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This work is aimed at assessing the presence of positive selection and/or shifts of the evolutionary rate in a fast-expanding HIV-1 subtype F1 transmission cluster affecting men who have sex with men in Spain. We applied Bayesian coalescent phylogenetics and selection analyses to 23 full-coding region sequences from patients belonging to that cluster, along with other 19 F1 epidemiologically-unrelated sequences. A shift in the overall evolutionary rate of the virus, explained by positively selected sites in the cluster, was detected. We also found one substitution in Nef (H89F) that was specific to the cluster and experienced positive selection. These results suggest that fast transmission could have been facilitated by some inherent genetic properties of this HIV-1 variant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

HIV-1 protease-reverse transcriptase sequences from 62 HIV-1-infected individuals recently diagnosed in Moscow were analyzed. Subtype A former Soviet Union (FSU) (A FSU ) variant was the predominant clade (62.9%), followed by subtype B (22.6%), unique recombinants (6.5%), subtype G (6.5%), and CRF01_AE (1.6%). A FSU predominated among people who inject drugs (88.9%) and heterosexually acquired infections (77.8%), while subtype B was the most prevalent genetic form among men who have sex with men (44%), although A FSU was also frequent in this population (36%). Forty-eight (77.4%) viruses branched within intrasubtype clusters, three of which, of subtype B, had a majority of viruses collected outside of FSU. The four subtype G viruses identified in this study belonged to the Portuguese-Spanish (Iberian) variant and, together with three from databases, formed a Russian cluster closely related to viruses from Denmark. This is the first report of the circulation of the Iberian subtype G variant in Russia.

In this study, the genetic diversity of HIV-1 in Tunisia was analyzed. For this, 193 samples were collected in different regions of Tunisia between 2012 and 2015. A protease and reverse transcriptase fragment were amplified and sequenced. Phylogenetic analyses were performed through maximum likelihood and recombination was analyzed by bootscanning. Six HIV-1 subtypes (B, A1, G, D, C, and F2), 5 circulating recombinant forms (CRF02_AG, CRF25_cpx, CRF43_02G, CRF06_cpx, and CRF19_cpx), and 11 unique recombinant forms were identified. Subtype B (46.4%) and CRF02_AG (39.4%) were the predominant genetic forms. A group of 44 CRF02_AG sequences formed a distinct Tunisian cluster, which also included four viruses from western Europe. Nine viruses were closely related to isolates collected in other African or in European countries. In conclusion, a high HIV-1 genetic diversity is observed in Tunisia and the local spread of CRF02_AG is first documented in this country.

This work was aimed to study the HIV-1 subtype B epidemics in the Basque Country, Spain. 1727 HIV-1 subtype B sequences comprising protease and reverse transcriptase (PR/RT) coding regions, sampled between 2001 and 2008, were analyzed. 156 transmission clusters were detected by means of phylogenetic analyses. Most of them comprised less than 4 individuals and, in total, they included 441 patients. Six clusters comprised 10 or more patients and were further analyzed in order to study their origin and diversification. Four clusters included men who had unprotected homosexual sex (MSM), one group was formed by intravenous drug users (IDUs), and another included both IDUs and people infected through unprotected heterosexual sex (HTs). Most of these clusters originated from the mid-1980s to the mid-1990s. Only one cluster, formed by MSM, originated after 2000. The time between infections was significantly lower in MSM groups than in those containing IDUs (P-value <0.0001). Nucleoside RT and non-nucleoside RT inhibitor (NRTI and NNRTI)-resistance mutations to antiretroviral treatment were found in these six clusters except the most recent MSM group, but only the IDU clusters presented protease inhibitor (PI)-resistance mutations. The most prevalent mutations for each inhibitor class were PI L90M, NRTI T215D/Y/F, and NNRTI K103N, which were also among the most prevalent resistant variants in the whole dataset. In conclusion, while most infections occur as isolated introductions into the population, the number of infections found to be epidemiologically related within the Basque Country is significant. Public health control measures should be reinforced to prevent the further expansion of transmission clusters and resistant mutations occurring within them., (Copyright © 2016 Elsevier B.V. All rights reserved.)

