Abstract

We describe human immunodeficiency virus type 1 (HIV-1) diversity in Western Brittany, France, and trace the dissemination of HIV-1 non-B subtype infection. The strategy for HIV-1 subtyping used involved subtype specific enzyme immunoassays, heteroduplex mobility assays and phylogenetic analysis of the sequences of env encoding the V3 loop region. Samples were obtained from 567 patients: 465 (82%) were of subtype B and 66 (11.6%) were not (20 were subtype A, 11 subtype C, four subtype D, seven subtype F, five subtype G and 19 others with circulating recombinant forms: 4CRF01_AE, 11CRF02_AG, 1H, 3CRF11_cpx). These findings are consistent with other studies of French populations. There is an epidemiological correlation between subtype B and homosexual or heterosexual contamination in France and between non-B subtype and heterosexual contamination in Africa.

Introduction

Extremely high genetic variability is an important feature of human immunodeficiency virus type 1 (HIV-1). It is a consequence of the high error rate of the reverse transcriptase (RT), the presence of viral RNA as a dimer allowing recombination during reverse transcription, and the high turnover of HIV-1 in vivo [1,2].

HIV-1 strains are classified into three phylogenetic groups: group M (main), which is prevalent world-wide, group O (outlier) and group N (non-M, non-O) [3,4]. Group M comprises the majority of the HIV-1 strains responsible for the AIDS epidemic. It includes at least nine envelope subtypes (A, B, C, D, F, G, H, J and K) and various circulating recombinant forms (CRFs) [5]. Subtype E was reclassified as an intersubtype recombinant called CRF01_AE after the first full genome sequences had been completed [6]. The highest diversity of HIV-1 strains is in sub-Saharan Africa where all subtypes have been found.

As in other countries of western Europe and North America, subtype B viruses predominate in France, but non-B strains are increasingly found. Virological tests for following HIV-1 patients are now available in developed countries, where B subtype is predominant. Their performance with non-B viruses, and particularly recombinant strains, remains to be fully assessed. Patients in developed countries have access to antiretroviral chemotherapy and therefore there is the possibility of the emergence of resistance to treatment. Guidelines for the interpretation of resistance mutations are based on what we know about B subtypes. Epidemiological studies associated with genotyping in developed countries are of great value, as non-B subtype HIV-1 viruses constitute a pool for analysing the behaviour of African viruses outside their country of origin. Furthermore, the appropriate tools are available.

We used three different techniques to study a large number of samples. The aim was to define HIV-1 subtype diversity in Finistere, in northwest France. It is relatively isolated from the rest of France, due to its geographical position at the western end of the Brittany peninsula; moreover, it is largely surrounded by the sea. Brest, the main town, is a naval port and many of the seamen often travel to diverse parts of the world. There are also many fishermen, some of who work as far away as the west coast of Africa. Therefore, the prevalence of non-B HIV-1 strains in Finistere may differ from that elsewhere in France [7].

Materials and methods

Clinical samples

Between 1989 and 2000, the Brittany Regional Health Observatory had an estimated 675 cumulated cases of HIV-1 infection per one million inhabitants in Finistere [8]. Blood specimens from 567 HIV-1 seropositive patients (406 men, 161 women) treated in four Finistere hospitals were collected for viral load quantification in two laboratories (Brest and Quimper hospitals) between January 1996 and January 2000. These samples were serotyped with an HIV-1 subtype specific enzyme immunoassay (SSEIA). The viruses scored by SSEIA as non-B were genotyped by heteroduplex mobility assays (HMA) or direct sequencing or both. The following epidemiological data were collected by clinicians with the consent of each individual: age, sex, country of birth, risk exposure, and probable place of contamination. Note that this was a retrospective study, and epidemiological data concerning 50% of the patients were missing. Patients were enrolled irrespective of their therapeutic regimen and response to treatment.

Serotyping

For each of the enrolled patients, one of the plasma samples with the lowest viral load was used for subtype determination by SSEIA, a peptide immunoassay based on the HIV-1 V3 synthetic consensus sequences (A, B, C, D, E and F subtypes), as previously described [9]. These peptides were purchased from Neosystem (Neosystem, Meylan, France). The peptide showing the highest percentage inhibition indicates the immunodominant subtype reactivity.

Genotyping

Specimens that were non-B or undetermined by SSEIA were genotyped by HMA with plasmids from clades A–H or by sequencing or both.

