National Retrovirus Reference Center, Department of Hygiene and Epidemiology, Athens University Medical School, M. Asias 75, Athens 115 27, Greece1
Rega Institute for Medical Research and University Hospitals, Katholieke Universiteit Leuven, Belgium2
Aaron Diamond AIDS Research Center, The Rockefeller University, New York 10016, USA3
Author for correspondence: Angelos Hatzakis. Fax +30 1 7719725. e-mail ahatzak{at}cc.uoa.gr
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Abstract |
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This study presents a re-analysis of the three full-length sequences of CRF04_cpx available from Cyprus and Greece (strains 94CY032.3, 97PVMY, 97PVCH) and includes all known HIV-1 subtypes AD, FH, J and K.
To determine any potential relationships of these strains with subtypes in addition to A and G, we performed a bootscanning plot using the Ray Simplot software, version 2.5 (http://www.med.jhu.edu/deptmed/sray/download/). All three available full-length strains of CRF04_cpx were each separately, and subsequently together, compared with reference isolates of subtypes AD, FH, J and K (Fig. 1). More specifically, trees were constructed for a window of 400 bp moving in steps of 50 bp and then the obtained bootstrap values supporting the clustering of the CRF04_cpx isolates with the different subtypes were plotted across the alignment (Gao et al., 1996
). The separate bootscanning plots showed similar patterns, suggesting that the three strains can indeed be considered a CRF. The bootscanning plot suggested that CRF04_cpx isolates consisted of genomic regions belonging to subtypes A, G, H and K as well as regions which showed no apparent similarity to these subtypes (Fig. 1
). Subtype classification for each region was confirmed by phylogenetic analysis by the neighbour-joining method with Kimuras two-parameter correction (Kimura, 1980
) using programs from the PHYLIP package, version 3.5 (Felsenstein, 1993
). The reliability of the phylogenetic trees produced was estimated by bootstrapping (100 replicates). Only representative trees are shown in Fig. 2
. More specifically, clustering of CRF04_cpx sequences in the middle of the pol gene, in the region spanning nt 20003000, was significantly favoured for both subtypes F and K (Fig. 1
). Using this region, CRF04_cpx isolates cluster on a deep branch defined by both subtypes F and K, but are more closely associated with subtype K (Fig. 2
). The clustering of subtype G in the region spanning nt 52515650 was not supported with high bootstrap values in the bootscanning plot (Fig. 1
). Phylogenetic analysis in this region revealed that CRF04_cpx sequences were similar to those of subtypes G and J, but were only distantly related to these subtypes (Fig. 2
).
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Re-analysis of the three available full-length sequences of CRF04_cpx suggested that the three isolates share a common pattern of complex mosaicism consisting of at least five distinct HIV-1 subtypes. The observed recombination pattern was quite similar to that presented in the HIV-1 nomenclature proposal (Robertson et al., 2000 ). More specifically, 3' pol/vif and 5' env are derived from subtype A and G, respectively, and not unclassified regions. To date, CRF04_cpx are unique in mosaicism complexity, representing the only recombinant HIV-1 strains consisting of more than four different HIV-1 subtypes which have spread successfully in several cases (Paraskevis et al., 1999a
, b
). More specifically, the 94CY032.3 strain initially designated as subtype I in 1995 was first identified in Cyprus in two heterosexual partners who presumably became infected in Greece (Kostrikis et al., 1995
; Nasioulas et al., 1998
). Until then, CRF04_cpx strains had been detected in several cases in Greece, accounting for approximately 2% of HIV-1-infected individuals (Paraskevis et al., 1999a
). According to epidemiological data, these strains have not been restricted to particular routes of HIV-1 transmission, as initially suggested (Nasioulas et al., 1999
), but were also found in different risk group categories such as heterosexuals, vertically infected children, intravenous drug users, as well as in a homosexual male (Paraskevis et al., 1999a
). Until now, CRF04_cpx sequences have been detected only in Cyprus and Greece, but they possibly originated from Africa, for two reasons. (a) CRF04_cpx isolates present a complex pattern of mosaicism consisting of subtypes A, G, H, K and U. These subtypes, and especially subtypes H and K, are very uncommon in geographical regions outside Africa, thus suggesting that the putative recombination event between these subtypes possibly occurred in Africa. (b) A partial genomic region clustering with the unclassified portion of CRF04_cpx has been documented for one other recombinant, Z321, from Africa (Getchell et al., 1987
; Srinivasan et al., 1989
; Gao et al., 1998
).
The unique complexity of the CRF04_cpx HIV-1 strains provides evidence for the great capability of the HIV-1 genome to recombine and to give rise to new viruses with an altered genetic make-up. The identification of these recombinant strains in several cases of HIV-1-infected individuals in Greece, infected through different routes, suggests the successful spread of these variants. However, the question that still remains unanswered is whether these strains have altered biological properties associated with their high genetic complexity.
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References |
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Received 17 July 2000;
accepted 25 October 2000.