour-joining method with Kimuras two-parameter correction (Kimura, 1980
) and programs of the PHYLIP package (Felsenstein, 1993
). The reliability of the phylogenetic trees produced was estimated by bootstrapping for 100 replicates. Representative trees of the analysis are shown in Fig. 2
. For the region spanning nucleotides 25002950, the clustering of the 99GR303 isolate with subtype K was not significantly supported (Fig. 1
). Phylogenetic analysis including an additional sequence of subtype K (accession no. AJ249235) showed that the 99GR303 isolate clusters significantly with subtype K for the above region (Fig. 2
). More specifically, the p17/p24 gag region, the 5' RT gene, a middle region of pol and most of env classified as subtype A. The 3' gag/pol region and the 3' portion of pol clustered with subtype G. The 3' pol/vif region, the first exons of tat and rev, as well as vpu/env and a middle portion of pol were classified as subtypes J and K, respectively. Nevertheless, the remaining genomic regions of vpr as well as the C2-V3 region of env could not be classified with any of the HIV-1 subtypes characterized previously and, thus, remain unclassified. Since all known HIV-1 subtypes comprise distinct clades in the C2-V3 region, the existence of a new branch represented by the 99GR303 isolate suggests the existence of an additional HIV-1 subtype (Fig. 2
).
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Analysis of the full-length HIV-1 sequence isolated from a Greek subject who was probably infected in Africa revealed a complex mosaic genomic organization comprising subtypes A, G, J, K and regions of indeterminate classification. Interestingly, the 99GR303 isolate formed an additional cluster in the C2-V3 region of env, where all HIV-1 subtypes characterized previously constitute distinct clades, suggesting that this isolate represents a novel HIV-1 subtype.
The C2-V3 region has been sequenced extensively (Kuiken et al., 1999) and constitutes one of the most highly recommended regions of sequence for use in subtyping HIV-1. The fact that several CRFs, such as CRF01 AE and CRF04 cpx, constitute distinct clades in this region renders it appropriate for the documentation of additional subtypes. Interestingly, analysis of HIV-1 mosaic sequences available to date revealed that there were no recombination breakpoints in this specific genomic region. The absence of recombination breakpoints may be explained by the fact that this region is the most variable region of the HIV-1 genome (Kuiken et al., 1999 ) and, thus, according to the proposed models for recombination, it is not an ideal region for RT jumps (Hu & Temin, 1990
).
The re-analysis of all previously partially characterized HIV-1 recombinants in env revealed that none of them was closely related to the 99GR303 isolate in the C2-V3 region. Nevertheless, the 99GR303 isolate clustered together with the HIM389775 isolate in the above region. Interestingly, HIM389775 was isolated from Nigeria, suggesting that an unrecognized HIV-1 subtype found in the partial genomic regions of the 99GR303 and HIM389775 isolates possibly existed in Western Africa or spread from other African regions.
The genome of the 99GR303 isolate is a complex recombinant comprising at least five distinct subtypes. Subtypes J and K are extremely rare in Western Africa (Heyndrickx et al., 1996 ; Ishikawa et al., 1996
; Takehisa et al., 1997
; Ellenberger et al., 1999
; McCutchan et al., 1999
), suggesting that putative recombination events involving these subtypes could have occurred in other geographical regions. The identification of additional mosaic HIV-1 viruses consisting of partial regions that show similarity to this novel sequence could provide further information on how these isolates originated.
Identification of this partial genomic region in a mosaic sequence, which probably originated from an additional and previously unrecognized HIV-1 subtype, suggests that complex recombinants embody partial genomic regions originating from HIV-1 clades that either existed in the past and became extinct or are extremely rare. Not surprisingly, unclassified regions have been found more frequently in complex recombinants isolated mainly from Central Africa where the greatest genetic heterogeneity of the virus has been documented (Louwagie et al., 1995 ; Takehisa et al., 1998
; Candotti et al., 1999
; Kuiken et al., 1999
; Mboudjeka et al., 1999
; Mokili et al., 1999
; Müller-Trutwin et al., 1999
; Kalish et al., 2001
). This observation may be explained by several reasons. Firstly, Central Africa is the geographical region where HIV-1 originated and evolved and, consequently, several other subtypes may have existed in the past and probably became extinct or are extremely rare (Korber et al., 2000
; Salemi et al., 2001
). Secondly, the greatest genetic variability of the virus has been documented in this region and, therefore, several recombination events among different subtypes in this region could have given rise to complex recombinants (Paraskevis & Hatzakis, 1999
; Kalish et al., 2001
).
The extinction of additional subtypes which may have existed in the past need not necessarily be explained by altered biological properties of the viruses, but may also have resulted from epidemiological circumstances. Further studies are required to clarify the issue of whether the various HIV-1 subtypes or CRFs differ significantly in their biological properties, such as infectivity, disease progression or response to therapy.
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References |
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Received 11 April 2001;
accepted 25 June 2001.