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Journal of Virology, January 2000, p. 363-370, Vol. 74, No. 1
Department of Microbiology, Institute of Tropical Medicine,
Antwerp,1 and The Flanders
Interuniversity Institute for Biotechnology (VIB),
Zwijnaarde,2 Belgium; Laboratoire de
Santé Hygiène Mobile, Ministère de la Santé,
Yaoundé, Cameroon3; Tropical
Diseases Research Centre, Ndola, Zambia4;
Programme National de Lutte contre le SIDA, Cotonou,
Bénin5; National
AIDS/STD Control Programme, Nairobi,
Kenya6; London School of Hygiene & Tropical Medicine, Department of Infections and Tropical
Diseases, London, United Kingdom7; Henry
M. Jackson Foundation, Rockville, Maryland8;
Swedish Institute for Infectious Disease Control,
Karolinska Institute, Stockholm, Sweden9;
Harvard Institute and the Department of Immunology
and Infectious Diseases, Harvard School of Public Health, Boston,
Massachusetts10; Department of Human
Retrovirology, Amsterdam, The Netherlands11;
National Centre for Research in Virology, University of
Bergen, Bergen, Norway12; and
Department of Chronic Viral Infections, National
Public Health Institute, Helsinki, Finland13
Received 10 May 1999/Accepted 22 September 1999
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
AGIbNG 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) AECM240, AGIbNG, AGICY032, and
ABKal153, 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 AECM240 and AGIbNG.
AECM240 is common in the Central African Republic,
Thailand, and other Asian countries (11). In Nigeria
AGIbNG recombinants (named after the first isolate from
Ibadan, Nigeria) were identified (4, 15, 22). Additional
AGIbNG-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
AGIbNG 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.
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
AECM240), 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).
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simplified Strategy for Detection of Recombinant
Human Immunodeficiency Virus Type 1 Group M Isolates by
gag/env Heteroduplex Mobility Assay
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
gag HMA
reference panela
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), AGIbNG (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), AECM240 (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), AECM240 (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), AECM240 (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 AECM240, and CRF AGIbNG, the gel composition was altered by adding 30% urea and extending the electrophoresis time up to 3 h.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 AECM240, and CRF AGIbNG.
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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; AGIbNG, n = 3; A/G, n = 2; AECM240, 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.
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Differentiation between subtype A, CRF AECM240, and CRF AGIbNG. CRFs AECM240 and AGIbNG 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 AECM240, and CRF AGIbNG (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 AGIbNG isolates, a pol gene fragment of 14 randomly chosen potential CRF AGIbNG variants (CM, n = 8; BJ, n = 6) was PCR amplified, sequenced, and phylogenetically analyzed (Fig. 4). Clustering of these variants with CRF AGIbNG representatives for this pol fragment supports the classification as CRF AGIbNG variants (4).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 AECM240, and CRF AGIbNG 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 AECM240, and CRF AGIbNG 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). AGIbNG/F and AGIbNG/G recombinants were identified in Cameroon and Bénin, respectively. The AGIbNG 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 AGIbNG 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 AGIbNG/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 AECM240 and AGIbNG isolated from individuals infected in the following different geographic regions: subtype A and/or AGIbNG, 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; AECM240, 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.
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ACKNOWLEDGMENTS |
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This work was supported by the Fonds voor Wetenschappelijk Onderzoek, Brussels, Belgium (grants 3-3301-96 and G.0134.97); the Flanders Interuniversity Institute for Biotechnology (VIB), Zwijnaarde, Belgium; the EC (grant IC18-CT97-02476); the Human Science Foundation (Tokyo, Japan); the United Kingdom Department for International Development (DFID, research project RD420); and UNAIDS. The work of the Study Group on Heterogeneity of HIV Epidemics in African Cities was supported by UNAIDS; the Agence Nationale de Recherche sur le SIDA/Ministère de la Coopération Française; DGXII of the EC (contract number ERBIC 18CT96-0109); SIDACTION (France); the Belgian Development Cooperation, Nairobi, Kenya; and the Fonds voor Wetenschappelijk Onderzoek (grant number K.V.E. 182, 1997), Brussels, Belgium. Linda Morison of the London School of Tropical Health and Medicine was supported by the British Medical Research Council.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Phone: 32-3-247-63-28. Fax: 32-3-247-63-33. E-mail: wjanssens{at}itg.be.
Members of the Study Group on Heterogeneity of HIV Epidemics in
African Cities include A. Buvé (coordinator), M. Laga, E. Van
Dyck, W. Janssens, and L. Heyndrickx (Institute of Tropical Medicine,
Antwerp, Belgium); M. Caraël (UNAIDS); S. Anagonou (Programme
National de Lutte contre le SIDA, Bénin); M. Laourou (Institut
National de Statistiques et d'Analyses Economiques, Bénin); L. Kanhonou (Centre de Recherche en Reproduction Humaine et en
Démographie, Bénin); L. Zekeng (Laboratoire de Santé Hygiène Mobile, Cameroon); E. Akam and M. de Loenzien (Institut de Formation et de Recherche en Démographiques, Cameroon);
S.-C. Abega (Université Catholique d'Afrique Centrale,
Cameroon); M. Kahindo (formerly National AIDS/STD Control
Programme, Kenya); J. Chege and N. Rutenberg (The
Population Council, Nairobi); V. Kimani (Department of Community
Health, University of Nairobi); R. Musonda, T. Sukwa, and F. Kaona
(Tropical Diseases Research Centre, Zambia); B. Auvert and E. Lagarde
(INSERM U88, Paris, France); N. J. Robinson (formerly INSERM
U88, Paris, France); B. Ferry and N. Lydié (Centre Français
sur la Population et le Développement, Paris, France); and R. Hayes, L. Morison, H. Weiss, and J. Glynn (London School of Hygiene and
Tropical Medicine, London, United Kingdom).
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Materials and Methods
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Discussion
References
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Journal of Virology, January 2000, p. 363-370, Vol. 74, No. 1
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