The aim of this study was to investigate the susceptibility to T20 and the dynamics of amino acid changes in HR1 and HR2 of gp41 of HIV-1 obtained from plasma, peripheral blood mononuclear cells (PBMC), and primary isolates (PI) in four highly antiretroviral-experienced patients. These patients received T20 plus an antiretroviral regimen and were followed-up over a period of 40-72 weeks. In one non-responder patient, N43D substitution was detected at 12 weeks of treatment, in association with a value of T20-IC50 of 10 microg/ml (10-fold increase). Double mutations N42T + N43D were observed in plasma RNA at 32 weeks and remained detectable up to 16 weeks after the withdrawal of the drug. The S138A substitution in HR2 was observed in plasma RNA at 32 weeks, and both in plasma RNA and in PI DNA at 40 weeks, associated with an increase of the T20-IC50 to 25 microg/ml (25-fold increase). Mutations V101G and E137K, not reported previously, were also observed in the HR2 region. Whether these new substitutions play a role in T20 resistance needs to be examined. In three temporary responders, coinciding with viral load rebound, G36D, and N42T substitutions were observed at 12, 24, and 40 weeks. G36D mutation was associated with a value of T20-IC50 of 5 microg/ml. The HR2 S138A mutation was detected after the detection of HR1 substitutions and was associated with an increase in the level of T20-IC50 to 125 microg/ml (125-fold increase) All these data reinforce the role of gp41 amino acids 36-45 and the potential influence of the HR2 S138A mutation in the genotypic/phenotypic resistance to T20.

The objectives of this study are to describe the incidence of non-B and recombinant HIV-1 strains in newly diagnosed HIV-1 infections in Galicia, northwest of Spain, during a 2-year period (May 2000 to June 2002), and the frequency of resistance-associated mutations in reverse transcriptase (RT) and protease (PR) genes, analysing the polymorphisms more frequently detected in non-B and recombinant viruses. All newly diagnosed HIV-1-infected patients attending the nine public hospitals of the seven main cities of Galicia were included in this study. RT, PR and V3 regions from HIV-1 RNA plasma were amplified and sequenced, being the corrected sequences sent to the Stanford HIV RT and Protease Sequence Database. Nineteen of 85 patients (22.3%) were infected by non-B or recombinant viruses: three subtype C, two G, one F1, one Dpol/A1V3, five CRF02_AG, one CRF14_BG, five BGpol/BV3 and one UKpol/UV3 (U, unknown fragment). Eleven of these 19 patients (57.9%) were foreign individuals living in Galicia infected through heterosexual contact, and the other eight (42.1%) were Spanish intravenous drug users who had shared injection equipment. Five of 85 patients (5.9%), all infected with B subtype viruses, showed resistance-associated mutations in RT (M184V, M41L, L210W, T215Y/D and K219Q). In one patient (1.2%) infected with a subtype G strain, resistance-associated mutations in PR (K20I+M36I+M46I+V82I) were detected. In subtype B viruses resistance mutations in PR were not detected. Several polymorphisms in RT: D123S, Q174K, D177E, T200A, V245Q, and PR: I13V, K20I, M36I, R41K, H69K, L89M were detected more frequently in non-B and recombinants than in B strains (P<0.01 to P<0.001). This study reports a high incidence (22.3%) of newly diagnosed patients infected by different non-B and recombinant HIV-1 strains, in a geographical area of Spain, showing also a high frequency of polymorphisms in RT and PR genes.