Nucleic acid extraction

Total nucleic acid was extracted according to the manufacturers’ protocols from ultracentrifuged aliquots of 500 µl of plasma using Amplicor Hepatitis C extraction reagents (Roche Diagnostics, Meylan, France), or from 140 µl plasma with the QIAmp viral RNA kit (Qiagen, Courtaboeuf, France) for HMA and sequencing respectively. Nucleic acids were stored at −80°C in 50 µl RNase-free water.

Peripheral blood mononuclear cells from samples with undetectable viral loads were separated on a Ficoll gradient, pelleted and stored at −70°C. DNA was extracted with QIAmp DNA Blood MiniKit (Qiagen), according to the manufacturer's recommendations.

HMA

Reverse transcription and the first PCR were carried out in one tube using the Titan One Tube RT-PCR kit (Roche Diagnostics), according to the manufacturer's recommendations. A 1.8-kb and then a 0.7-kb internal fragment of the HIV-1 env (encoding the V3–V5 region of the gp120) were amplified by nested PCR with ED5/ED12 as outer primers and ES7/ES8 as inner primers, as previously described [10]. Heteroduplexes were generated with analogous PCR products from a set of reference HIV-1 strains of known env subtype as described by Delwart et al. [10]. Electrophoresis in 5% polyacrylamide gels revealed HIV-1 env subtype [10–12].

Sequencing

RNA was reverse-transcribed and complementary DNA was amplified using the Access one tube RT-PCR kit (Promega, Charbonnieres, France) according to the manufacturer's recommendations, with upstream 5′-ATGGGATSAAAGCCTAARGCCATGTG-3′ and downstream 5′-AGTGCTTCCTGCTGYGCCYAAGAACCCAA-3′ primers corresponding to nucleotides positions 6557–6582 and 7811–7783 respectively in strain HXB2 HIV-1.

Direct sequencing was performed using fluorescent dye primer technology with 7-deaza-dGTP kit (Visible Genetics, Epinay sur Orges, France) according to the instructions of the manufacturer. JA168 and JA169 primers [13] were labelled with Cy5.5 and Cy5 respectively. An OpenGene automated sequencer (Visible Genetics) was used for electrophoresis and data collection. The automated Long-Read Tower was used for electrophoresis. Data were acquired with the Gene Librarian module of Gene Object software by combination of the forward and reverse sequences [14].

Phylogenetic analysis

Sequences were compared to the HIV-BLAST database (http://hiv-web.lanl.gov) to find similarities with known subtypes. They were further aligned using the profile alignment option of CLUSTAL X [15]. This alignment was also submitted to Dambe [16]. V3 protein-coding nucleotide sequences were aligned according to their amino acid sequences. Final alignment was manually edited with the alignment editor GENEDOC [17]. Positions where most of the sequences had gaps and regions that could not be aligned unambiguously were omitted from the analysis. The genetic distances between isolates were computed using DNADIST from the PHYLIP package [18], pairwise evolutionary distances were estimated using the Kimura two-parameter model [19] with a transition/transversion ratio of 2. Phylogenetic trees were constructed using the neighbour-joining method [20] and the reliability of tree topologies assessed by bootstrap analysis with 1000 replicates using SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE modules of the PHYLIP package [18]. HIV-1 reference sequences for various subtypes were taken from the HIV sequence database (see above). Treeview was used to visualise the phylogenetic trees [21]. Sequences were submitted to GenBank under accession numbers AF461904–AF461995.

Statistical analysis

The χ2 test was used to test for associations between subtypes and epidemiological data. Statistical significance was defined as α<0.05.

Results

SSEIA

All of the 567 specimens were serotyped by SSEIA. Four hundred samples (70.5%) were conclusively assigned as subtype B without subsequent genotyping analysis, as SSEIA validity and specificity to differentiate B from non-B subtypes has been confirmed for isolates in countries where B subtype predominates [9,22–24].

Of the 167 non-B reactive samples, 21.6% reacted with one non-B V3 peptide, 22.8% were multireactive. For 20.3% results were consistent with immunoreactivity to subtypes other than A–F, they were called ‘other’. The remaining 35.3% could not be determined and were called ‘undetermined–. In this group, a second sample from each of the corresponding patients on a different date was tested and gave the same result. This could have been due to an immunodeficient state leading to low affinity, or no V3 antibody in these patients [25,26].

Genotyping

The 167 non-B SSEIA samples were genotyped using either HMA (60 samples) or direct sequencing (107 samples). Twenty-six samples were tested by both techniques.

HMA

The 60 samples genotyped by HMA exhibited the following SSEIA reactivities: four A, five C, one D, two E, three F, 19 ‘other–, 19 multireactive and seven ‘undetermined–.

