Emerging genetic diversity of HIV-1 in South America

Objective: Genotype determination and risk group analysis of HIV-1 infected individuals in selected regions of South America.

Design: Cross-sectional convenience sampling of HIV-1-positive individuals in Peru, Ecuador, Uruguay and Paraguay from March, 1994 through September, 1998.

Cited Here...: HIV-1-positive subjects were identified through the national AIDS surveillance program in each country. A standardized questionnaire was used to obtain demographic, clinical and risk factor data on each study subject. Viral DNA was extracted from participants' peripheral blood mononuclear cells either directly or after co-cultivation. A nested PCR was used to obtain selected fragments of the envelope genes for genotyping by the heteroduplex mobility assay (HMA). A 600 bp sequence encompassing the V3 loop was sequenced from a selection of 23 of these samples for phylogenetic analysis and confirmation of HMA genotype.

Cited Here...: Among the 257 successfully genotyped HIV-1-positive samples, genotype B was found in 98.3% (228/232) of those obtained from subjects in Peru, Ecuador, and Paraguay. In contrast, 56% (14/25) of the samples from Uruguay were genotype F, and the remainder were genotype B. Genotype F was detected for the first time in Peru (2/224) and Paraguay (1/4), and genotype A for the first time in Peru (1/224). Phylogenetic analysis confirmed the genotype identified by HMA in the 23 samples sequenced. There was no detectable genetic clustering of HIV-1 within the different high-risk groups or geographic locations.

Conclusions: These findings verify and extend the presence of several different HIV-1 genotypes in South America.

Introduction

Currently, at least 10 different genotypes of HIV-1 have been found throughout the world. While only a few systematic studies have been conducted in South America, available literature suggests that new HIV-1 genotypes are being introduced into this region. Genotypes B and F were detected in Brazil during 1989 and 1990 ^[1], and later a genotype D was identified from samples collected in 1993-1996 ^[2]. A B/F recombinant from Brazil was described in 1994 and recently heterogeneic B/F or B/D genotypes were again described from HIV-positive blood donors in Rio de Janeiro, Brazil ^[3,4]. Genotypes A and B were reported from Chile ^[5], genotypes B, F, and a B/F env recombinant from Argentina ^[6,7], and genotype B from Paraguay ^[8], Venezuela ^[9,10] and Colombia ^[11]. In Uruguay, genotype B was reported from civilians and genotype E from military personnel returning from deployment in Southeast. Asia ^[12,13]. No published information is currently available on HIV-1 genotypes in the countries of Peru, Ecuador or Bolivia.

As genotypes of HIV evolve or are introduced into new populations, epidemics may occur ^[14]. In addition, it is likely that a global understanding of the genetic diversity of HIV will be critical for developing and targeting vaccines and therapeutic drugs. Thus, the objective of this study was to genotype a large number of specimens from selected countries to provide a better understanding of the circulating HIV-1 genotypes and phylogenetic relationships among these genotypes in South America.

Materials and methods

Study subjects and clinical samples

Surveys were conducted from March 1994 through September 1998 to obtain isolates of HIV-1 from infected individuals in Peru, Ecuador, Uruguay and Paraguay for genotyping. The infected subjects were previously identified in the respective Ministry of Health's ongoing national AIDS surveillance programs. Written informed consent was obtained and a standardized questionnaire implemented to obtain demographic, clinical and risk factor data on each study subject. The research protocol using human subjects in this study was reviewed and approved by the Naval Medical Research Center's Committee for the Protection of Human Subjects.

Heparinized blood samples were obtained from HIV-1-infected individuals and peripheral blood mononuclear cells (PBMC) were isolated from the whole blood with a Ficoll gradient method (LSM, Lymphocyte Separation Medium; ICN/Cappel, Aurora, OH, USA). DNA was extracted directly from approximately 1 × 10⁶ infected PBMC or from a similar number of co-cultivated phytohemagglutinin-stimulated donor PBMC by the QIAmp DNA extraction technique (Qiagen, Valencia, CA, USA).

