Simplified Strategy for Detection of Recombinant Human Immunodeficiency Virus Type 1 Group M Isolates by gag/env Heteroduplex Mobility Assay

Abstract

We developed a heteroduplex mobility assay in the gag gene (gag HMA) for the identification of group M subtypes A to H. The assay covers the region coding for amino acid 132 of p24 to amino acid 20 of p7 (according to human immunodeficiency virus type 1 [HIV-1] ELI, 460 bp). The gag HMA was compared with sequencing and phylogenetic analysis of an evaluation panel of 79 HIV-1 group M isolates isolated from infected individuals from different geographic regions. Application of gag HMA in combination with env HMA on 252 HIV-1- positive plasma samples from Bénin, Cameroon, Kenya, and Zambia revealed a high prevalence of a variety of intersubtype recombinants in Yaoundé, Cameroon (53.8%); Kisumu, Kenya (26.8%); and Cotonou, Bénin (41%); no recombinants were identified among the samples from Ndola, Zambia. The AG_IbNG circulating recombinant form, as determined by gag HMA, was found to be the most common intersubtype recombinant in Yaoundé (39.4%) and Cotonou (38.5%). Using a one-tube reverse transcriptase PCR protocol, this gag HMA in combination with env HMA is a useful tool for rapidly monitoring the prevalence of the various genetic subtypes as well as of recombinants of HIV-1. Moreover, this technology can easily be applied in laboratories in developing countries.

The Human immunodeficiency virus type 1 (HIV-1) exhibits an extremely high genetic variation, which is driven by a high error rate of the reverse transcriptase (RT), the presence of viral RNA as a dimer (recombination occurs frequently during reverse transcription) (29), the high turnover of HIV-1 in vivo (14), and selective immune responses.

By genetic analyses, HIV strains collected from around the world have been shown to have substantial diversity (19). Two types have been characterized in humans: HIV-1, the predominant HIV type throughout the world, and HIV-2, which is less widespread and still primarily found in West Africa. Three different HIV-1 groups are distinguished: group M (major), which is globally prevalent; group O (outlier) (5); and group N (non-M/non-O) (27). HIV-1 groups and subtypes are unevenly distributed throughout the world (2, 16, 17, 23). Over the years the definition of group M subtypes has been adapted in the light of emerging recombinants. Representatives of different “pure” (nonrecombinant) subtypes A, B, C, D, F, G, H, and J and circulating recombinant forms (CRFs) AE_CM240, AG_IbNG, AGI_CY032, and AB_Kal153, were proposed based on near-full-length genome analysis, as determined by the HIV Sequence Database (HSB; J. K. Carr, B. T. Foley, T. Leitner, M. Salminen, B. Korber, and F. McCutchan [http://hiv-web.lanl.gov/]). These are needed as a framework to identify new subtypes and intersubtype recombinants, which are an important source of HIV-1 genetic variation. The most prevalent CRFs are AE_CM240 and AG_IbNG. AE_CM240 is common in the Central African Republic, Thailand, and other Asian countries (11). In Nigeria AG_IbNG recombinants (named after the first isolate from Ibadan, Nigeria) were identified (4, 15, 22). Additional AG_IbNG-like full-length sequences were also obtained from Djibouti (4) and Côte d'Ivoire (per HSB). Reanalysis of partial env sequences from Côte d'Ivoire (9) and Cameroon (28), initially documented as subtype A, indicates that strains of HIV-1 similar to AG_IbNG are the most common form of subtype A in Côte d'Ivoire and Cameroon (per HSB and J. K. Carr, personal communication).

To date, there have been few systematic large-scale attempts to characterize HIV isolates from different parts of the world. Thus, our knowledge about the distribution of HIV strains in different populations and about changes in that distribution over time is rather limited. env heteroduplex mobility assay (HMA) is much less cumbersome and less expensive than nucleic acid sequencing and phylogenetic analysis (6, 7). Since there is a strong correlation between the subtyping result obtained by HMA and that obtained by sequencing and phylogenetic analysis (6, 13), HMA has been introduced by UNAIDS in several developing countries as a tool for monitoring subtype distribution. Adding gag HMA to env HMA would allow one to provide a “minimum estimate” of the prevalence of the various genetic subtypes as well as some of the recombinants of HIV-1.

