Virus Research 60 (1999) 191 – 197 Short communication HIV-1 subtype B in Honduras Boris Renjifo a,1, Jason T. Blackard a,1, Winslow Klaskala b, Beth R. Chaplin a, Pulin Shah a, Mary Frances McLane a, Frances Barin c, Jose Esparza d, Jose Enrique Zelaya e, Saladin Osmanov e, Ramon Soto e, Jorge Alberto Fernandez e, Marianna K. Baum b, Myron E. Essex a,* a Har6ard School of Public Health, Boston, MA, USA Uni6ersity of Miami School of Medicine, Miami, FL, USA c Laboratoire de Virologie, Uni6ersité de Tours, Tours, France d United Nations Programme on HIV/AIDS, Gene6a, Switzerland e Ministry of Health, Tegucigalpa, Honduras b Received 13 October 1998; received in revised form 29 January 1999; accepted 1 February 1999 An estimated 1.5 million people are currently infected with the Human Immunodeficiency Virus Type (HIV-1) in Central and South America (Mertens and Low-Beer, 1996). Nearly 60% of Central America’s AIDS cases have occurred in Honduras despite the country accounting for only 17% of the region’s population. The number of cumulative HIV/AIDS infections in Honduras reached 8,217 in 1997 (WHO, 1998) with greater than 80% attributable to heterosexual contact (Trujillo-Garcia et al., 1998). UNAIDS estimates the adult HIV prevalence rate in Honduras to be 1.46% with approximately 41 000 seropositive individuals between the ages of 15 and 49 (Baltner, 1998). Despite the apparent magnitude of this epidemic, little information is available about * Corresponding author. Boris Renjifo and Jason T. Blackard contributed equally to this Short communication paper. 1 HIV-1 subtypes prevalent in this area of the world. HIV-1 subtype B predominates in North America and Western Europe and is thought to be the predominant subtype in South America, although other non-B subtypes have been identified in Brazil, Argentina, and Uruguay (Csillag, 1994; Artenstein et al., 1995; Campodonico et al., 1996; Sabino et al., 1996). Peptide serotyping and cDNA sequencing of the third variable (V3) region of the envelope gene were previously used to determine the subtypes present in a population of HIV-1 seropositive Hondurans (Lara et al., 1997). Serotyping indicated that 95 of 120 samples belonged to subtype B, one sample belonged to subtype A, 21 samples showed multiple reactivities (indeterminate), and three samples were not typeable as a result of lack of reactivity with any V3 peptide. Of the 21 indeterminate samples, 18 were associated with heterosexual modes of transmission. However, 0168-1702/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 9 9 ) 0 0 0 1 4 - 3 192 B. Renjifo et al. / Virus Research 60 (1999) 191–197 when genotyping was performed, 12 of 12 samples belonged to subtype B. Similar results were obtained by sequence analysis of the C2V3 region of envelope from HIV-1 infected patients in San Pedro Sula and Tegucigalpa (Candal et al., 1997). Because of the large number of indeterminate heterosexual samples and the limited phylogenetic data available from one study (Lara et al., 1997) and the restricted geographic distribution of samples from the other (Candal et al., 1997), we were interested in further defining HIV-1 subtype(s) present in various locations throughout Honduras as determined by both serology and sequence analysis of multiple HIV-1 genes. In this report, we describe full-length and partial envelope sequences and 3% Long Terminal Repeat (LTR) sequences and/or peptide serological results of 62 HIV-1-infected Hondurans from several locations in Honduras. Study patients were recruited from known HIVpositive persons attending major public health centers in San Pedro Sula (S), Comayagua/ Siguatepeque (C), Tela (P), La Ceiba (L), and Tegucigalpa (T). Only those individuals who either seroconverted or had an initial HIV-positive test carried out within the past two years were eligible for participation. Risk categories enrolled in this study include female commercial sex workers (n = 18), women representing the general population (n= 35), and men who have sex with men (n= 9). Participants were predominantly single (72%) with less than 6 years of education. Their median age was 28 years for females (range 18– 45 years) and 26 years