Genotyping of HIV-1 isolates

Two rounds of PCR were performed beginning with 10 μl of PBMC DNA in a total reaction volume of 50 μl. The first round was performed with primers ED14 and ED3 (to amplify an approximately 2.0 kb fragment of the env gene). Using 2 μl of the first round product in a total reaction volume of 100 μl, the second round PCR was performed with primers ED12 and ED5 (to amplify an approximatley 1.25 kb fragment of V1-V5 of gp120) ^[15]. If a PCR product was not visible, the second round PCR was repeated using primers ES7 and ES8 (to generate an approximatly 0.64 kb fragment of the V3-V5 of gp120). If a product was again not seen, a final second round amplification attempt was made with primers ED31 and ED33 (to amplify an approximately 0.56 kb fragment of the C2-C3 region). A heteroduplex mobility assay (HMA) was performed with the PCR fragments as described previously using nine reference standards in the formation of the heteroduplexes ^[15]. The reference genotypes included in the HMA analysis were one genotype A (RW20), three genotype B (BR20, TH14, SF162), one genotype C (ZM18), one genotype D (UG21), one genotype E (TH22), and two genotype F (BZ162, BZ163) ^[16].

Amplification and sequencing of HIV-1 isolates

DNA harvested from HIV-1-infected cells (PBMC or co-cultivated cells) of 23 of the 263 study subjects was also used for sequence analysis. A 600 bp fragment of gp120 (C2-V5) was amplified by nested PCR using AA750 and ED12 as outer primers, and AAV3 and AA1400 as inner primers ^[17,15]. Bulk PCR product was sequenced using fluorescent dye terminators and an Applied Biosystems Inc. (Foster City, CA) Model 373A DNA sequencer. DNA sequences were assembled using Sequencher Software (GeneCodes Inc., Ann Arbor, MI, USA) on Macintosh computers.

Phylogenetic analysis

A multiple alignment of the 23 new South American sequences was made with reference sequences from each of the HIV-1 genotypes ^[16]. Phylogenetic trees were constructed and the consistency of branching order was evaluated using SEQBOOT, DNADIST, NEIGHBOR, CONSENSE and DNAPARS modules of the Phylip Package (V3.52c) ^[18]. A phylogenetic tree was built using neighbor-joining based on the Kimure two-parameter distance ^[19] and the stability of the nodes was assessed with the bootstrap ^[20].

Nucleotide sequence accession numbers

The sequences were submitted to Genbank and are available under the following accession numbers: AF268914 through AF268936.

Results

A total of 263 HIV-1 isolates were obtained from individuals in Peru (n = 230), Uruguay (n = 25), Ecuador (n = 4) and Paraguay (n = 4). Of these 263, 257 were successfully genotyped by the HMA method (97.3%). Although the six samples with undetermined genotypes by HMA were Western blot-positive (Cambridge Biotech, Rockville, MD, USA) no PCR product was detectable with any of the primer pair combinations. Among the remaining 257 samples, genotype B was predominant in Peru (98.7%; 221/224), Paraguay (75%; 3/4) and Ecuador (100%; 4/4). In Uruguay, 56% (14/25) of the isolates were genotype F and the remainder were genotype B. Two genotype F and one genotype A in Peru and one genotype F in Paraguay were also identified.

The relative distribution of the HIV genotypes B and F by sex, country of origin and risk behavior groups are presented in Table 1. Overall, 70.7% (181/256) of the samples were from males. In the self-identified risk behavior groups, genotype B was found more frequently than genotype F among men who had sex with men (99%; 96/97), the heterosexual groups of men who had sex with women and women who had sex with men (92%; 110/119), and female commercial sex workers (86%; 12/14). Genotype F was found more commonly among injecting drug users (IDU) (62%; 5/8). In addition, genotype F was more common than genotype B in Uruguay (56%; 14/25) where most of the IDU were found (75%; 6/8). Of those who responded, 13% of the individuals with genotype B reported sexual contact with foreigners, whereas 35% of genotype F individuals reported having sexual contact with foreigners (P = 0.03).

Table 1

Demographic and risk behavior information for the 23 sequenced HIV-1 samples is shown in Table 2. Samples from each country and risk group were chosen for sequencing. The individual in Peru from whom a genotype F was obtained and sequenced (97PE0155) reported sexual contact with an individual from Argentina. The genotype A from Peru (97PE0223) categorized himself as a man who had sex with women and denied same-gender sex or sexual contact with individuals from another country, although he had traveled to the USA. A wide range of clinical disease was seen among the study subjects. The last two columns of Table 2 list the genotypes based on the phylogenetic analysis of C2-V5 of env and the GeneBank accession numbers. In all cases, the genotype was the same as that identified by the HMA analysis.

