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Journal of General Virology (2001), 82, 575–580. Printed in Great Britain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHORT COMMUNICATION

Re-analysis of human immunodeficiency virus type 1 isolatesfrom Cyprus and Greece, initially designated ‘subtype I’,reveals a unique complex A/G/H/K/? mosaic pattern

D. Paraskevis,1 M. Magiorkinis,1 A. M. Vandamme,2 L. G. Kostrikis3 and A. Hatzakis1

1 National Retrovirus Reference Center, Department of Hygiene and Epidemiology, Athens University Medical School, M. Asias 75,Athens 115 27, Greece2 Rega Institute for Medical Research and University Hospitals, Katholieke Universiteit Leuven, Belgium3 Aaron Diamond AIDS Research Center, The Rockefeller University, New York 10016, USA

Human immunodeficiency virus type 1 (HIV-1) hasbeen classified into three main groups and 11distinct subtypes. Moreover, several circulatingrecombinant forms (CRFs) of HIV-1 have beenrecently documented to have spread widely causingextensive HIV-1 epidemics. A subtype, initiallydesignated I (CRF04jcpx), was documented inCyprus and Greece and was found to compriseregions of sequence derived from subtypes A and Gas well as regions of unclassified sequence. Re-analysis of the three full-length CRF04jcpx se-quences that were available revealed a mosaicgenomic organization of unique complexity com-prising regions of sequence from at least fivedistinct subtypes, A, G, H, K and unclassifiedregions. These strains account for approximately2% of the total HIV-1-infected population inGreece, thus providing evidence of the great capa-bility of HIV-1 to recombine and produce highlydivergent strains which can be spread successfullythrough different infection routes.

According to the updated proposed nomenclature system,human immunodeficiency virus type 1 (HIV-1) has beendivided into groups, subtypes, sub-subtypes and circulatingrecombinant forms (CRFs) (Robertson et al., 2000). CRFsrepresent intersubtype recombinant viruses that have beenspread successfully and share an identical pattern of mosaicism(Robertson et al., 2000). For the designation of a new CRF,representative strains must be identified in at least threeindividuals with no direct epidemiological linkage (Robertsonet al., 2000 ; http :}}hiv-web.lanl.gov}). There are currently

Author for correspondence: Angelos Hatzakis.

Fax ­30 1 7719725. e-mail ahatzak!cc.uoa.gr

four CRFs : (a) CRF01jAE, representing AE recombinantsoriginating from Central Africa and having been extensivelyspread in Asia (Carr et al., 1996 ; Gao et al., 1996) ; (b) CRF02jAG, representing AG recombinants circulating in West andCentral Africa (Carr et al., 1998, 1999) ; (c) CRF03jAB,representing AB recombinants, which originated and spread inKaliningrad (Bobkov et al., 1998 ; Liitsola et al., 1998) ; and(d) CRF04jcpx, representing recombinant viruses found inCyprus and Greece that were initially characterized as A}G}I(Gao et al., 1998 ; Nasioulas et al., 1999) but which wererecently found to be amore complex recombination of genomicsequences derived from subtypes A, G, H, K as well as regionsof unclassified sequence (Robertson et al., 2000). To date,Cyprus and Greece are the only geographical regions wherethese CRFs have been detected. These strains account forapproximately 2% of the total HIV-1-infected individuals inGreece (Paraskevis et al., 1999a).

This study presents a re-analysis of the three full-lengthsequences of CRF04jcpx available from Cyprus and Greece(strains 94CY032.3, 97PVMY, 97PVCH) and includes allknown HIV-1 subtypes A–D, F–H, J and K.