HMA classified all seven samples undetermined by SSEIA as B env subtypes. They were all from AIDS stage patients: low antibody titres may be the reason why they did not show any reactivity to the synthetic B peptide used in SSEIA.

The four SSEIA serotype A results were confirmed to be genotype A by HMA. Only one of the five C serotypes was confirmed as C by HMA; two were found to be genotype A and two were undetermined. Unlike previous authors, we did not observe any cross reactivity between C and A subtypes [9,23,27], possibly because of the small number of samples. One D serotype was shown to be B by HMA; these two subtypes are phylogenetically close. HMA identified 19 other samples as subtype B, which by serotyping were F (n=1), ‘other’ (n=4), multireactive (n=7) and ‘undetermined’ (n=7). Of the 18 samples scored as subtype A by HMA only four were detected as A by SSEIA. Non-B SSEIA serotypes were not all genotyped by HMA because we decided to replace these tests by sequencing analysis. Of 26 samples which were genotyped by both genotyping methods, six showed env homologies with recombinant forms and 16 gave concordant results (data not shown).

Sequencing

We determined the sequences of a 330-bp region within the env gene for 97 non-B SSEIA samples, and they were aligned with CLUSTAL [15]. Five were not included in phylogenetic analysis because they were too short. However, they were submitted to BLAST and were assigned recombinant A/E (two sequences), recombinant A/G, B and C env subtypes according to the best matches. The topology of the resulting neighbour-joining tree (Fig. 1) shows an overall branching order consistent with phylogenies reported elsewhere for full-length env sequences [28]. Subtype assignment was established on the basis of phylogenetic clustering with reference sequences supported by a bootstrap value above 75% [29]. The 92 strains fell into eight different clusters; the seven major clusters (B, D, C, F1, G, H, and recombinent A/E) with high bootstrap values, and one cluster which we called ‘A–, covering various subclusters (Fig. 1). This ‘A’ cluster included 10 sequences matching CRF02_AG and three matching CRF11_cpx. CRF11_cpx strains are mosaics of four HIV-1 subtypes: A, G, E and J. The env region encompassing the V3 loop is of the A type [30], explaining why our three sequences are in the ‘A’ subcluster. The presence of recombinant strains could be the reason why this cluster ‘A’ is not as well supported as the others (52% versus more than 70% of bootstrap replicates). Moreover, eight of the 10 env subtype A sequences close to CRF02_AG had the hexameric GPGQTF amino acid sequence in their V3 loop (data not shown). This pattern has been previously described in A/G mosaic strains [31]. Except for CRF01_AE, which shares a common mosaic pattern and is subtype E in the region examined [32,33], we did not include reference recombinant HIV-1 sequences in the tree, as their recombinant origin would have disturbed the analysis. The branching of our two CRF01_AE like sequences was well supported by a high bootstrap value (100%). Some strains branched dependently of each other (two in cluster B, two in A, two in F1 and two in C). This is consistent with the epidemiologically linked infections documented for three heterosexual couples (indicated with an asterisk in Fig. 1).

Figure 1

Phylogenetic analysis based on alignments of the sequences of 330 nucleotides from the env region of 92 HIV-1 isolates from Western Brittany and reference strains representing 10 genetic subtypes. The neighbour-joining tree was built using the Kimura two-parameter method of estimating genetic distance. Branch lengths are drawn to scale (the scale bar represents 10% nucleotide divergence). Numbers at nodes of the tree indicate the bootstrap values (numbers higher than 75% are shown) obtained from 1000 replicates. The following reference strains were included in the phylogenetic analysis: A1.SE.94.SE7253, A2.CY.94.94CY017.41, D.CD.83.NDK, B.US.90.WEAU160, B.FR.83.HXB2, C.ET.86.ETH2220, C.BR.92.92BR025, J.SE.93.SE7887, G.BE.96.DRCBL, G.NG.92.92NG083, 01_AE.CF.90.90CF11697, F2.CM.95.MP255, F1.FR.96.MP411, K.CD.97.EQTB11C, H.CF.90.90CF056. R: reference strains, ■: CRF02_AG BLAST matching strains, ♦: CRF 11_CPX BLAST matching strains, *: strains with an epidemiological link.

