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Primary resistance mutations to fusion inhibitors and polymorphisms in gp41 sequences of non-B subtypes and recombinant HIV-1 isolates were analysed. L91H to RPR103611 was detected in one DGpol/Denv/Dgp41 recombinant; L9F and K144R, rarely reported previously, were frequent in the B region of CRF14_BG recombinants. V194I and V318A, not described in the G subtype, were detected in the G region of BG recombinants and in G subtype viruses that also show the rare mutations T115L, M118V and K90R.

Background: HIV-1 subtype B is largely predominant in the Caribbean, although other subtypes have been recently identified in Cuba., Objectives: To examine HIV-1 genetic diversity in Cuba., Methods: The study enrolled 105 HIV-1-infected individuals, 93 of whom had acquired the infection in Cuba. DNA from peripheral blood mononuclear cells was used for polymerase chain reaction amplification and sequencing of pol (protease-reverse transcriptase) and env (V3 region) segments. Phylogenetic trees were constructed using the neighbour-joining method. Intersubtype recombination was analysed by bootscanning., Results: Of the samples, 50 (48%) were of subtype B and 55 (52%) of diverse non-B subtypes and recombinant forms. Among non-B viruses, 12 were non-recombinant, belonging to six subtypes (C, D, F1, G, H and J), the most frequent of which was subtype G (n = 5). The remaining 43 (78%) non-B viruses were recombinant, with 14 different forms, the two most common of which were Dpol/Aenv (n = 21) and U(unknown)pol/Henv (n = 7), which grouped in respective monophyletic clusters. Twelve recombinant viruses were mosaics of different genetic forms circulating in Cuba. Overall, 21 genetic forms were identified, with all known HIV-1 group M subtypes present in Cuba, either as non-recombinant viruses or as segments of recombinant forms. Non-B subtype viruses were predominant among heterosexuals (72%) and B subtype viruses among homo- or bisexuals (63%)., Conclusion: An extraordinarily high diversity of HIV-1 genetic forms, unparalleled in the Americas and comparable to that found in Central Africa, is present in Cuba.

We recently reported the finding of phylogenetically related HIV-1 BG intersubtype recombinant and G subtype nonrecombinant viruses circulating among injecting drug users in the region of Galicia in northwestern Spain. Here, we report the characterization of near full-length genome sequences of nine of these viruses (seven BG recombinant and two of nonrecombinant G subtype), obtained from epidemiologically unlinked individuals. Bootscan analysis reveals that six recombinant viruses share an identical mosaic structure, with two intersubtype breakpoints delimiting a B subtype segment comprising most of Env gp120 and the external portion of Env gp41, with the remaining portions of the genome being of subtype G, thus mimicking a pseudotype virion structure. The seventh BG recombinant virus exhibits breakpoints in env coincident with the other BG viruses but contains additional B subtype segments in gag and pol. In phylogenetic trees of complete genomes and of the B subtype segment of env, all seven BG viruses group in a monophyletic cluster. G subtype portions of the BG viruses group uniformly with the newly derived nonrecombinant G subtype viruses of Galicia in bootscan analysis, which points to the locally circulating G subtype strain as parental of the recombinants. These results allow us to define a new HIV-1 circulating recombinant form (CRF14_BG), the first reported to originate in Western Europe.

The findings that BF intersubtype recombinant human immunodeficiency type 1 viruses (HIV-1) with coincident breakpoints in pol are circulating widely in Argentina and that non-recombinant F subtype viruses have failed to be detected in this country were reported recently. To analyse the mosaic structures of these viruses and to determine their phylogenetic relationship, near full-length proviral genomes of eight of these recombinant viruses were amplified by PCR and sequenced. Intersubtype breakpoints were analysed by bootscanning and examining the signature nucleotides. Phylogenetic relationships were determined with neighbour-joining trees. Five viruses, each with predominantly subtype F genomes, exhibited mosaic structures that were highly similar. Two intersubtype breakpoints were shared by all viruses and seven by the majority. Of the consensus breakpoints, all nine were present in two viruses, which exhibited identical recombinant structures, and four to eight breakpoints were present in the remaining viruses. Phylogenetic analysis of partial sequences supported both a common ancestry, at least in part of their genomes, for all recombinant viruses and the phylogenetic relationship of F subtype segments with F subtype viruses from Brazil. A common ancestry of the recombinants was supported also by the presence of shared signature amino acids and nucleotides, either unreported or highly unusual in F and B subtype viruses. These results indicate that HIV-1 BF recombinant viruses with diverse mosaic structures, including a circulating recombinant form (which are widespread in Argentina) derive from a common recombinant ancestor and that F subtype segments of these recombinants are related phylogenetically to the F subtype viruses from Brazil.