HIV-1 exhibits a characteristically high genetic diversity, with the M group, responsible for the pandemic, being classified into nine subtypes, 72 circulating recombinant forms (CRFs) and numerous unique recombinant forms (URFs). Here we characterize the near full-length genome sequence of an HIV-1 BG intersubtype recombinant virus (X3208) collected in Galicia (Northwest Spain) which exhibits a mosaic structure coincident with that of a previously characterized BG recombinant virus (9601&#95;01), collected in Germany and epidemiologically linked to Portugal, and different from currently defined CRFs. Similar recombination patterns were found in partial genome sequences from three other BG recombinant viruses, one newly derived, from a virus collected in Spain, and two retrieved from databases, collected in France and Portugal, respectively. Breakpoint coincidence and clustering in phylogenetic trees of these epidemiologically-unlinked viruses allow to define a new HIV-1 CRF (CRF73&#95;BG). CRF73&#95;BG shares one breakpoint in the envelope with CRF14&#95;BG, which circulates in Portugal and Spain, and groups with it in a subtype B envelope fragment, but the greatest part of its genome does not appear to derive from CRF14&#95;BG, although both CRFs share as parental strain the subtype G variant circulating in the Iberian Peninsula. Phylogenetic clustering of partial pol and env segments from viruses collected in Portugal and Spain with X3208 and 9691&#95;01 indicates that CRF73&#95;BG is circulating in both countries, with proportions of around 2-3% Portuguese database HIV-1 isolates clustering with CRF73&#95;BG. The fact that an HIV-1 recombinant virus characterized ten years ago as a URF has been shown to represent a CRF suggests that the number of HIV-1 CRFs may be much greater than currently known.

We recently reported the rapid expansion of an HIV-1 subtype F cluster among men who have sex with men (MSM) in the region of Galicia, Northwest Spain. Here we update this outbreak, analyze near full-length genomes, determine phylogenetic relationships, and estimate its origin. For this study, we used sequences of HIV-1 protease-reverse transcriptase and env V3 region, and for 17 samples, near full-length genome sequences were obtained. Phylogenetic analyses were performed via maximum likelihood. Locations and times of most recent common ancestors were estimated using Bayesian inference. Among samples analyzed by us, 100 HIV-1 F1 subsubtype infections of monophyletic origin were diagnosed in Spain, including 88 in Galicia and 12 in four other regions. Most viruses (n = 90) grouped in a subcluster (Galician subcluster), while 7 from Valladolid (Central Spain) grouped in another subcluster. At least 94 individuals were sexually-infected males and at least 71 were MSM. Seventeen near full-length genomes were uniformly of F1 subsubtype. Through similarity searches and phylogenetic analyses, we identified 18 viruses from four other Western European countries [Switzerland (n = 8), Belgium (n = 5), France (n = 3), and United Kingdom (n = 2)] and one from Brazil, from samples collected in 2005-2011, which branched within the subtype F cluster, outside of both Spanish subclusters, most of them corresponding to recently infected individuals. The most probable geographic origin and age of the Galician subcluster was Ferrol, Northwest Galicia, around 2007, while the Western European cluster probably emerged in Switzerland around 2002. In conclusion, a recently expanded HIV-1 subtype F cluster, the largest non-subtype B cluster reported in Western Europe, continues to spread among MSM in Spain; this cluster is part of a larger cluster with a wide geographic circulation in diverse Western European countries.

Our objectives were to carry out an epidemiological surveillance study on transmitted drug resistance (TDR) among individuals newly diagnosed of HIV-1 infection during a nine year period in Spain and to assess the role of transmission clusters (TC) in the propagation of resistant strains. An overall of 1614 newly diagnosed individuals were included in the study from January 2004 through December 2012. Individuals come from two different Spanish regions: Galicia and the Basque Country. Resistance mutations to reverse transcriptase inhibitors (RTI) and protease inhibitors (PI) were analyzed according to mutations included in the surveillance drug-resistance mutations list updated in 2009. TC were defined as those comprising viruses from five or more individuals whose sequences clustered in maximum likelihood phylogenetic trees with a bootstrap value ≥90%. The overall prevalence of TDR to any drug was 9.9%: 4.9% to nucleoside RTIs (NRTIs), 3.6% to non-nucleoside RTIs (NNRTIs), and 2.7% to PIs. A significant decrease of TDR to NRTIs over time was observed [from 10% in 2004 to 2% in 2012 (p=0.01)]. Sixty eight (42.2%) of 161 sequences with TDR were included in 25 TC composed of 5 or more individuals. Of them, 9 clusters harbored TDR associated with high level resistance to antiretroviral drugs. T215D revertant mutation was transmitted in a large cluster comprising 25 individuals. The impact of epidemiological networks on TDR frequency may explain its persistence in newly diagnosed individuals. The knowledge of the populations involved in TC would facilitate the design of prevention programs and public health interventions.