To determine any potential relationships of these strainswith subtypes in addition to A and G, we performed abootscanning plot using the Ray Simplot software, version 2.5(http :}}www.med.jhu.edu}deptmed}sray}download}). Allthree available full-length strains of CRF04jcpx wereeach separately, and subsequently together, compared withreference isolates of subtypes A–D, F–H, J and K (Fig. 1). Morespecifically, trees were constructed for a window of 400 bpmoving in steps of 50 bp and then the obtained bootstrapvalues supporting the clustering of the CRF04jcpx isolateswith the different subtypes were plotted across the alignment(Gao et al., 1996). The separate bootscanning plots showedsimilar patterns, suggesting that the three strains can indeed beconsidered a CRF. The bootscanning plot suggested thatCRF04jcpx isolates consisted of genomic regions belongingto subtypes A, G, H and K as well as regions which showedno apparent similarity to these subtypes (Fig. 1). Subtype

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Fig. 1. Bootscanning plot of 94CY032.3, 97PVMY and 97PVCH sequences compared with representative isolates of subtypesA, U455 and 92UG037.1; B, LAI and RF; C, C2220 and 92BR025; D, ELI and NDK; F, FIN9363 and 93BR025.1; G,92NG083.2 and HH8793; H, 90CR056, VI991 and VI997; J, SE7022 and SE7887; and K 96CMMP535C, with a slidingwindow of 400 bp moving in steps of 50 bp. The starting positions of gag, pol, vif, vpr, env and nef reading frames are shown.

classification for each region was confirmed by phylogeneticanalysis by the neighbour-joining method with Kimura’s two-parameter correction (Kimura, 1980) using programs from thePHYLIP package, version 3.5 (Felsenstein, 1993). The reliabilityof the phylogenetic trees produced was estimated by boot-strapping (100 replicates). Only representative trees are shownin Fig. 2. More specifically, clustering of CRF04jcpx sequencesin the middle of the pol gene, in the region spanning nt2000–3000, was significantly favoured for both subtypes F andK (Fig. 1). Using this region, CRF04jcpx isolates cluster on adeep branch defined by both subtypes F and K, but are moreclosely associated with subtype K (Fig. 2). The clustering ofsubtype G in the region spanning nt 5251–5650 was notsupported with high bootstrap values in the bootscanning plot(Fig. 1). Phylogenetic analysis in this region revealed thatCRF04jcpx sequences were similar to those of subtypes G andJ, but were only distantly related to these subtypes (Fig. 2).

On the basis of these results, the suggested recombinationpattern of the full-length sequences of CRF04jcpx is shown inFig. 3. According to this scheme, CRF04jcpx presents anextremely complex pattern of mosaicism, including at least 15breakpoints that occurred between subtypes A, G, H, K and the

parental HIV-1 isolate from which the unclassified regionsoriginated. More specifically, the p17}p24 gag region, the 3«pol}vif region, the first exons of tat and rev and the 5« terminusof gp41 classified as subtype A. The p7}p6 gag region, the polregion and the 5« portion of gp120 clustered with subtype Gand the 3« region of gp41 as well as a middle portion of pol andthe 3« region of gp120 fell within subtypes H and K,respectively. On the other hand, the remaining genomicregions could not be classified with any of the previouslycharacterized HIV-1 subtypes and thus remained unclassified.These regions will be classified if non-recombinant ‘parental ’subtype I sequences become available.

Furthermore, we re-examined the subtype classification ofthree other recombinant HIV-1 strains MAL, Z321 and BFP90,which were found to cluster with the unclassified portionsof CRF04jcpx in partial genomic regions (Alizon et al.,1986 ; Getchell et al., 1987 ; Srinivasan et al., 1989 ; Gao et al.,1998 ; Montavon et al., 1999). A bootscanning plot wasperformed for the complete pol gene of MAL and BFP90isolates as well as for the genomic region spanning pol, vif}vprand tat}vpu of the Z321 sequence, with a sliding window of400 bp moving in steps of 50 bp. According to the boot-

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Re-analysis of HIV ‘subtype I ’ isolatesRe-analysis of HIV ‘subtype I ’ isolates

Fig. 2. Representative phylogenetic trees of different genomic regions. Positions in the alignments are shown above each tree.The number at the nodes indicates bootstrap values. Only values greater than 75 are shown. 94CY032.3, 97PVMY and97PVCH sequences are boxed.