Figure 1

Phylogenetic analysis based on alignments of the sequences of 330 nucleotides from the env region of 92 HIV-1 isolates from Western Brittany and reference strains representing 10 genetic subtypes. The neighbour-joining tree was built using the Kimura two-parameter method of estimating genetic distance. Branch lengths are drawn to scale (the scale bar represents 10% nucleotide divergence). Numbers at nodes of the tree indicate the bootstrap values (numbers higher than 75% are shown) obtained from 1000 replicates. The following reference strains were included in the phylogenetic analysis: A1.SE.94.SE7253, A2.CY.94.94CY017.41, D.CD.83.NDK, B.US.90.WEAU160, B.FR.83.HXB2, C.ET.86.ETH2220, C.BR.92.92BR025, J.SE.93.SE7887, G.BE.96.DRCBL, G.NG.92.92NG083, 01_AE.CF.90.90CF11697, F2.CM.95.MP255, F1.FR.96.MP411, K.CD.97.EQTB11C, H.CF.90.90CF056. R: reference strains, ■: CRF02_AG BLAST matching strains, ♦: CRF 11_CPX BLAST matching strains, *: strains with an epidemiological link.

For 36 patients, genotype results were not available for several reasons: no plasma sample with detectable viral load, and no lymphocyte DNA was available (18 patients: two A, one C, four other, two multireactive and nine undetermined SSEIA serotypes), amplification failure or repeatedly unreadable sequences (18 patients: two A, two multireactive, three other, one C, one E, one F and eight undetermined SSEIA serotypes). Amplification failure may be due to mismatches between PCR primers and the corresponding region of proviral DNA. The primers were designed to incorporate variation, but nevertheless are not necessarily compatible with all virus strains [13,34].

Overall, in our samples, the B subtype was the most prevalent (82% from 567 patients) and the other subtypes found were substantially rarer: A subtype (3.5%), CRFs (3.2%), C subtype (2%), D (0.7%), F1 (1.2%), G (0.9%), and H (0.2%) subtypes.

Epidemiological analysis

Subtyping results were tested for association with risk group and presumed geographic place of contamination (Tables 1 and 2). The B subtype was significantly associated with infection through homosexual contact (a<0.001), heterosexual contact in France (a<0.001) and intravenous drug addiction (a<0.01), consistent with previous findings in France [7]. Most non-B HIV-1 subtype viruses were detected in patients who had heterosexual contacts abroad, mostly in Africa (a<0.001). Of the 12 B subtype viruses resulting from contamination in Africa, six could be French strains because contamination took place in Djibouti (French military area); four of these 12 patients were homosexual.

Table 1

HIV-1 subtype and presumed origin of contamination for the 567 patients

Presumed origin of contaminant strain HIV-1 subtype Total 
 CRF AE CRF AG CPX Und  
France 211       222 
Europe           
Africa 12   44 
Asia          
Brasil          
USA           
Guyana           
Others         
Unknown 226   25 282 
Total 20 465 11 11 36 567 
Presumed origin of contaminant strain HIV-1 subtype Total 
 CRF AE CRF AG CPX Und  
France 211       222 
Europe           
Africa 12   44 
Asia          
Brasil          
USA           
Guyana           
Others         
Unknown 226   25 282 
Total 20 465 11 11 36 567 

Regions of Africa (when known) are: sub-Saharan Africa: two A, one B, one C, one CRF02_AG, one G, one H; West Africa: one B, two G; Abidjan: one CRF02_AG, two Und; Bangui: one A, one CRF01_AE; Burundi: one A, one C; Cameroon: one CRF02_AG; Djibouti: one A, six B, two C, one Und; Gabon: two C; Guinea: one B; Madagascar: one B; Mauritania: one CRF01_AG; Nigeria: one CRF02_AG; Rwanda: three A; Senegal: one Und; Zaire: two D. Others: Guadeloupe (1), foreign travel (5). Und: undetermined subtype. CRF: circulating recombinant form. CPX: complex CRF.

Table 2

HIV-1 subtype and transmission groups

Transmission group HIV-1 subtype Total 
 CRF AE CRF AG CPX Und  
Homosexual/bisexual  137        145 
Heterosexual 11 94  140 
Haemophyliac/transfused  12          12 
Injected drug user 64        68 
Mother to child           
Unknown 155   19 199 
Total 20 465 11 11 36 567 
Transmission group HIV-1 subtype Total 
 CRF AE CRF AG CPX Und  
Homosexual/bisexual  137        145 
Heterosexual 11 94  140 
Haemophyliac/transfused  12          12 
Injected drug user 64        68 
Mother to child           
Unknown 155   19 199 
Total 20 465 11 11 36 567 

Abbreviations: refer to Table 1.

Discussion

We used a sequential strategy to define HIV-1 subtype diversity in Finistere. We obtained 93.7% subtype determination by combining three techniques. The finding of non-B subtypes for more than 11.6% of the HIV-1 positive patients was confirmed by genotyping.