for males (range 21– 39 years). Although all participants were aware of condoms, only 32% reported their consistent use. Intravenous drug use is not a major risk factor for HIV transmission in Honduras. Plasma (300-600 ml) from 62 HIV-1 infected individuals were serotyped using peptides corresponding to the envelope V3 region of HIV-1 subtypes A, B, C, D, and E (Barin et al., 1996). Laboratory control plasmas from the United States (n=5), Botswana (n= 5), and Thailand (n= 5) previously subtyped as HIV-1 subtypes B, C, and E, respectively, by envelope sequence analysis were serotyped correctly using this assay (data not shown). Peripheral blood mononuclear cells (PBMC) were separated by Ficoll gradients and genomic DNA was extracted by the proteinase K/phenol method. The polymerase chain reaction (PCR) was used to amplify the full-length envelope (gp120) molecule (1500 bp), the C2V3C3 region of envelope (210 bp), and the 3% Long Terminal Repeat (620 bp). Individual clones (for LTR and gp120) and PCR products (C2V3C3) were sequenced using dye terminator chemistry and an ABI 373 sequencer. Nucleotide and amino acid sequences from each clone were assembled using the ClustalW (Thompson et al., 1994) software package and phylogenetic trees of envelope and LTR sequences were obtained by comparison with HIV-1 subtypes A, B, C, D, and E reference sequences (Korber et al., 1997). Serological results indicated that 55 of 62 samples from Honduras showed preferential reactivity with the subtype B-specific peptide, two (LF001 and SF030) reacted preferentially with the subtype A peptide, and five (CF017, CF018, SF005, SF031, and TF021) were dually reactive with the subtype B peptide and either the subtype A or subtype C peptide (Table 1). All 7 samples that were dually reactive or reacted with a non-B-specific peptide were obtained from females. These women showed no significant differences in sociodemographic or risk behavior characteristics when compared to other females enrolled in the study. Because V3 serotyping involves a small region of the entire viral genome, the nature of other regions of the genome cannot be determined. To note the presence or absence of intersubtype recombination as well as the conservation of important structural motifs within the envelope gene, full-length gp120 sequences were obtained from 17 Honduras isolates. All sequences belonged to HIV-1 subtype B. Analysis of these sequences revealed that all 24 cysteine residues, critical for proper glycoprotein folding, were conserved in all samples (with the exception of a C-S change at C7 in sample TM013). The GPGR motif of the V3 crown was present in 13 of 17 samples with the other four being GPGS (1), GLGG (1), or GPGK (2). N-glycosylation sites (Asn-X-Ser/Thr) of HXB2 were also conserved and an additional glycosylation site was identified in the V1 region in 13 patients. One to three extra glycosylation B. Renjifo et al. / Virus Research 60 (1999) 191–197 Table 1 HIV-1 subtype classification as determined by serotyping and genotyping of envelope and LTRe. Sample CF002 CF003 CF004 CF005 CF006 CF008 CF012 CF015 CF016 CF017 CF018 LF001 LF003 LF004 LF006 LF008 LF014 LF015 LM001 PF002 PF004 SF005 SF006 SF007 SF008 SF009 SF010 SF011 SF014 SF018 SF020 SF026 SF028 SF029 SF030 SF031 SF035 SF038 SF049 SF050 SF051 SF052 SM002 SM003 SM005 SM007 SM008 SM009 TF001 TF002 Serology B B B B B B B B B B/C B/A A B B B B B B B B B B/A B B B B B B B B B B B B A B/A B B B B B B B B B B B B B B Envelope gp120a LTRc C2V3C3 B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B Serology TF003 TF006 TF007 TF009 TF013 TF014 TF021 TF022 TF024 TF028 TM013 TM017 B B B B B B C/B B B B B B Envelope gp120a LTRc C2V3C3 B B B B B B B B B b,d B B B 1500 bp from V1 to V5 of gp120 (n= 17). 