MATERIALS AND METHODS

Reference panel.

A panel of 23 plasmids containing the gag gene of HIV-1 strains belonging to group M subtypes A to H was available (20). This panel was extended by adding plasmids containing part of the gag gene of CA10 (CRF AE_CM240), VI991 and VI997 (subtype H). Following evaluation of this panel, gag HMA reference plasmids for subtype B (PIC63 and PIC335) and subtype D (VI761 and PIC49) were added (Table 1).

TABLE 1.

gag HMA reference panel^a

Isolate	AGIbNG	A	B	C	D	AECM240	F	G	H
1	LBV23-10	VI310	TB132	DJ259	K31	TN238	BZ162	LBV21-7	VI525
2	DJ258	CI4	UG280	VI313	VI203	CA10c	VI174	RU520	VI991
3	CI51	VI57	PIC63b	UG268	VI205		VI69	RU131	VI997
4		K29	PIC335b		VI761c
5					PIC49c

^aUnless otherwise indicated, all clones have a 1,500-bp insert (20). 

^bgag fragment (830 bp); nucleotides 822 to 1652 according to ELI (accession number K03454). 

^cgag fragment (1,216 bp); nucleotides 436 to 1652 according to ELI. 

Evaluation panel.

There were 10 supernatants from cultures for which full-length HIV-1 sequences were available: subtypes A (n = 6; SE6594, SE7253, SE7535, SE8131, SE8538, and SE8891), AG_IbNG (n = 1; SE7812), G (n = 1; SE6165), and J (n = 2; SE9173 and SE9280) (per HSB). There were five supernatants from cultures for which the gag/env subtype was determined by sequencing and phylogenetic analysis: subtype F (n = 5; 93R26, 96R85, 96R95, 97R102, and 97R110) (E. Op de Coul, R. van den Burg, B. Asjö, A. Cupsa, R. Pascu, C. Usein, J. Goudsmit, and M. Cornelissen, unpublished data). There were 4 plasmids containing full-length HIV-1 sequences of subtype C (n = 4; 96BW01, 96BW04, 96BW05, and 96BW15) (26); 13 plasmids containing (part of) the gag gene of subtypes A (n = 7; K98, K7, K88, K112, K89, VI415, and VI32), D (n = 3; UG274, UG270, and SE365), AE_CM240 (n = 1, CM240), and A/G (n = 2; CI20 and CI32) (20); and 11 peripheral blood mononuclear cell cultures for which the gag/env subtypes were A/A (n = 2; VI537 and VI1139), A/D (n = 1; VI1308), B/B (n = 1; VI1663), B/ND (n = 2; PH136 and PH153), C/C (n = 2; ZM18 and ZM20), AE_CM240 (n = 2; CM235 and CM243), and F/F (n = 1; BZ126) (3, 20, 21). Plasma of 36 HIV-1 group M (A to H) infected patients, from different geographic regions, for which the gag/env subtype was determined by sequencing and phylogeny, was also tested: subtypes A/A (n = 2; PIC349 and PIC382), A/D (n = 1; PIC25), AE_CM240 (n = 4; PIC6, PIC807, PIC231, and PIC1047), A/G (n = 2; PIC436 and VI1197), B/B (n = 10; PIC63, PIC55, PIC135, PIC1626, PIC1852, PIC271, PIC1622, PIC143, PIC335, and PIC1585), B/A (n = 1; PIC364), C/C (n = 5; VI 882, VI1044, VI1144, VI1233, and PIC146), D/D (n = 4; VI205, VI761, PIC797, and PIC1018), D/F (n = 3; PIC49, PIC179, and PIC885), F/F (n = 1; PIC132), G/A (n = 1; PIC201), and H/H (n = 2; VI997 and PIC450).

Study subjects.

Between July 1997 and January 1998 serum samples and corresponding data were collected from roughly 1,000 men and 1,000 women, who were randomly selected from the general population, as well as 300 commercial sex workers, in each of four sub-Saharan African cities (Cotonou, Bénin [BJ]; Yaoundé, Cameroon [CM]; Kisumu, Kenya [KE]; and Ndola, Zambia [ZM]). Among the HIV-positive subjects, plasma samples were analyzed from 39 HIV-1-infected individuals from the general population; among HIV-infected commercial sex workers from Cotonou, 66 were from Yaoundé, 61 were from Ndola, and 86 were from Kisumu.