Objectives: This study aimed to develop a genotypic method to predict HIV-1 coreceptor usage by employing the nucleotide sequence of the env gene in a tree-augmented naive Bayes (TAN) classifier, and to evaluate its accuracy in prediction compared with other available tools., Methods: A wrapper data-mining strategy interleaved with a TAN algorithm was employed to evaluate the predictor value of every single-nucleotide position throughout the HIV-1 env gene. Based on these results, different nucleotide positions were selected to develop a TAN classifier, which was employed to predict the coreceptor tropism of all the full-length env gene sequences with information on coreceptor tropism currently available at the Los Alamos HIV Sequence Database., Results: Employing 26 nucleotide positions in the TAN classifier, an accuracy of 95.6%, a specificity (identification of CCR5-tropic viruses) of 99.4% and a sensitivity (identification of CXCR4/dual-tropic viruses) of 80.5% were achieved for the in silico cross-validation. Compared with the phenotypic determination of coreceptor usage, the TAN algorithm achieved more accurate predictions than WebPSSM and Geno2pheno [coreceptor] (P<0.05)., Conclusions: The use of the methodology presented in this work constitutes a robust strategy to identify genetic patterns throughout the HIV-1 env gene differently present in CCR5-tropic and CXCR4/dual-tropic viruses. Moreover, the TAN classifier can be used as a genotypic tool to predict the coreceptor usage of HIV-1 isolates reaching more accurate predictions than with other widely used genotypic tools. The use of this algorithm could improve the correct prescribing of CCR5 antagonist drugs to HIV-1-infected patients.

We analyzed RNA splice site usage in three HIV-1 subtype B primary isolates through reverse transcriptase polymerase chain reaction (RT-PCR) amplification of spliced RNAs using a fluorescently labeled primer, with computerized size determination and quantification of PCR products, which were also identified by clone sequencing. In one isolate, P2149-3, unusual and unreported spliced transcripts were detected. This isolate preferentially used for rev RNA generation a 3' splice site (3'ss) located five nucleotides upstream of A4a, previously identified only in a T cell line-adapted virus and in a group O isolate, and designated A4d. P2149-3 also used an unreported 3'ss for rev RNA generation, designated A4h, located 20 nucleotides upstream of 3'ss A4c. Additionally, unusual nef RNAs using 3'ss A5a and A7a and with exon composition 1.3.7 were identified. The identification of several unusual and unreported spliced transcripts in an HIV-1 primary isolate suggests a greater diversity of splice site usage in HIV-1 than previously appreciated.

Due to the extraordinary degree of genetic diversity of HIV-1 and the structural complexity of its envelope glycoproteins, designing an effective vaccine is difficult, requiring the development of viral reagents to assess vaccine-elicited neutralizing antibodies. The aim of this study was to improve on our previously developed panel of HIV-1 strains of different genetic forms, focusing on strains from acute and recently acquired infections as the most representative of the transmitted viruses. HIV-1 primary isolates were expanded in peripheral blood mononuclear cells. Viral stocks of 40 ml each were produced. Syncytium-inducing (SI) phenotype, coreceptor use, and TCID(50)/ml were determined. Near full-length HIV-1 genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Forty-four HIV-1 strains were included in the panel. Twenty-four (54.1%) strains were from early infections (16 acute and 8 recent); of them, 21 (87%) were sexually transmitted. NSI/R5 phenotype was detected in 37 (84.1%) viruses and SI/R5,X4 in another 7 (15.9%). TCID(50)/ml ranged between 10(4) and 10(6.6). Twelve different genetic forms constituted this panel: subtypes A1, B, C, F1, and G; circulating recombinant forms CRF02&#95;AG, CRF14&#95;BG, and CRF24&#95;BG; and unique recombinant forms CRF02&#95;AG/A3, BF1, CRF12&#95;BF/B, and DF1G. In conclusion, in this study, we report the development of a comprehensive and well-characterized panel of HIV-1 isolates for assessing neutralization in HIV vaccine research. This panel is available for distribution through the Programme EVA Centre for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC).