The HIV-1 epidemic in western Europe is dominated by B-subtype viruses, but non-B and intersubtype recombinants are being identified with increasing frequency [1]. Little is known about the biological characteristics of the recombinant viruses and the relationship with their transmissibility and clinical progression [2,3]. The V3 loop of B-subtype viruses has been shown to play a role in the syncytium-inducing (SI) phenotype, viral tropism and in the differential usage of the chemokine receptors (CCR5, CXCR4) [4]. Although all subtypes can use co-receptors CXCR4 or CCR5, some preferences relative to subtypes have been described [5–8]. In general, non-SI variants display low V3 net charge (≤ +4), whereas SI variants emerge late in the disease course and display higher V3 net charges (≥ +5) [9,10]. In order to determine the biological characteristics of the newly described BG recombinants [11–13] we sequenced the V3 loop from plasma RNA of eight BG strains and eight B-subtype viruses as controls, obtained from HIV-1-infected patients who were attending the same hospitals at the time the study was carried out. Moreover, recombinant BG virus was isolated from the peripheral blood mononuclear cells (PBMC) of one patient and its in-vitro biological properties were studied, including the cell tropism, replication capacity, SI and co-receptor usage. Epidemiological data are shown in Table 1. All patients were native Spanish, except one from Cabo Verde (X-623). The V3 net charge was calculated on the basis of the difference between the number of positively charged amino acid residues, arginine (R) or lysine (K), and the number of negatively charged residues, aspartic acid (D) or glutamic acid (E) [14]. The prediction of SI phenotype and CXCR4 or CCR5 co-receptor usage was made considering the net charge and by analysing the presence of positively charged amino acids within specific positions that have previously been shown to influence the biological in-vitro phenotype: 306, 320 and 324 positions [15,16].Table 1: V3 loop amino acid sequences in BG recombinants viruses in Spain (Galicia). Virus isolation and biological characterization from PBMC were performed as previously described [17]. Co-receptor usage was determined by infecting, with cell-free viral stock, the human glioma cell line, U87.CD4, stably expressing a chemokine receptor CCR1, CCR2b, CCR3, CCR5 or CXCR4, and the human osteosarcoma cell line, GHOST, expressing either CCR3, CCR5, CXCR4, BOB/GPR15 or BONZO(STRL33) [18,19]. Cell cultures were observed daily for cytopathic effects, and supernatant was harvested on day 7 for the detection of p24 antigen (Innotest HIV antigen monoclonal antibody; Innogenetics, Zwijndrecht, Belgium). GHOST cells were analysed using a FACScan flow cytometer. The V3 region was sequenced from patient PBMC proviral DNA and from in-vitro HIV-1 primary isolate. V3 sequences are shown in Table 1. The estimated V3 net charge was +5.1 (with a range of +4 to +6) for BG recombinant strains, and +4 (with a range of +3 to +5) for B subtypes. The presence of a basic amino acid (R) at the 306 phenotype predictive position was observed in three BG recombinants and in three B-subtype V3 sequences. Other basic amino acid substitutions at phenotype-predictive positions were not detected. However, the net charge in the majority of the BG recombinants was predictive of SI/CXCR4, but they lacked phenotype-associated signature amino acids. Similar findings have been described in some subtype-F isolates [20], suggesting that multiple basic substitutions within the V3 loop, associated with an overall net charge, may be a general requirement for generating the SI/CXCR4 phenotype. It should be noted that most BG recombinant sequences showed several characteristic mutations, corresponding to: 308T, 309M, 314V, 315L, 320Q, 327K (Table 1). These changes were not detected in the B-subtype viruses analysed in this study. Moreover, the frequency of these mutations in the B-subtype sequences published in the Los Alamos Data Base is low, being observed at a range of 5–16%. An important aspect will be to determine whether these divergences between the BG recombinant strains and B-subtype viruses will result in biological differences. One BG recombinant primary isolate (X-421) was obtained from a patient in A1 clinical stage. Its biological characteristics were: rapid/high replication, SI phenotype, and the usage of CCR2b, CCR3, CCR5 and CXCR4 co-receptors. A V3 amino acid sequence, corresponding to proviral DNA obtained from patient PBMC, was compared with those obtained from primary isolate and MT2 cell lysates. The sequences derived from in-vitro samples showed a positively charged amino acid (K) at the 320 position, resulting in a net charge of +7. This mutation was not observed either in proviral DNA from patient PBMC or in plasma RNA, indicating its emergence under culture-selective pressure. We have described, for the first time, the biological characteristics of the BG recombinant primary isolate. Interestingly, the