Objectives: To develop an assay for the early detection and quantification of minor human immunodeficiency virus-1 populations bearing multiple drug resistance (MDR) mutations., Study Design/methods: The oligonucleotide ligation assay (OLA) is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay. In OLA-MDR, oligonucleotides were designed to detect MDR mutations. The method was validated with wild-type and MDR mutant clones mixed at different proportions., Results: K103N mutants were detected as minor populations (5%-30%) by OLA in 6 of 18 samples from patients treated with nonnucleoside reverse transcription inhibitors and classified as wild type by sequencing. In one patient, the kinetics of the increase of MDR mutants could be followed in sequential samples, with K103N being detected earlier by OLA than by sequencing. Q151M mutants were detected as minor populations (13%-24%) by OLA but not by sequencing in 4 samples., Conclusions: Oligonucleotide ligation assay MDR exhibits higher sensitivity than sequencing for detection of minor MDR mutant populations.

Objectives: To determine the prevalence of drug resistance and to analyze the subtyping in HIV-1 samples from Cuba., Methods: From an estimated total number of 1,950 HIV-1-infected persons in Cuba, a sample of 103 patients were studied, 76 of whom had received drug treatment for HIV and 27 who had not. The RNA plasma viral load was measured, and automated sequencing was used to assess resistance mutations to reverse transcriptase inhibitors (RTIs) and to protease inhibitors (PIs). Subtyping in the V3 region was performed using heteroduplex mobility assay (HMA). In order to corroborate the HMA results, sequencing of env (C2-V3-C3) was done with one-third of the samples in each of the subtype groups detected by HMA., Results: Out of the 103 samples, 81 of them (78.6%) were classified as subtype B, 19 (18.5%) as subtype A, and 3 (2.9%) as subtype C. The prevalence of resistance mutations was 26.2% to RTIs, none to PIs alone, and 3.9% to both categories of drugs. The prevalence of resistance to nucleoside RTIs (NRTIs) was 27.6% in treated patients and 7.4% in the untreated patients, and for nonnucleoside RTIs (NNRTIs) it was 5.3% and 0%, respectively. Among treated patients a low frequency (2.6%) of dual resistance to zidovudine (ZDV) plus lamivudine (3TC) and abacavir (ABC) was detected, and multidrug resistance to NRTIs was not found. In relation to PIs together with RTIs, the prevalence of resistance was 5.3% for treated patients and 0% for untreated patients., Conclusions: Even though Cuba is generally considered an area where subtype B is dominant, we detected a high proportion of non-B subtype viruses. The low prevalence of resistance mutations to RTIs and PIs reflects the delay in introducing these drugs to Cuba. Multidrug resistance to RTIs was not found, so, as of now, the use of these drugs continues to be an option for Cuban patients.

We report the first study on prevalence of antiretroviral drug-associated resistance mutations in Venezuela. Protease and reverse transcriptase (RT) coding regions were analyzed in DNA samples obtained from 100 HIV-1-infected individuals. Primary resistance mutations to RT inhibitors were identified in 26% of patients treated with these drugs. Transmission of HIV-1-resistant strains was detected in a drug-naive patient (3%). Primary resistance mutations to protease inhibitors (PIs) were present in 9% of the 44 PI-treated patients and in 1 PI-naive individual. Phylogenetic analysis of these samples has resulted in the most extensive survey, to date, of HIV-1 genetic forms circulating in Venezuela. Ninety-nine samples clustered with subtype B, and 1 individual harbored the first B/F recombinant virus reported in Venezuela, with protease clustering with subtype F and RT with subtype B. In addition, this isolate had a new insertion (Glu-34 duplication) in the protease gene.