We report the identification of a new HIV-1 circulating recombinant form (CRF47&#95;BF) derived from subtypes B and F. It was initially identified in protease-reverse transcriptase sequences from nine individuals from three separate regions of Spain who acquired HIV-1 infection via sexual contact. All nine sequences formed a strongly supported phylogenetic cluster, branching apart from all known CRFs, and in bootscan analyses were BF mosaics with two coincident breakpoints. Two epidemiologically unlinked viruses were sequenced in near full-length genomes, which exhibited identical mosaic structures, with 16 intersubtype breakpoints in a genome predominantly of subtype B. Subtype F segments of the new CRF failed to cluster with any of the near full-length genome subtype F sequences available in public databases. Recent dates of HIV-1 diagnoses and short genetic distances suggest a recent origin of this CRF. This is the tenth reported CRF&#95;BF, the first apparently having originated outside of South America.

An increase in HIV transmission among men who have sex with men (MSM) has been reported in eight regions of Spain from 2003 to 2007. In order to study the incidence of HIV-1 genetic forms in Galicia, northwest of Spain, in particular the spread of HIV-1 variants among MSM, 93 newly diagnosed HIV-1 patients, including those with acute and recently acquired infections, were studied for a year from August 2008 to August 2009. Thirty eight (41%) were MSM. Of them, nine (24%) were infected by non-B viruses, including seven different genetic forms. The analysis of transmission clusters showed that 23 (60%) MSM grouped in different clusters and mostly in large clusters. Resistance mutations were detected in six (16%) MSM.

We report the near full-length genome characterization of an HIV-1 subtype F virus (D88&#95;845) collected in St. Petersburg, Russia, from a 25-year-old Russian woman perinatally infected in 1982. In a Bayesian phylogenetic analysis, the genome sequence branched basally to the subsubtype F1 clade. In partial sequences, D88&#95;845 clustered with 13 other subtype F sequences from Russia, corresponding to gag (n = 2), pol (n = 3), and env (n = 8) segments. At least 11 of these sequences are from samples collected in St. Petersburg from heterosexually infected Russian individuals. In each of these segments, the Russian viruses formed a monophyletic cluster that branched as a sister clade of the F1 subsubtype. One sequence from Belgium branched with D88&#95;845 with a posterior probability of 0.99. This is the first report on the identification and near full-length genome characterization of the subtype F variant circulating in St. Petersburg, which is closely related to, but distinct from, the F1 subsubtype.

Objectives: To examine HIV-1 genetic diversity in St. Petersburg., Methods: Partial HIV-1 pol sequences from 102 plasma samples collected in 2006 were analyzed with a Bayesian phylogeny inference method., Results: Subtype A, former Soviet Union (FSU) variant (AFSU), was the predominant clade (89.3%); other clades were subtypes B (9.7%) and F1 (1%). AFSU was predominant both among injecting drug users (98.2%) and heterosexually infected individuals (91.4%), whereas subtype B was more prevalent among homosexual men (75%). Within the AFSU variant, most sequences (93.5%) branched within 1 of 4 strongly supported subclusters. The largest comprised 63% AFSU viruses and was uncommon outside St Petersburg. A second subcluster (17.4% AFSU viruses) corresponds to the variant with the V77I substitution in protease, which is widely circulating in different FSU countries. Two minor subclusters comprised 8.7% and 6.5% AFSU viruses, respectively. There was no correlation between risk exposure and AFSU subclusters. Six of 8 subtype B sequences, 4 of them from homosexual men, grouped in a monophyletic subcluster., Conclusions: The results of this study show a great predominance of AFSU viruses in St Petersburg and point to a few phylogenetically identifiable introductions as the origin of most current HIV-1 AFSU infections in the city.