Table 2

A phylogenetic tree showing the genetic relatedness of the sequenced samples is presented in Fig. 1. The genotype assignment by HMA was well confirmed by the distinct clustering of the sequences with either genotype B (n = 12), genotype F (n = 10) or genotype A (n = 1). It was also clear from the analysis that the viral samples from Uruguay (pink) were most commonly genotype F, while those from men who had sex with men (triangles) were more commonly genotype B. All genotype F samples sequenced were of the F1 sub-subtype as opposed to the F2 sub-subtype described recently from Central Africa ^[21].

Fig. 1

The amino acid sequences of the HIV-1 V3 loop are provided in Table 3. The common tip seen in Brazil (GWGR) was not seen among the genotype B samples sequenced from Peru. Several unusual genotype B sequences are represented here. The crown sequence 'GPGS' and 'GPGK' are typically seen in fewer than 10% of genotype B sequences ^[16], but were not uncommon here (33%; 4/12); in addition, GTGS (96PE0121) has never been reported in genotype B. The majority of the genotype F sequenced have the GPGQ crown that characterizes the genotype. The crown GPGK (95UY0091) has never been reported in this genotype, though the C2-V5 regions of env of this isolate is not atypical (Fig. 1). It is likely that the 97PE0081 virus is defective as it lacks one cysteine of the V3 loop.

Table 3

Discussion

With the exception of Brazil, little published data exist on the prevalence or distribution of HIV-1 genotypes in South America. This report describes the genotypes of 257 HIV-1 isolates from Peru, Ecuador, Uruguay, and Paraguay. In addition, confirmatory sequencing was performed for 23 of these isolates; in all cases, the genotypes assigned by HMA were correct. Among these, genotype F was described for the first time in Peru and Paraguay, and genotype A was described for the first time in Peru.

Within the surveyed hospital in Uruguay, genotype F was found with a higher frequency than in all of the other countries (14/25, 56% versus 3/242, 1.2%). In addition, six of the eight identified IDU in this study were also from Uruguay; of these six, five were identified with genotype F infections. Excluding two of the 25 individuals in Uruguay for whom risk category of infection was unknown, a trend towards an association between IDU and genotype F is suggested (odds ratio, 4.44; 95% confidence interval, 0.42-46.55). However, because the participants in this study were a convenience sample from the participating countries and included patients from only one hospital in Uruguay, this relationship cannot be generalized into the entire country of Uruguay or to other countries.

The association of genetic clusters with different risk categories within a genotype, especially within genotype B, has rarely been seen in populations in North America and Europe. Likewise, in this sample from South America, no significant genetic clustering was observed within the B genotype among MSM versus MSW/WSM (Fig. 1). A previous study using 94 of the same samples presented here and calculating genetic distance from the mobility on an HMA gel also showed no significant clustering with transmission risk category (C. Carcamo, unpublished data). These are not, however, population-based samples and therefore may not represent the risk categories accurately.

Sequencing of selected HIV-1 isolates confirmed that the HMA is a highly useful tool for identifying genotypes of HIV-1 in South America as it has been shown previously elsewhere. Our success with the HMA technique (257 from 263 positive samples assigned a genotype) might reflect our perseverance in using all primer pairs before calling a sample 'undetermined'. Current studies are aimed at transferring this HMA capability to local researchers and laboratories. This will be an invaluable tool in allowing continued analysis of genotype composition of HIV-1 infections throughout South America.

Although the published data are limited, HIV-1 genotype B appears to be the predominant genotype circulating in South America. However, there are certain populations where genotype F may be the predominant genotype. In addition, new genotypes are emerging in populations where they were not recognized previously. These factors reinforce the need for continued studies to monitor the changing dynamics of the HIV-1 epidemic in South America.

Acknowledgements

The authors thank G. Chauca, G. Carrion and C. Guevara for their invaluable field and laboratory support of this study.