Background: The HIV-1 epidemics in Western Europe are dominated by B subtype viruses. Non-B subtype is largely restricted to individuals infected outside of Europe and to their direct contacts and is generally acquired by the heterosexual route., Methods: Protease and a segment of reverse transcriptase were amplified and sequenced from plasma RNA in 451 individuals from seven cities of Galicia, north-western Spain. Subtype sequence homologies were determined using the BLAST algorithm. Non-B sequences were examined by phylogenetic analysis and intersubtype recombination by bootscanning. The env V3 region was analysed in all non-B and in 38 B subtype viruses., Results: Ten different non-B genetic forms were identified in 20 (4.4%) individuals. Subtypes were concordant between pol and V3 in five viruses; 14 (70%) infections were with intersubtype recombinant viruses, and one individual had a dual B+G infection. Seven recombinant viruses were phylogenetically related to five reported recombinant forms. Three non-recombinant G and six recombinant BG viruses formed a monophyletic cluster for pol. All but three individuals with non-B infections were native Spanish. Only 6 of 16 individuals referred to sexual contacts with sub-Saharan Africans. Twelve (60%) non-B subtype infections, including all with G and BG viruses, were in injecting drug users (IDU)., Conclusions: Non-B subtype viruses were identified in 4.4%, with a high diversity of genetic forms, including 70% infections with intersubtype recombinant viruses. The majority of individuals with non-B infections were IDU, most of them without known contacts with non-European sources, and among whom BG recombinant viruses are circulating.

Objectives: To describe the prevalence of genotypic resistance mutations, including single and multidrug resistance (MDR) to reverse transcriptase (RT) and protease (PR) inhibitors in treated and untreated patients from two geographical areas in Spain (Madrid and Galicia)., Study Design/methods: Resistance mutations to RT inhibitors were studied by line probe assay (LiPA) or by automated sequencing in 468 patients (Madrid, 268; Galicia, 200), and resistance mutations to PR inhibitors were studied by automated sequencing in 295 patients (Madrid, 85; Galicia, 210)., Results: The proportion of resistance mutations in treated and untreated patients results were higher by the LiPA method than by sequencing. By sequencing, we detected resistance mutations to nucleoside analogue RT (NRT) inhibitors and NRT inhibitors plus nonnucleoside RT (NNRT) inhibitors in 35.4% and 17.2% of treated patients, respectively. We also detected MDR to zidovudine plus lamivudine in 13.9% of treated patients from Galicia, in 1.7% from Madrid (p < 0.001), and in 1.5% of untreated patients from Galicia. Also, we detected MDR to NRT inhibitors in 3.8% and to NNRT inhibitors in 9.1%. We found resistance mutations to PR inhibitors in 38.1% of treated patients and in 0.9% of untreated patients., Conclusions: These findings reinforce the usefulness of testing for resistance mutations in some cases to evaluate their prevalence in a given population and in the follow-up of treated patients.

Objectives: We attempted to define optimal conditions for amplification of low copy number HIV-1 RNA sequences in plasma samples, applying improved conditions for nucleic acid extraction and amplification., Methods: Several methodologic parameters were evaluated, including methods of RNA extraction, volumes of plasma samples, proportion of extracted RNA used as a template for amplification, and reverse transcriptase-DNA polymerase enzyme combination employed in cDNA synthesis and polymerase chain reaction amplification., Results: With this improved assay, we were able to obtain sufficient amounts of amplified material for direct sequencing in 97% of all plasma samples in our study, including 88% of samples with viral loads <80 copies/mL, 78% of samples with viral loads <50 copies/mL, and even 2 (67%) of 3 samples with <20 copies/mL., Conclusions: This procedure could be useful for testing resistance mutations in patients undergoing highly active antiretroviral therapy, in which the viral load is commonly <400 copies/mL, and even if it is <20 RNA copies/mL.

We report a statistical analysis of genetic heterogeneity of the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1. Both newly determined sequences and sequences contained in the data banks have been examined. For the calculations, the viral samples and the regions within the RT molecule were divided in two groups. The viral samples were split into those from patients not subjected to antiretroviral therapy and those from patients treated with zidovudine (AZT, 3'-azido-3'-deoxythymidine) alone or in combination with other RT inhibitors. The RT-coding region was divided into segments encoding beta-strands and segments encoding alpha-helices. A significantly lower heterogeneity was observed in beta-strands relative to the alpha-helix coding segments. Application of the D test of Tajima has provided evidence of operation of negative (or purifying) selection in sequences from viruses of patients not subjected to antiretroviral treatment as well as in treated patients. In the group of untreated individuals, regions encoding beta-strands are subjected to stronger negative selection than those encoding alpha-helices. It is likely that the observed differences reflect stronger functional constraints in beta-strands than in alpha-helices of RT.