RNA extractions and RT-PCR.

RNA extractions were performed as previously described (1). One-tube RT-PCR (Access RT-PCR; Promega, Leiden, The Netherlands) was performed according to the manufacturer's recommendations.

(i) gag one-tube RT-PCR.

The primers were H1G777 (5′-TCACCTAGAACTTTGAATGCATGGG-3′) and H1P202 (5′-CTAATACTGTATCATCTGCTCCTGT-3′), and the cycle protocol was 45 min at 48°C (cDNA reaction), followed by 2 min at 94°C and 40 cycles for 30, 30, and 90 s at, respectively, 94, 50, and 68°C; and 1 cycle for 7 min at 68°C. For nested PCR, the primers were H1Gag1584 (5′-AAAGATGGATAATCCTGGG-3′) and g17 (5′-TCCACATTTCCAACAGCCCTTTTT-3′), and the cycling conditions were 1 cycle for 2 min at 94°C; 35 cycles for 30, 30, and 60 s at, respectively, 94, 50, and 72°C; and 1 cycle for 7 min at 72°C. A larger fragment (830 bp) containing the entire gag HMA fragment was amplified by using H1G822 (5′-GCTTTCAGCCCAGAAGTAATACC-3′) and H1GHMA1317 (5′-CCAAATTCTCCCTAAAAAATTAGCCT-3′) during the nested PCR and by using the same cycling conditions as for H1Gag1584 and g17.

(ii) env one-tube RT-PCR.

The primers ED5 and ED12 (6) were used, and the cycle protocol was 45 min at 48°C (cDNA reaction), 1 cycle for 2 min at 94°C; 40 cycles for 30, 30, and 120 s at, respectively, 94, 55, and 68°C; and 1 cycle for 7 min at 68°C. For nested PCR, the primers were ES7 and ES8 (6).

(iii) pol one-tube RT-PCR.

The primers H1P4235 (10) and H1P5155as (5′-CTCTGTGGCCCCTGGTCTTCT-3′) were used. The cycle protocol was 45 min at 48°C (cDNA reaction), followed by 2 min at 94°C and 40 cycles for 30, 30, and 90 s at, respectively, 94, 55, and 68°C; and 1 cycle of 7 min at 68°C. For nested PCR the primers H1P4327 (10) and H1P5128as (5′-CTCTTTCCATCTGTCTTCTGCTA-3′) were used, and the cycling conditions were 1 cycle for 2 min at 94°C; 35 cycles for 30, 30, and 60 s at, respectively, 94, 50, and 72°C; and 1 cycle for 7 min at 72°C.

Sequence analysis.

Sequencing of both DNA strands was performed by cycle sequencing and 5′-fluorescein isothiocyanate (FITC)-labeled primers on an automated laser fluorescence sequencer (Amersham Pharmacia Biotech, Roosendaal, The Netherlands). Sequences were aligned with CLUSTAL W (30), and the resulting alignments were refined manually with the dedicated comparative sequence editor (8). The software package TREECON was used for distance calculations (Jukes and Cantor), tree construction (neighbor-joining method), and bootstrap analysis (31). The HIV-1 gag nucleotide sequence data were deposited in the EMBL, GenBank, and DDBJ nucleotide sequence databases under the following accession numbers: AF184346 to AF184587.

Heteroduplex formation and mobility analysis.

HMA was largely performed as described by Delwart et al. (6, 7). Briefly, the following methodology was used. Heteroduplex molecules were obtained by mixing 4.5 μl from two second-round PCRs and adding 1 μl of 10× annealing buffer (1 M NaCl, 100 mM Tris-HCl [pH 7.8], 20 mM EDTA). The DNA fragments were denaturated at 94°C for 2 min and reannealed by rapid cooling on wet ice.

Electrophoresis was done on a 5% polyacrylamide gel (29:1 acrylamide:bisacrylamide) that included 20% urea in 1× TBE buffer (88 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) at 250 V for 2.5 h. Detection of heteroduplexes was done by staining with ethidium bromide and visualization under UV light.

