Sample collection. A total of 131 HIV-1 samples whose envelopes (V3-V5) were genetically characterized were analyzed. Fifty samples were obtained from in- or outpatients, mainly African, attending the Bichat Hospital in northern Paris (European samples). The genetic subtypes of these samples were identified by HMA (n = 47) or sequencing (n = 3). Among these 50 samples, the genetic subtype distribution was as follows: 15 were subtype A-like, 10 were subtype B, 6 were subtype C, 5 were subtype D, 4 were subtype F, 4 were subtype G, 3 were subtype H, and 3 were CRF01_AE. Discrimination between subtype A and CRF02_AG in the envelope was not possible since the genetic subtypes were identified by HMA.
An additional 81 samples (African samples) were obtained from patients attending the Military Hospital in Yaounde, Cameroon (n = 36), and patients attending one of the three major hospitals in Dakar, Senegal (n = 45). For all these samples the genetic subtype was identified by sequence analysis; and the subtype distribution was as follows: 19 were subtype A, 3 were subtype B, 3 were subtype C, 9 were subtype D, 7 were subtype F, 12 were subtype G, and 28 were CRF02_AG.
Peripheral blood mononuclear cell isolation, DNA extraction, and reference subtyping. Peripheral blood mononuclear cells were separated on a Ficoll gradient. They were washed twice in phosphate-buffered saline (PBS), pelleted, and stored at −80°C. DNA was extracted as described previously (
32).
The
env subtypes of 47 European samples were determined by
env HMA as follows: 1μg of DNA was used for PCR amplification with primers ED3 and ED12. The second round of amplification was performed with 2 μl of the first-round product and primers ES7 and ES8, as described by Delwart et al. (
15). Subtype reference samples were amplified with second-round primers and 10 ng of plasmids A to H as templates. HMA was performed as described previously (
14).
Three European samples and all African samples were genotyped by sequencing of the V3-V5 region of the
env gene (700 bp, amplified with HMA primers ED5 and ED12 as outer primers and primers ES7 and ES8 as inner primers), followed by phylogenetic analysis, under the conditions described previously (
62).
DEIA genotyping. (i) Choice of subtyping region and selection of identification probes. The subtype-specific probes were selected by analyzing the entire consensus sequences of the
env genes of pure subtypes A to D and F to G and the two CRFs, CRFs AE and AG (HIV Sequence Database). The criteria for probe specificity were set on the basis of previous experience with the DEIA genotyping of HCV (
25,
31,
61). Probes were 20 to 30 bases long and were designed such that the melting temperatures (
Tms) of all probes were close, with intersubtype differences of at least 20% (about 4 different bases), and the region corresponding to the probes was conserved for a given subtype. The most suitable region presenting these characteristics comprised the first codon of the
env coding sequence (
env cds) to the middle of conserved region C1 of gp120 (DEIA region).
A set of different probes specific for this region was defined. Single subtype-specific probes for subtypes C, F, and G were easily identified (Table ). Unique probes for subtypes B and D could not be found. The lack of specificity of the subtype D probe, which also detected non-B, non-D subtypes, was associated in part with a high level of sequence similarity between subtypes B and D. Therefore, samples of subtypes B and D (called subtype BD) were first distinguished with a common subtype BD-specific probe (probe SBD3), followed, if a positive result was obtained, by specific identification with subtype B- and subtype D-specific probes (Table ).
| TABLE 1.Characteristics of subtype-specific probes |
It was difficult to find a specific probe for subtype A due to the close relationship between this subtype, CRF01_AE, and CRF02_AG. The 5′ end of the env gene of the two CRFs is indeed partly subtype A-like (HIV Sequence Database). A common probe (probe SA2) that identifies subtype A, CRF01_AE, and CRF02_AG was therefore identified, with discrimination between subtype A and the two CRFs being achieved with additional CRF-specific probes, designed on the basis of CRF-specific signature sequences (Table ).
After preliminary assays, nine probes (probes SA2, SAE1, SAG1, SBD3, SB2, SD1.1, SC1, SF6, and SG1) were selected for the detection and identification of samples of subtype A, CRF01_AE, and CRF02_AG; samples belonging to either subtype B or D; samples of subtype B; samples of subtype D; and samples of subtypes C, F, and G (Table ). They were synthesized, 5′ biotinylated, and purified by high-pressure liquid chromatography (Eurogentec, Seraing, Belgium). All probes were 20- to 29-base oligonucleotide probes. The probe sequences and Tms are presented in Table . Nine oligonucleotides with sequences complementary to those of the nine probes were used as positive controls for hybridization.
