IDENTIFICATION OF A NEW B/G RECOMBINANT HUMAN IMMUNODEFICIENCY VIRUS TYPE 1
IN PORTUGAL

Nuno Taveira^1,2, Inês Bártolo^1,2, Rute Antunes^2, Sofia Figueiredo², Maria Helena Lourenço², Ricardo Camacho³, Victor Bezerra⁴, Manuel Pinheiro⁵, José Gonçalo⁵, Helena Barroso^1,2
and José Moniz-Pereira².
¹ISCSS, Caparica; ²URIA, CPM, Faculdade de Farmácia de Lisboa; ³Hospital de Egas Moniz, Lisboa; ⁴Hospital Distrital de Santarém; ⁵Infecciologia Pediátrica,
Hospital de Santa Maria, Lisboa, Portugal.

Introduction

The current AIDS epidemic is mainly caused by HIV-1 subtypes A, B, C, and D, and by the circulatory recombinant forms (CRFs) CRF01_AE and CRF02_AG. Eight other CRFs, CRF03-CRF11, composed of up to four subtypes, have been described, causing distinct epidemics in different geographic regions. Some recently identified CRFs have been disseminated very rapidly by injecting drug users (IDUs), e.g. CRF03_AB, CRF07_BC and CRF08_BC. A new BG recombinant virus has recently been detected among IDUs in Galicia, north of Spain.
As of December 31, 2000, the total number of reported HIV-1 infections in Portugal was 16731. About 50% of these infections affected injecting drug users. The second most important mode of HIV-1 transmission is by heterosexual contact. In Portugal, like in all Western European countries, HIV-1 subtype B strains predominate. However, several non-B subtypes, mainly subtype G strains, also circulate among the heterosexual community. In this study, we have identified and characterized at the molecular and phylogenetic levels, several BG recombinant strains detected in Portuguese patients living in Lisbon.

Material and Methods

Clinical samples

Blood samples were collected from 105 HIV-1 infected individuals belonging to all transmission groups, residing in Lisbon, Portugal. PBMCs were separated by Ficoll-Hypaque gradient centrifugation.

PCR amplification, cloning and sequencing

DNA was extracted from PBMCs by standard procedures. A nested PCR was performed to amplify a 409 bp fragment from the C2-C3 env gene region and a 582 bp fragment from the p17 gag gene region. When PBMCs were not available, virus RNA was isolated by using the Boom’s method. cDNA was produced using the Titan One Tube RT-PCR System (Roche). Amplified DNA fragments were cloned into pCR2.1 using TOPO TA Cloning Kit (Invitrogen). DNA sequencing was carried out by the dideoxy chain termination method with an automated DNA sequencer. Templates were sequenced on both strands, and all ambiguities were resolved.

Phylogenetic analysis and identification of recombination breakpoints

DNA sequences were aligned with reference sequences using the program Clustal X. The alignment was optimized with GeneDoc. Genetic distances were calculated using the Kimura two-parameter model. Phylogenetic trees were constructed by the neighbor-joining method with 1000 bootstrap replications. To identify recombination breakpoints, bootscanning and informative site analysis were performed using Simplot Version 2.5.

Results and discussion

The molecular epidemiology of HIV-1 infection in Portugal was studied by sequencing the C2-C3 env and/or p17 gag gene regions
of 166 patients living in Lisbon and Oporto and surrounding areas, and belonging to all transmission groups, races and genders (Fig. 1).
No group O or N HIV-1 were found. Subtype B viruses were found to predominate in all transmission groups, in particular in the IDU
group. Among the non-B subtypes, HIV-1G predominated both in Oporto and Lisbon.




Figure 1. HIV-1 subtypes circulating in Portugal


Six strains displayed discordant subtype classification in the gag and env genes. Five strains were gag subtype G and env subtype B(Fig. 2); one strain, 00PTHGO21S, was gag subtype B or G, depending on the analysis, and env subtype G. The epidemiological and demographic characteristics of the subjects harbouring recombinant viruses are shown in Table 1.

Table 1. Epidemiological and demographic characteristics of the individuals infected with B/G recombinant viruses.





Figure 2. Phylogenetic analysis of the BG recombinant virus infecting a Portuguese patient


Determination of the recombination breakpoint of BG virus

Phylogenetic analysis suggested the p17 gag gene region of the 00PTHGO21S strain was composed of more than one subtype. Bootscanning analysis was used to determine the subtype composition (Fig. 3). The 5’ half of p17 was assigned to subtype B whereas the remaining sequence was subtype G. By informative site analysis, the recombination breakpoint was mapped to a region that lies between nucleotides 233 and 291. This was confirmed by phylogenetic analysis of the 1-269 and 269-463 sub-fragments.



Figure 3. Recombination breakpoint analysis of the gag p17 region of a BG recombinant virus


The BG recombinant virus is transmitted heterosexually and causes AIDS and death

Two patients, 98PTHEM103 and 98PTHEM104, were thought to be an heterosexual transmission pair. Phylogenetic analysis confirmed the epidemiological linkage between both patients.These results demonstrate that the BG recombinant strain is efficiently transmitted by the heterosexual route.Both patients died of AIDS. Patient 98PTHEM103 died of AIDS ten months after infection. These results indicate that this BG recombinant strain may be highly pathogenic.



Figure 4. The BG recombinant virus is transmitted by heterosexual contacts.Phylogenetic analysis of a known transmission pair.


Conclusions

Co-circulation of B and G subtypes has generated the inter-subtype recombinant HIV-1BG
HIV-1 BG is already disseminated in all transmission groups in Portugal : a new CRF?
The presence of multiple molecular variants of the BG virus indicates that it is evolving rapidly
The potential widespread dissemination of this new recombinant virus in Western Europe may have important therapeutic and vaccination implications




Acknowledgements: This work was supported by Comissão Nacional de Luta contra a SIDA (Portuguese Ministry of Health) through project CRIA CR-3751.