In order to better discriminate between subtype A, CRF AE_CM240, and CRF AG_IbNG, the gel composition was altered by adding 30% urea and extending the electrophoresis time up to 3 h.

RESULTS

Design of gag HMA.

Based on sequences available for HIV-1 group M subtypes A through H, primers were designed to amplify a fragment of approximately 460 bp covering a gag gene region coding for amino acid 132 of p24 to amino acid 20 of p7 (according to HIV-1 ELI; accession number KO3454). The experimental conditions were mainly as described for env HMA (ED31-ED33) (7), initially with a reference panel including 26 plasmids containing the gag gene of HIV-1 group M subtypes A to H. The choice of different subtype references was guided by their availability and by their phylogenetic classifications with respect to subtype representatives in distinguished subtype clusters for the gag HMA fragment analyzed (Table 1 and Fig. 1). Experimental conditions were obtained whereby reference isolates could be amplified by PCR and unambiguously subtyped by HMA. Altered experimental conditions relative to the gel composition (30% urea instead of 20%) allowed better distinction between gag subtype A, CRF AE_CM240, and CRF AG_IbNG.

FIG. 1.

Phylogenetic tree based on a gag gene region (460 bp; nucleotides 1123 to 1589; according to HIV-1 ELI) encoding amino acid 132 of p24 to amino acid 20 of p7. References of the gag HMA panel are indicated in italic. A total of 2,000 bootstrap samples were analyzed. Bootstrap values are given in percentages at the internodes if they exceed the 70% level. The distance between two sequences is obtained by summing the lengths of the connecting horizontal branches by using the scale on top. The tree is rooted arbitrarily.

Evaluation of gag HMA.

An evaluation of gag HMA was done on a reference panel of 79 genetically confirmed HIV-1 group M isolates, isolated from individuals infected in different geographic regions, with the following gag/env subtypes: A/A, n = 17; A/D, n = 2; AG_IbNG, n = 3; A/G, n = 2; AE_CM240, n = 7; B/B, n = 11; B/A, n = 1; B/ND, n = 2; C/C, n = 11; D/D, n = 7; D/F, n = 3; F/F, n = 7; G/A, n = 1; G/G, n = 1; H/H, n = 2; and J/J, n = 2. For all of these isolates a positive PCR product was obtained. As indicated by phylogenetic analysis of the gag HMA references, two subtype D clusters were distinguished, which together with subtype B form one cluster (Fig. 1). Additional reference isolates were added to optimize subtype B (B/B, PIC63; B/B, PIC335) and D (D/D, VI761; D/F, PIC49) differentiation (Table 1). gag HMA was successful in determining the correct genetic subtype of 76 of 79 isolates (96%). Strains that could not be classified due to an intersubtype migration pattern belonged to subtype D (n = 1) and subtype J (n = 2). For the latter subtype no references were included.

Validation of gag/env HMA.

gag HMA in combination with env HMA was validated on HIV-1-positive plasma samples from individuals who participated in the above-mentioned population-based survey.

A positive gag HMA PCR product was obtained for 242 of 252 (96%) of the analyzed plasma samples. The result obtained by gag HMA was confirmed by sequencing and phylogenetic analysis of the gag HMA fragment. For the 10 gag HMA fragment PCR-negative samples, an overlapping gag fragment was amplified. These samples were classified into subtypes A (CM, n = 1; KE, n = 1; and BJ, n = 5), C (KE, n = 1), and D (KE, n = 2) by sequencing and phylogenetic analysis of the gag HMA fragment. Of 242 samples, 6 could not be classified by gag HMA. These samples belonged to subtypes A (KE, n = 1), C (ZM, n = 1), D (KE, n = 2), dual D+C (KE, n = 1), and U (unclassified) (CM, n = 1, env subtype F) based on sequencing and phylogenetic analysis of the gag HMA fragments. gag subtype distribution results are presented in Table 2.