(ii) Subtype-nonspecific PCR. A nested PCR for the region that spans the 5′ end of the
env gene (DEIA region) of all genetic subtypes A through H and the two CRFs (CRFs AE and AG) was performed to amplify a 250-bp fragment encompassing the sequences of the identification probes (Fig. ) (HIV Sequence Database). The primer sets used were primers ED3 and ED14 as outer primers (as described for HMA) and primers ES4 and ES5 as inner primers (designed for DEIA), (see the sequences in Fig. ). For the first round, 10 μl of DNA template was added to a 90-μl reaction mixture consisting of 10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl
2, each deoxynucleoside triphosphate at a concentration of 0.2 mM, 20 pmol of each primer, and 3.5 U of Expand high-fidelity
Taq polymerase (Roche Diagnostics, Mannheim, Germany). Ten microliters of product from the first round was used for the second round in 100 μl of the same mixture used for the first-round PCR and 2.5 U of
Taq polymerase (Promega, Madison, Wis). The first-round PCR was performed as previously described by Delwart et al. (
14); a first denaturation step for 2 min at 94°C was followed by 3 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min and 32 cycles of 94°C for 15 s, 55°C for 45 s, and 72°C for 1 min, with a final extension of 5 min at 72°C. The conditions for the second round were a denaturation step of 2 min at 94°C, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension of 7 min at 72°C.
(iii) DEIA procedure. The DEIA was based on the hybridization of amplified single-stranded DNA (ssDNA) with oligonucleotide subtype-specific probes attached to the surfaces of microtiter plate wells by a streptavidin-biotin bond. Hybrids of the probe and the ssDNA were detected with a mouse monoclonal anti-DNA antibody. Antibody binding was detected by means of a colorimetric immunoenzymatic reaction.
The DEIA was performed with a GEN-ETI-K PS0001 DEIA kit, according to the manufacturer's instructions (DiaSorin, Saluggia, Italy). Three parameters had to be defined: the concentration of the identification probe used to coat the microplates, the concentration of the complementary oligonucleotides, and the hybridization temperature. After calibration, the coating concentrations of the identification probes were defined according to the probes used. The concentrations were 0.25 ng/μl for probes SA2, SAE1, SBD3, SD1.1, SC1, and SG1; 0.75 ng/μl for probe SB2; and 0.05 ng/μl for probes SAG1 and SF6. The optimal concentration of the complementary oligonucleotides was defined as 2.5 ng/μl, and the hybridization temperature was 45°C.
Three formats of the assay, one for each sample collection (i.e., a Europe, a Cameroon, and a Senegal format) were evaluated (Fig. ). The number and type of probes were adapted to the predominant circulating subtypes.
(a) Europe format. The Europe format used subtype-specific probes SA2, SAE1, SBD3, SB2, SD1.1, SC1, SF6, and SG1 to identify subtype A-like samples (subtype A and CRF02_AG samples), CRF01_AE samples, and subtype B, D, C, F, and G samples. A first step discriminated six subtypes (subtype A-like, CRF01_AE, and subtypes BD, C, F, and G), as follows (Fig. ). Wells of a streptavidin-coated microplate were sensitized with 100 μl of probes appropriately diluted in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8]; one probe per strip for strips 1 to 6 and 7 to 12) by incubation for 20 h at 4°C. The wells were washed five times with PBS containing Tween 20 (PBS-TW; DiaSorin). Before the assay, the products of the nested PCR were denatured for 15 min at 97°C. They were cooled on ice for at least 10 min before DEIA analysis. Hybridization buffer (100 μl, containing Denhardt's solution, SSC [0.15 M NaCl plus 0.015 M sodium citrate], Tris-HCl, and EDTA; DiaSorin) was added to each well. We then added 10 μl of a negative control (NC) consisting of Tris-HCl, MgCl2, and bovine serum albumin (DiaSorin) to wells 1 to 6 of the first line. Ten microliters of each of the six complementary oligonucleotides was added as a positive control to wells 1 to 6 of the second line. Ten microliters of the ssDNA sample (one sample per six wells) was added to each well of the next lines and to each well of all lines on strips 7 to 12. A cardboard lid was placed over the plate to prevent evaporation, and the microplate was incubated for 1 h at 45°C (±1°C). The wells were then washed five times with PBS-TW. One hundred microliters of an anti-double-stranded DNA antibody solution (1:50 dilution in PBS and fetal calf serum; DiaSorin) was added, and the plates were incubated for 30 min at 30°C (±1°C) after they were covered with a cardboard lid. The wells were then washed five times with PBS-TW. One hundred microliters of the enzyme tracer solution (protein A conjugated to horseradish peroxidase [1:50 dilution in PBS-fetal calf serum]; DiaSorin) was added, and the plates were incubated for 30 min at room temperature after they were covered with a cardboard lid. The wells were washed five times with PBS-TW, and the reaction was detected by incubation with 100 μl of a 1:1 chromogen-substrate solution of tetramethylbenzidine and hydrogen peroxide for 30 min at room temperature in the dark. Color development was stopped by adding 1 N H2SO4 (200 μl), and the A450 and A630 were read. After preliminary experiments, the cutoff value was defined as the absorbance for the NC + 0.400.