HIV-1 serotyping using V3 synthetic consensus peptide antigens has proven its reliability to discriminate between B and non-B HIV-1 subtypes, particularly in areas where subtype B predominates. Serological differentiation of non-B subtypes is however relatively poor. The antigenic V3 loop domain differs by only a few amino acids between subtypes, so cross reactivity is possible [24]. This serologic assay is useful for preliminary subtyping as it is less expensive and easier to handle than genetic sequencing for large scale screening. We checked the validity of SSEIA by sequencing three serotype B African strains including one from Djibouti and two from elsewhere. In all cases sequencing confirmed the SSEIA results (data not shown).

Phylogenetic analysis of the sequences obtained was an attractive tool not only for its further subtyping approach but also for the epidemiological interest of distributing them in clusters where common relevant elements can be found (contamination link, region of origin, …).

There are various weaknesses in our study. The HMA version we used did not detect recombinant genotypes. HMA is a suitable technique but, as recombinant viruses are becoming more prevalent, there is a need to extend HMA subtyping to other distinct HIV-1 genome regions. Heyndrickx et al. [35] implemented a gag/env HMA which gave a more accurate estimate of the true prevalence of HIV-1 subtypes and intersubtype recombinants in areas like Africa where sequencing technology is not readily available. The subtype assignment used in our study only targeted the V3 region: it was based on a 300–400-nucleotide-long env sequence PCR product. It is therefore possible that the viruses detected had regions outside the amplified V3 region with sequence similarity to different subtypes [6,36,37]. This is particularly true for patients contaminated in geographic regions where multiple HIV-1 strains co-circulate allowing dual infection with different HIV-1 subtypes and HIV-1 intersubtype recombinants to arise and spread.

We report the presence of non-B subtypes in Finistere. Sixty-six samples (11.6%) were non-B HIV-1 subtypes on the basis of V3 analysis. However, this is probably an underestimate as some of the 36 unclassified samples are likely to be non-B subtypes: (i) five infections were contracted through heterosexual contacts in Africa, (ii) of the 25 patients for which we did not obtain any data on the probable place of contamination, 14 gave non-B SSEIA profiles, (iii) the failure of sequencing reactions with uncommon recombinant due to mismatches between primers and templates is possible. The percentage of non-B HIV-1-infected patients in Finistere could therefore be as high as 18% therefore. Eighty-eight percent of those non-B-infected patients were born in France and had acquired the virus from African partners. Our data are in agreement with those of the many studies reporting that CRF_02 AG is prevalent in West Africa, in African immigrants in Europe and among European people who have travelled and/or have lived in Africa [33,38–41]. They are also in agreement with published French national data which describe 15% of HIV-1 infections as non-B subtype [7]. HIV-1 diversity in France is largely due to African immigrants. There are few African immigrants in Finistere and non-B viruses are brought to the area by returning travellers.

Our data constitute a valuable tool for adapted follow up of Finistere's HIV-1 patients, and in particular for interpreting viral load and genotyping resistance tests. The partially env sequences obtained are an interesting starting point for further studies such as investigations into HIV-1 recombination.

The presence of non-B subtypes must be taken into consideration in diagnostic tests such as measurement of viral load and antiretroviral drug resistance testing. Current antiretroviral drug resistance genotyping assays have been designed on the basis of the prevalent sequence patterns circulating in the USA and Europe, which belong to the subtype B. However, recent studies seem to indicate that those tests may be unsatisfactory for some non-B viruses [42,43]. Once tests adapted to non-B subtypes become available, the clinical implications of subtype for phenotypic and genotypic resistance should be studied. Twenty-seven percent (18/66) of the env non-B viruses in our study seem to be CRFs. The simultaneous presence of multiple subtypes is now common in many regions outside Africa and determining the role of these recombinant strains in the HIV-1 pandemic is of great importance.

Acknowledgements

This work was supported by grants from the Communaute Urbaine de Brest. We thank Dr Yann Dorval for providing HIV-1 plasma samples from Quimper and Dr Florence Damond for providing HMA plasmids. We express our gratitude to Dr Granier, Hôpital d'instruction des Armées de Brest, Dr Roge, Morlaix hospital, Marie-Christine Derrien, Jocelyne Guevel, Andre Blouët for their help in collecting epidemiological data. We also thank the Visible Genetics company for their technical support and Stephanie Gouriou for her help with submitting sequence data. We are grateful to the organisers of the VIIth workshop on virus evolution and molecular epidemiology, particularly to Anne-Mieke Vandamme and Joke Snoeck for their advice on phylogenetic analysis.

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