TABLE 2.

gag subtypes^a

Origin	No. of subtypes
	a/bb	A	AECM240	AGIbNG	C	D	F	G	Uc
ZM	61/61	1			59				1d
CM	65/66	24	1	26		3	1	9	1e
KE	82/86	50			9	19			4f
BJ	34/39	11		15				8
Total	242/252	86	1	41	68	22	1	17	6
(%)	(96)	(35.5)	(0.4)	(16.9)	(28.1)	(9.1)	(0.4)	(7)	(2.5)

^aSubtyping by HMA, sequencing, and phylogenetic analysis of a fragment of ca. 460 bp covering a gag gene region coding for amino acid 132 of p24 to amino acid 20 of p7 (according to HIV-1 ELI; accession number K03454). 

^ba, number of gag HMA PCR positive samples; b, number of tested samples. 

^cU, not classified by gag HMA. 

^dSubtype C sample. 

^eSample remains unclassifiable by sequencing and phylogeny. 

^fSubtypes by sequencing and phylogeny: A, n = 1; D, n = 2; dual D+C, n = 1. 

A positive PCR product for the env HMA ES7-ES8 fragment (6) was obtained for 238 of 252 (94.4%) of the analyzed plasma samples. For 13 of 14 PCR-negative samples a C2V3 coding env fragment could be amplified, sequenced, and phylogenetically analyzed. These samples were classified into subtypes A (CM, n = 5; KE, n = 2), D (KE, n = 2), AE_CM240 (CM, n = 1), F (CM, n = 1), and U (KE, n = 2, gag subtype A) (Table 2). For the remaining sample no env fragment could be amplified (gag subtype G). Of 238 samples, 29 could not be unambiguously subtyped by env HMA. These samples were classified into subtypes A (CM, n = 1; KE, n = 5), C (KE, n = 4; ZM, n = 1), D (CM, n = 4; KE, n = 3), F (CM, n = 1), G (CM, n = 3; KE, n = 1), and U (ZM, n = 1, gag subtype C; CM, n = 1, gag subtype AG_IbNG; KE, n = 1, gag subtype A), as based on sequencing and phylogenetic analysis of the env C2V3 coding region. For three samples evidence of dual infection was obtained by cloning prior to subtyping by HMA and/or sequencing and phylogenetic analysis of the env C2V3 coding region: dual A+C (ZM, n = 1); dual A+D (KE, n = 2). env subtype distribution results are presented in Table 3.

TABLE 3.

env subtypes^a

Origin	No. of subtypes
	a/bb	Ac	C	D	AECM240	F	G	H	Ud	MIe
ZM	61/61		59						1	1
CM	65/66	48		4	2	4	5	1	1
KE	86/86	48	7	26			1		2	2
BJ	39/39	29					10
Total	251/252	125	66	30	2	4	16	1	4	3
(%)	(99.6)	(49.8)	(26.3)	(12.0)	(0.8)	(1.6)	(6.4)	(0.4)	(1.6)	(1.2)

^aSubtyping by HMA (ES7-ES8 fragment) (6) or by sequencing and phylogenetic analysis of a C2V3 encoding env fragment if samples could not be amplified for the ES7-ES8 fragment or could not be classified by HMA. 

^ba, number of env PCR positive samples; b, number of tested samples. 

^cA, subtype A and AG_IbNG could not be differentiated by env HMA. 

^dU, unclassifiable by HMA and C₂V₃ sequence analysis. 

^eMI, multiple infections. Three cases of dual infection were found: ZM, A+C (n = 1); KE, A+D (n = 2). 

The HIV-1 gag and env subtyping results from samples collected in Zambia, Cameroon, Kenya, and Bénin are presented in Table 4 and Fig. 2. Altogether, the frequency of HIV-1 intersubtype recombinants in this study was 53.8% (35 of 65) in Yaoundé, Cameroon; 41% (16 of 39) in Cotonou, Bénin; 26.8% (23 of 86) in Kisumu, Kenya; and 0% in Ndola, Zambia.