A second step was performed for the BD-specific probe-reactive samples to distinguish between subtypes B and D. The same conditions used for the first step was applied, except that the cut-off value for the well coated with probe D1.1 was defined as the absorbance for the NC + 0.150.
In this Europe format, we were able to carry out an initial screening of 14 samples per microplate.
(b) Cameroon format. The Cameroon format involved two steps: the first used probes SA2, SAG1, SF6, and SG1, corresponding to the predominant genotypes in this country. The nontypeable (NT) samples (those with absorbance values below the cutoff) were then analyzed with probes SBD3 and SC1 (Fig. ). The same experimental conditions described above for the Europe format were applied for the Cameroon format. In the Cameroon format, we were able to carry out an initial screening of 22 samples per microplate.
(c) Senegal format. The Senegal format was performed as described above, except that the subtype BD-specific probe replaced the subtype F-specific probe for the first step; the NT samples were further analyzed with subtype C- and F-specific probes (Fig. ). In this format, we were able to carry out an initial screening of 22 samples per microplate.
(iv) Probe reactivity. The intersubtype relationships discussed above precluded direct simple subtyping with single reactivity to the specific probe. However, a reproducible profile of probe reactivity including cross-hybridization made it possible to define a chart representing a specific pattern of hybridization for each subtype or CRF (Table ). A genotype was then assigned according to this chart, with any reactivity profile different from those in this chart being classified as indeterminate.
DNA sequencing of the env cds-C2 region. DNA sequencing and phylogenetic analysis of the env 5′ end of NT, indeterminate (similar absorbances with two probes), and discordant samples were performed to assign a subtype to the DEIA region. An 820-bp fragment was amplified from proviral DNA by nested PCR with primers ED3 and ED14 as the outer primers and primers ES4 and PSA2 (5′-CCATTTAACAGCAGTTGAGTTG-3′) as the inner primers. The first round was performed with the Expand Long Template PCR system (Roche Diagnostics), according to the manufacturer's instructions. The second round was performed with Taq DNA polymerase (Promega), as follows: 1 to 5 μl from the first-round amplification was used for the second round with the inner primers, the reaction mixture consisting of 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 1.8 mM MgCl2, each deoxynucleoside triphosphate at a concentration of 0.2 mM, 2.5 U of Taq polymerase, and 20 pmol of each primer. The PCR conditions were 92°C for 5 min, followed by 38 cycles at 92°C for 20 s, 50°C for 30 s, and 72°C for 2 min, with a final extension step at 72°C for 10 min. The amplified fragments were purified with the QIAquick gel extraction kit (QIAGEN, Courtaboeuf, France) and were directly sequenced with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (Perkin-Elmer, Roissy, France) on an Applied Biosystems 373A automatic DNA sequencer (Stretch model).
Phylogenetic analysis. Nucleotide sequences were aligned with the CLUSTAL W program with minor manual adjustments to take into account the protein sequences (
58). Regions that could not be aligned unambiguously due to length or sequence variability were omitted from the analysis. Phylogenetic trees were produced by the neighbor-joining method, and the reliability of the branching orders was assessed by the bootstrap approach with the CLUSTAL W program. Genetic distances were calculated by Kimura's two-parameter method (
27).
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