210 bp including C2V3C3 (n = 41). c 620 bp including U3R of 3% LTR (n= 15). d Some sequence data derived from direct sequencing of PCR products. e Nucleotide sequences were deposited in GenBank under accession numbers AF096658-AF096689. b B B B B B B B Sample a B B Table 1 (continued) HIV-1 subtype classification as determined by serotyping and genotyping of envelope and LTRe. b,d B 193 B sites were identified in a fraction of patients. The mean gp120 interpatient divergence for these 17 sequences was 13.1% (range 9.5– 18.1%). This was considerably higher than the 8.8% divergence noted by Candal et al., (1997) when analyzing a 333-bp fragment corresponding to the C2V3 region of envelope. To determine the diversity of Honduran isolates as compared to other HIV-1 subtype B isolates, 17 database (Korber et al., 1997) subtype B sequences from nine countries – P896 (Jamaica), WEAU160 (United Kingdom), JH32 (Japan), OY1 (Gabon), 3202A21 (Netherlands), NY5 (United States), MN (United States), RF (Haiti), LAI (France), YU2 (United States), HXB2 (France), HAN (Germany), JRFL (United States), BCSG3C (United States), CAM1 (United Kingdom), SF2B13 (United States) and BRVA (United States) – were aligned and their pairwise distances determined. The mean interpatient divergence for these database sequences was 10.3% (range 1.8– 13.5%), significantly lower (pB 0.001, t-test) than that seen with envelope sequences from Honduras described here. The phylogenetic 194 B. Renjifo et al. / Virus Research 60 (1999) 191–197 Fig. 1. 1445 bp sequences containing C1– C5 of the envelope gene were aligned in Clustal W (Thompson et al., 1994) with Los Alamos database references (conA, 92UG037, U455, conB, OY1, RF, JRFL, conC, 92BR025, 93MW965, conD, NDK, Z2Z6, conE, 93TH253, and 90CF402).100 bootstraps were performed. Sequences from Honduras are indicated in bold italics. tree comparing Honduras full-length envelope sequences with database references shows no significant clustering of Honduras subtype B envelopes separate from other HIV-1 subtype B sequences or by geographic region. (Fig. 1). The Long Terminal Repeat region of HIV-1 is essential for proviral synthesis, integration of the proviral DNA into the host cell’s genome, and regulation of HIV-1 transcription. Because determinants of cellular tropism (Chen et al., 1984; Speck et al., 1990a) and disease specificity (Chatis et al., 1983; DesGroseillers et al., 1983; Speck et al., 1990b) have been investigated in the LTRs of other retroviruses, the 3% Long Terminal Repeat sequences from 15 patients were analyzed to note changes within critical regulatory regions that may further characterize HIV-1 in Honduras. All sequences belonged to HIV-1 subtype B. Alignment of 15 Honduras LTR sequences demon- strated that the consensus Honduras LTR differed from the database consensus B LTR (Korber et al., 1997) at several positions. However, motifs such as NF-kB enhancers, Sp1 sites, and a TATA box were highly conserved in all Honduran LTRs analyzed (data not shown). These egulatory elements are present in representative database subtype B LTR sequences and are important for regulation of HIV-1 gene expression (Gaynor, 1992). Conserved COUP sites were also identified as well as putative NF-AT and USF sites. Interestingly, four of the patient consensus sequences contained an additional putative TATA box located between the two NF-AT binding sites. The sequence TATAAG found at this position was identical to the hexameric TATA box located at − 30 relative to the transcription start site in the consensus B LTR. This sequence was not noted in other laboratory samples belonging B. Renjifo et al. / Virus Research 60 (1999) 191–197 to HIV-1 subtype B (data not shown). Although the functional relevance of this extra TATA box and other nucleotide differences with the consensus B sequence have not been explored, these represent areas for future study. Pairwise comparisons show that the mean 3% LTR interpatient divergence is 7.8% (range 0.2– 12.5%). The phylogenetic tree of Honduras 3% long terminal repeat sequences shows no significant subclustering of these samples within the subtype B branch. (Fig. 2) Although LF001 and SF030 were determined to be subtype A isolates by serology, analysis of the C2– C3 region of the envelope gene revealed these samples to cluster phylogenetically with subtype B references. Samples SF005, SF031, and TF021 were dually reactive by serology but also clustered with subtype B references when the C2– C3 region of envelope was analyzed phylogenetically. Sam- 195 ples CF017 and CF018 were shown to be dually reactive by