TABLE 4.

gag/env genetic subtyping of HIV-1 from plasma samples collected in Zambia, Cameroon, Kenya, and Bénin

Origin (total samples)a	Subtypec		nb
	gag	envd
Zambia (61)	C	C	60
	A	A+Cd	1 (dual infection)
Cameroon (66)	A	A	21
	D	D	3
	F	F	1
	G	G	5
	AGIbNG	A	24
	AGIbNG	F	1
	AGIbNG	U	1
	A	D	1
	AECM240	AECM240	1
	A	AECM240	1
	A	F	1
	A	H	1
	G	A	3
	G	PCR−	1
	U	F	1
Kenya (86)	A	A	39
	C	C	5
	D	D	17
	A	C	2
	A	D	7
	A	G	1
	A	A+D	1 (dual infection)
	A	U	3
	C	D	2
	C	A	3
	D	A	5
	D+C	A+D	1 (multiple infection)
Bénin (39)	A	A	15
	G	G	8
	A	G	1
	AGIbNG	A	14
	AGIbNG	G	1

^aTotal of samples tested per country. 

^bn, number of samples per gag/env subtype. 

^cThe nonrecombinant isolates (for the examined regions) are indicated in boldface, i.e., the same subtype in gag and env. 

^dA, subtype A and AG_IbNG could not be differentiated by env HMA. 

FIG. 2.

Pie chart subtype representation for the four different countries based on gag/env regions. The pie is subdivided into pieces representing nonrecombinants (NR), recombinants (R), CRFs plus other recombinants, and multiple infections (MI). A, subtype A and AG_IbNG could not be differentiated by env HMA.

Differentiation between subtype A, CRF AE_CM240, and CRF AG_IbNG.

CRFs AE_CM240 and AG_IbNG each consist of variants that are genetically subtype A for the analyzed gag HMA fragment. Experimental gag HMA conditions as described for group M subtypes A to H to some extent also allow differentiation between subtype A, CRF AE_CM240, and CRF AG_IbNG (Fig. 3A). However this specific differentiation is much clearer when analyzed under altered gel conditions (30% urea) (Fig. 3B). In order to confirm correct classification of potential CRF AG_IbNG isolates, a pol gene fragment of 14 randomly chosen potential CRF AG_IbNG variants (CM, n = 8; BJ, n = 6) was PCR amplified, sequenced, and phylogenetically analyzed (Fig. 4). Clustering of these variants with CRF AG_IbNG representatives for this pol fragment supports the classification as CRF AG_IbNG variants (4).

FIG. 3.

HMAs of HIV-1 subtype A, AE_CM240, and AG_IbNG gag sequences on a 5% polyacrylamide gel containing 20% urea (A) and 30% urea (B). Isolate numbers are according to the references in Table 1.

FIG. 4.

Phylogenetic tree based on a pol gene region (750 bp; nucleotides 4377 to 5128; according to HIV-1 ELI) encoding integrase. Samples newly identified in this study are indicated in italic. A total of 2,000 bootstrap samples were analyzed. Bootstrap values are given in percentages at the internodes if they exceed the 70% level. The distance between two sequences is obtained by summing the lengths of the connecting horizontal branches by using the scale at the top. The tree is rooted arbitrarily.

DISCUSSION

The development of an efficacious vaccine against HIV-1 still remains one of the biggest challenges in the worldwide fight against HIV and AIDS. Major obstacles to vaccine development include the huge HIV variability and the fact that we do not know the correlates of protection in a vaccinated individual. The principle still holds that a vaccine should contain immunogenic characteristics of the prevalent HIV-1 subtype(s) circulating in a geographic region. We may need cocktails of different immunogens that are representative of each genotype and phenotype, in combination, or cocktails that are easily adapted to different geographic regions or over time. Up-to-date information on circulating HIV strains may thus be of crucial importance. Although sequencing remains the most accurate approach for characterizing virus genomes, this method is time-consuming and requires a considerable investment in terms of equipment and reagents, as well as a lot of experience. This precludes the use of sequencing for monitoring the distribution of HIV strains in populations. env HMA has been evaluated as a reliable alternative subtyping method compared to sequencing and phylogenetic analysis (6, 13) and is sensitive, cost-effective, and applicable on a relatively large scale. It has already been successfully introduced by UNAIDS in several developing countries. Since intersubtype recombination is an important source of HIV-1 genetic variation, there was a need to extend HMA subtyping to cover two distinct HIV-1 regions: gag and env.