Different methods for studying the concurrent effects of two linear inhibitors on a single enzyme have been published, including the fractional product of Webb, the Yonetani-Theorell plot or the method of Chou and Talalay. Recently the use of combination plots has also been advocated for this purpose. We have evaluated the applicability of these methods and found that most of them depend on assumptions about the mechanism of action of the inhibitors. If the mechanism of action is not completely understood, or if some assumptions about the mechanism are unfounded, the parameters obtained may be meaningless. Even if these assumptions are correct, the interaction can be advantageously measured using an alternative representation that does not require a knowledge of the inhibition constants and allows experimental data to be retrieved from the plot. In other cases it is the interpretation of the results rather than the validity of the method that is misleading. A common mistake is to take the exclusivity of the effects of two inhibitors as exclusivity of their binding. We show that this assumption is seldom justified. In any case, it is possible to decide whether the combination of two or more inhibitors is more effective than their individual use by means of isobolographic analysis, even when no information about their mechanism of action is available.

The presence of resistance-related mutations in 185 serial proviral DNA samples from 108 HIV-infected patients was monitored using the line probe assay (LiPA). The proportions of wild-type and mutant virus in each sample were determined. Subsequent samples from the same patient were analysed. Resistance mutations were detected in 58 of 108 patients studied (53.7%), 53 of 73 (72.6%) treated with antivirals and 5 of 35 (14.2%) untreated. The mutations were against zidovudine (51), lamivudine (1), zidovudine and lamivudine (4), zidovudine and zalcitabine (1) and zidovudine and didanosine (1). Among the 58 patients with resistant virus, 168 related mutations were observed: 161 to zidovudine (90 in codon 70, 25 in codon 41 and 46 in codon 215), 5 to lamivudine (codon 184), 1 to zalcitabine (codon 69) and 1 to didanosine (codon 74). Mixtures of wild-type and resistant mutants were detected in 76 of 90 (84.4%) mutated at codon 70, 28 of 46 (60.8%) mutated at codon 215 and in 21 of 25 (84%) mutated at codon 41. The mutations at codon 184 were mixtures of wild-type and resistant in 4 of 5 samples. The agreement between LiPA and sequencing was 96.5%. LiPA was more sensitive for the detection of mutants that were present at low frequency. The analysis of sequential samples from the same patient allowed evaluation of the dynamics of appearance of the resistant mutants.

Synergistic inhibition of HIV replication in cell culture has been reported for many combinations of reverse transcriptase inhibitors. However, the biochemical basis underlying this interaction is in most cases unknown. It has been previously shown that combinations of L-697,661 or U-90152s with AZT or ddC synergistically inhibit HIV-1 replication in cell culture. The combination of AZT with ddC is also favorable with respect to the inhibition of viral replication. However, the corresponding combinations showed no synergy in inhibiting enzyme activity when tested on conventional polymerase assays using homo- or heteropolymeric RNA and DNA as template. Data obtained suggest that amplification of the effect of chain terminators, a consequence of the high potential number of termination sites present on the template, override the synergistic effect expected for the combination of two independent nucleotide analogs. When a saturating amount of enzyme over template:primer was used, and a single site on the template was available for each chain terminator, the combination of AZTTP and ddCTP synergistically inhibited enzyme activity, whereas, as expected, the combination of AZTTP and ddTTP behaved as merely additive. Under similar conditions the combination of U-90152s and AZTTP was also synergistic. These results suggest that synergy found in antiviral assays with combinations having nucleosidic inhibitors is not related to the synergistic inhibition of reverse transcriptase and might be due to the presence in the viral population of virus strains with different sensitivity to the inhibitors.

A method to evaluate interactions between biologically active agents is presented. Synergism, zero interaction, and antagonism were easily detected with the three-dimensional approach proposed herein. This method is compatible with a checkerboard design to diagnose the interaction between agents and obviate the need to test their mixtures in a fixed concentration ratio as proposed by Chou and Talalay. Dose-response curves for individual agents were obtained, and experimental data fitted to appropriate equations by nonlinear regression. If zero interaction was present, the predicted effect could be calculated for each combination using the classical isobole equation with any spreadsheet having a command to solve mathematical equations by iteration. This allowed the selection of appropriate concentrations for the combination of two or more agents. Interaction between agents could be assessed in two ways: by comparing experimental with expected effects, if zero interaction is present; or by analyzing the reduction or increase in total dose found as a consequence of the interaction. The applicability of both approaches is discussed and, for purposes of comparison with other methods, examples based on published data are analyzed and commented upon.