serology; however, envelope sequences were not obtained from these isolates. The 3% LTR was sequenced from CF017 and shown to belong to subtype B. Gag sequences (800 bp) were also obtained from samples LF001, TF021, and TF022; all belonged to HIV-1 subtype B (data not shown). We were unable to amplify gag, en6, or LTR sequences from sample CF018. Epidemiological data suggests that HIV-1 subtype B is commonly associated with homosexual transmission and intravenous drug use; however, HIV-1 C and E – the prevalent subtypes in southern Africa/India and southeast Asia – are transmitted largely through heterosexual contact (Essex, 1994). This has led to the suggestion that certain subtypes may have different phenotypic properties resulting in a selective advantage for a given route of transmission (Van Harmelen et al., Fig. 2. 490 bp sequences containing U3-R of the 3% Long Terminal Repeat were aligned in Clustal W (Thompson et al., 1994) with Los Alamos database references. 100 bootstraps were performed. Sequences from Honduras are indicated in bold italics. 196 B. Renjifo et al. / Virus Research 60 (1999) 191–197 1997). In Thailand, for example, where both subtypes B and E are present, subtype E seems to be more readily transmitted via heterosexual contact than subtype B (Kunansont et al., 1995). In South Africa, subtype B is associated with homosexual transmission while subtype C is associated with heterosexual transmission (Van Harmelen et al., 1997). Although this association between HIV-1 subtypes and a particular mode of transmission is controversial (Mastro et al., 1997; Kitayaporn et al., 1998), HIV-1 transmission in Honduras is predominantly heterosexual (Trujillo-Garcia et al., 1998) and involves subtype B. In the current study, we use serotyping and genotyping of partial (V3) and full-length envelope and the 3% LTR to determine the subtype distribution of HIV-1 in Honduras. Serological results suggested that 55 of 62 (88.7%) of the Honduran samples belonged to HIV-1 subtype B, 2 (3.2%) belonged to subtype A, and 5 (8.1%) were dually reactive with the subtype B peptide and either the subtype A or subtype C peptide. However, phylogenetic analyses of multiple HIV-1 genes indicate that all Honduras isolates analyzed in the current study belonged to HIV-1 subtype B. Of the 49 samples for which both peptide serotyping and DNA sequencing (of full-length gp120 or C2V3C3) results were obtained, there was 89.8% concordance between the two methods. Serotyping alone may be misleading in this particular population leading one to suspect the presence of non-B subtypes in Honduras; however, genotypic results demonstrate the presence of only HIV-1 subtype B in Honduras. The reliability of serotyping is highly dependent upon the viral diversity within the studied population (European Commission and Joint United Nations Programme, 1997), thus any discrepancy between serotyping and genotyping can likely be explained by the high diversity of full-length envelope sequences from Honduras noted in the current study. Because envelope sequence data was not available for samples CF017 and CF018 that were dually reactive by serology, we could not rule out the possibility of HIV-1 intersubtype recombinant viruses circulating in Honduras. However, this seemed unlikely for several reasons. First, phylogenetic data showed no evidence of discordant subtype classification of different HIV-1 genes sequenced from the same sample that may indicate recombinant viruses. Second, for five of the samples that were dually-reactive or non-B by serology, the envelope gene was confirmed as subtype B by genotypic analyses. Third, despite analysis of multiple gene loci, only HIV-1 subtype B sequences were detected from several sites throughout Honduras. Although the isolates collected here represent those from a single country, the envelope sequences appear to be more diverse than other HIV-1 subtype B isolates from nine other countries. Interestingly, full-length envelope sequences from Honduras are also considerably more diverse (13.1% vs 8.8%) than the V3 envelope sequences previously studied (Candal et al., 1997). 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