A reference panel was assembled based on available plasmids containing gag gene inserts and then evaluated with a panel of 79 genetically characterized samples. Two subtype B and two subtype D references were added to the reference panel in order to optimize differentiation among subtype B and D variants. The gag HMA subtype results correlated well with sequence and phylogenetic analysis, except for one subtype D isolate that was unclassified by gag HMA and two subtype J samples for which no representatives were included in the reference panel. Experimental conditions were found to distinguish between subtype A, CRF AE_CM240, and CRF AG_IbNG variants in subtype A, which was supported by sequencing and phylogenetic analysis of a pol gene fragment (4). The latter conditions only improve differentiation among subtype A, CRF AE_CM240, and CRF AG_IbNG and cannot be applied to subtyping other group M subtype strains. Subtyping by gag HMA correlated with sequencing and phylogenetic analysis of the same fragment.

The combination of gag and env subtyping results obtained on HIV-1-positive plasma samples from Bénin, Cameroon, Zambia, and Kenya revealed a high prevalence of a variety of intersubtype recombinants in Cameroon (53.8%), Kenya (26.8%), and Bénin (41%). These data indicate the relevance of subtyping two different gene fragments. Contrary to the lack of recombinants in Zambia, a high frequency of intersubtype recombinants documented in Cameroon, Kenya, and Bénin correlates with the distribution of multiple subtypes, as previously documented for Cameroon (25, 28), Kenya (24; E. M. Songok, H. Ichimura, P. M. Tukei, K. Kakimoto, P. Orege, N. Sakagami, and T. Kurimura, Abstr. 12th World AIDS Conf., abstr. 11188, 1998), and Bénin (12). AG_IbNG/F and AG_IbNG/G recombinants were identified in Cameroon and Bénin, respectively. The AG_IbNG recombinant subtype, as determined by gag HMA, is prevalent in Yaoundé, Cameroon (39.4%), and Cotonou, Bénin (38.5%), and can therefore be considered as an epidemiologically important CRF. Reanalysis of previously documented env sequences encoding the region C2V3 to the start of gp41 (900 bp) of subtype A samples from Côte d'Ivoire (18) and Cameroon (25), in combination with gag HMA, indicates that CRF AG_IbNG strains were already prevalent in these countries (10 of 13 and 9 of 12, respectively) in the early 1990s. Furthermore, one isolate from Côte d'Ivoire was identified as an env AG_IbNG/gag A recombinant (data not shown). These results suggest that dual infection with different subtypes of HIV-1 may occur frequently in geographic regions where multiple HIV-1 subtypes cocirculate, giving rise to HIV-1 intersubtype recombinants that can be transmitted and spread in the population.

A different classification based on gag and env HMA obtained from the same sample is a strong indication for intersubtype recombination. However, no assumptions can be made regarding genome regions other than those analyzed in gag and env HMA. An isolate that is classified in the same subtype by gag and env HMA can still be an intersubtype recombinant. These results may thus have to be considered minimal estimates of the prevalence of recombinant strains. In the light of recent efforts to document full-length sequences of “pure” subtype and CRF references, the proposed reference panel is subject to improvement. Knowledge of subtype variants circulating in a particular geographic region may be used to add or replace subtype references for those variants most frequently encountered. The described gag HMA reference panel so far has allowed the subtyping of genetically confirmed HIV-1 group M gag subtype A to H strains and CRFs AE_CM240 and AG_IbNG isolated from individuals infected in the following different geographic regions: subtype A and/or AG_IbNG, Côte d'Ivoire, Bénin, Kenya, Cameroon, Democratic Republic Congo, and Zambia; B, Belgium and the Phillipines; C, Kenya, Djibouti, Uganda, Democratic Republic Congo, Zambia, and Botswana; D, Kenya, Cameroon, and Democratic Republic Congo; AE_CM240, Cameroon and Thailand; F, Romania, Democratic Republic Congo, and Cameroon; G, Russia, Gabon, Bénin, and Cameroon; and H, Democratic Republic Congo and Gabon.

We believe that the development of a gag HMA makes an important contribution to subtyping strategies in surveillance, in studies of biological characteristics of strains, and in surveys for the preparation of vaccine trials. Transfer of HIV-1 gag/env HMA to the developing countries may contribute to obtaining a more accurate estimate of the real prevalence of HIV-1 subtypes and intersubtype recombinants in those parts of the world where the technology for sequencing is not readily available. This will aid in the development and evaluation of HIV vaccines more suitable for use in developing countries.