Objective: To determine the introduction of HIV-1 genetic forms and to examine transmission clusters and resistance to antiretroviral inhibitors among newly diagnosed patients from the Basque Country, Spain, during 2004-2007., Methods: A total of 261 samples, corresponding to 47.5% heterosexuals, 37.9% men who have sex with men (MSM), and 11.1% intravenous drug users were analyzed in protease and reverse transcriptase to examine phylogenetic relationships and drug resistance-associated mutations., Results: Subtype B was detected in 220 (84.3%) samples and non-B subtype variants in 41 (15.7%) samples. Nearly half (47%) of the sequences grouped in transmission clusters. One of these comprised 14 individuals, 12 of them MSM, with the T215D revertan mutation. In largest transmission clusters, the percentage of MSM was higher than heterosexuals (P < 0.001). Resistance mutations were detected in 29 (11.1%) patients: 20 (7.6%) of them to nucleoside reverse transcriptase inhibitor; 6 (2.3%) to nonnucleoside reverse transcriptase inhibitor (NNRTI); and 1 each to protease inhibitors, protease inhibitor plus NNRTI, and nucleoside reverse transcriptase inhibitor plus NNRTI, respectively., Conclusions: Our findings underscore recommendations for HIV-1 genotyping in newly diagnosed patients not only to provide information on transmitted drug resistance as an issue in public health and as a guide to future therapy but also to document transmission clusters and to increase the necessary preventive measures.

The aim of this study was the development of a panel constituted by well-defined HIV-1 strains of different genetic forms, with a particular focus on isolates from acute and recent infections. Fourteen HIV-1 isolates, including four from acute and five from recent infections, were expanded in peripheral blood mononuclear cells. SI phenotype, coreceptors use, and TCID(50)/ml were determined. V3 net charge was calculated. Near full-length genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Analysis of cysteine residues, lengths of variable regions, and potential N-linked glycosylation sites in gp120 and gp41 was performed. Viral stocks were produced. Thirteen strains were NSI/R5 and one SI/R5,X4. TCID(50)/ml ranged between 10(4.6) and 10(6). V3 net charge was <+5 in 12 sequences and +5 in two sequences. Near full-length HIV-1 genomes analysis identified viruses of the following genetic forms: eight subtype B, three subtype C, two CRF02&#95;AG, and one subtype G. Cysteine residues that form the V1,V2,V3, and V4 loops were highly conserved. The number of potential N-linked glycosylation sites in gp120 and gp41 ranged between 24-29 and 4-6, respectively. Seven potential N-linked glycosylation sites in gp120 and three in gp41 were conserved. V1, V2, V4, and V5 variable regions exhibited substantial length variation. In addition, an analysis of transmitted and natural resistance to current antiretroviral drugs in these strains was performed. It is worth mentioning that the 13S mutation in the V3 sequence, associated with resistance to maraviroc, was observed in a subtype B strain that harbored resistance mutations to nucleoside reverse transcriptase inhibitors and to T20. The availability of a panel including strains from acute and recent infections should be a valuable resource for optimizing and standardizing vaccine candidate assessment. Near full-length genome characterization may be necessary for evaluating clade-specific reactivities.

Dichloromethane, methanol and aqueous extracts from the leaves of Terminalia triflora were investigated for their inhibitory effect on polymerase and ribonuclease activities of HIV reverse transcriptase.The most potent activity was found in the aqueous extract, which inhibited both polymerase and ribonuclease activities of the enzyme with an IC50 of 1.6 micro g/mL and 1.8 micro g/mL respectively. The antiinfective activity of the extract was demonstrated in HLT4LacZ-IIIB cell culture with an IC50 of 1.0 micro g/mL. The extract was submitted to a purification process by extractive and chromatographic methods. The activity remained in the hydrophillic fraction. Tannins present in this active purified fraction, as determined by TLC and HPLC methods, could account for the anti HIV-RT activity found in the aqueous extract., (Copyright 2002 John Wiley & Sons, Ltd.)

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&#95;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.