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(a)

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Fig. 3. (a) Mosaic structure of CRF04jcpx genome organization. Partial regions named as subtypes A, G, H, K and unclassifiedregions are shown in red, green, yellow, pink and white, respectively. Regions with uncertain classification are shown in grey.(b) Phylogenetic trees of BFP90 (nt 2070–3120) and MAL (nt 2070–2970) where both sequences clustered with subtype K,and Z321 (nt 4372–4711) where it clustered with CRF04jcpx sequences.

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Re-analysis of HIV ‘subtype I ’ isolatesRe-analysis of HIV ‘subtype I ’ isolates

scanning plot, MAL and BFP90 showed a high degree ofsimilarity to subtype K in these regions, which was initiallyclassified as ‘ subtype I’. Phylogenetic analysis further con-firmed these observations, as shown in Fig. 3. Interestingly,the above regions coincided with the partial pol region ofCRF04jcpx classified as subtype K (Fig. 3). On the other hand,the Z321 isolate was found to cluster with the CRF04jcpxsequences in partial vif}vpr regions as initially described (Fig.3) (Gao et al., 1998). According to these results, Z321 is theonly recombinant to date that has been found to cluster withthe unclassified regions of the CRF04jcpx in partial genomicregions.

Re-analysis of the three available full-length sequences ofCRF04jcpx suggested that the three isolates share a commonpattern of complex mosaicism consisting of at least five distinctHIV-1 subtypes. The observed recombination pattern wasquite similar to that presented in the HIV-1 nomenclatureproposal (Robertson et al., 2000). More specifically, 3« pol}vifand 5« env are derived from subtype A and G, respectively, andnot unclassified regions. To date, CRF04jcpx are unique inmosaicism complexity, representing the only recombinantHIV-1 strains consisting of more than four different HIV-1subtypes which have spread successfully in several cases(Paraskevis et al., 1999a, b). More specifically, the 94CY032.3strain initially designated as ‘ subtype I ’ in 1995 was firstidentified in Cyprus in two heterosexual partners whopresumably became infected in Greece (Kostrikis et al.,1995 ; Nasioulas et al., 1998). Until then, CRF04jcpx strainshad been detected in several cases in Greece, accounting forapproximately 2% of HIV-1-infected individuals (Paraskevis etal., 1999a). According to epidemiological data, these strainshave not been restricted to particular routes of HIV-1transmission, as initially suggested (Nasioulas et al., 1999), butwere also found in different risk group categories such asheterosexuals, vertically infected children, intravenous drugusers, as well as in a homosexual male (Paraskevis et al., 1999a).Until now, CRF04jcpx sequences have been detected only inCyprus and Greece, but they possibly originated from Africa,for two reasons. (a) CRF04jcpx isolates present a complexpattern of mosaicism consisting of subtypes A, G, H, K and U.These subtypes, and especially subtypes H and K, are veryuncommon in geographical regions outside Africa, thussuggesting that the putative recombination event betweenthese subtypes possibly occurred in Africa. (b) A partialgenomic region clustering with the unclassified portion ofCRF04jcpx 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 CRF04jcpx HIV-1 strainsprovides evidence for the great capability of the HIV-1genome to recombine and to give rise to new viruses with analtered genetic make-up. The identification of these recom-binant strains in several cases of HIV-1-infected individuals inGreece, infected through different routes, suggests the suc-

cessful spread of these variants. However, the question thatstill remains unanswered is whether these strains have alteredbiological properties associated with their high genetic com-plexity.

We wish to acknowledge Gikas Magiorkinis for producing thefigures. This study was supported by the Hellenic Society for the Studyof AIDS and Sexually Transmitted Diseases. D.P. was supported by theHellenic Center for Infectious Diseases. L.G.K. was supported by grantspartly provided by the National Institutes of Health under ROI AI43868.

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Received 17 July 2000; Accepted 25 October 2000

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