SI/X4 phenotype was obtained from a patient in the early clinical stage. The follow-up of this patient will allow us to establish whether these findings are associated with a faster progression of the disease. The Spanish Group for Antiretroviral Resistance Studies in Galicia A. Agulla, A. Mariño, Hospital Arquitecto Marcide, Ferrol (La Coruña); S. López-Calvo, J.D. Pedreira, Hospital Juan Canalejo (La Coruña); A. Aguilera, E. Losada, A. Prieto, Complejo Hospitalario Universitario de Santiago (La Coruña); J. Corredoira, M.J. López- Alvarez, A. Rodríguez, Hospital Xeral-Calde (Lugo); M. Bustillo, J. García-Costa, R. Fernández-Rodríguez, Hospital Nuestra Señora del Cristal (Orense); R. Rodríguez, Hospital Provincial Santa María Madre (Orense); C. Miralles, A. Ocampo, Hospital Xeral-Cíes, Vigo (Pontevedra); R. Ojea de Castro, Hospital Montecelo (Pontevedra); L.E. Morano, R. Pérez-Rodríguez, A. Rodríguez, J. Torres, Hospital Meixoeiro, Vigo (Pontevedra); J. Díz, R. Rodríguez.-Real, Hospital Xeral Provincial (Pontevedra). Lucía Pérez-Alvareza Elena Delgadoa María Luisa Villahermosaa María Teresa Cuevasa Valentina Garcíaa Elena Vázquez de Pargaa Michael M. Thomsona Arturo Prietob Laureano Cuevasa Leandro Medranoa José A. Taboadac Rafael Nájeraa and the Spanish Group for Antiretroviral Resistance Studies in Galicia Acknowledgements The authors would like to thank Dr José María Hernández Cochón, Conselleiro de Sanidade e Servicios Sociais, and Dra Pilar Farjas Abadía, Directora Xeral de Saúde Pública, Consellería de Sanidade e Servicios Socias, Xunta de Galicia for their support in the development of the study in Galicia. The technical assistance of Milagros Pinilla and Concepción González-Troncoso is gratefully acknowledged.

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 gp 120 and the external port ion 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.

HIV-1 subtype B is the predominant genetic form in western Europe, but non-B subtype and intersubtype recombinant strains are being identified with increasing frequency, mainly in natives of African countries [1]. It is of particular interest to know which are the genetic forms circulating in an area in order to know the potential for spread of any new variant, as has happened in Thailand [2], Kaliningrad [3] or Argentina [4]. Previous studies in Spain have described the presence of non-B subtype HIV-1 strains in inmigrants [5] and in natives [6]. Linked with the last study, we have reported the prevalence of drug resistance mutations in B, non-B subtypes and recombinant forms of HIV-1 in infected individuals in Spain [7]. In order to describe the patterns of reverse transcriptase (RT) and protease-associated mutations in non-B subtypes and recombinant forms of HIV-1, we analysed all the different RT and protease amino acid substitutions in HIV-1 of the Spanish patients previously reported [6,7], together with 115 newly identified viruses. In total, 556 HIV-1-infected individuals were included. RNA extraction from plasma was performed by the Boom method and RT and protease genes were amplified and sequenced as reported [8]. Subtype sequence homologies were examined using the BLAST algorithm with ‘Genotyping Tool’ (http://www. ncbi.nln.gov/retroviruses/subtype/subtype.htlm). Non-B subtype sequences were further examined by neighbouring phylogenetic trees, and intersubtype recombination was analysed by bootscanning using Simplot. Primary and secondary resistance mutations were defined following the criteria reported [9,10]. The statistical analysis was performed by McNemar test (RSIGMA, Babel statistical computer programme). Non-B subtype and recombinant viruses were identified in 31 (5.6%) of the 556 samples analysed, 20 of these had been reported previously [6] and 11 correspond to newly identified viruses. In one specific province (Pontevedra), the prevalence was 8.9% most probably because of its large fishing port with intensive contact with African ports, mainly in the Guinea Gulf, and close contact with Portugal. It is important to stress that 25 patients were native Spanish (80.6%), three were Portuguese, two were African and one was Argentinian. Seventeen (54.8%) were injecting drug users (IDU) (nine also had heterosexual exposure), and 14 were infected by heterosexual contact. Seven men had had sexual contacts with African women, three women were prostitutes, and five men were sailors who had multiple heterosexual contacts, but 10 (32%) were Spanish without any contact with Africans. The different non-B and recombinant HIV-1 viruses detected were: G, 11 (36%); A, two (6%); C, one (3%); H, one (3%); BG, four (13%); dual B + G infection, one (3%); UG, four (13%); GH, one (3%); UK, one (3%); DF, one (3%); BF, one (3%); and UAJ, three (10%). The proportion of primary resistance mutations was similar among B and non-B samples, as we have previously described [7], and as documented in other studies [11–13], in relation with some resistance mutations. Twenty-one (67%) of these 31 non-B strains were G subtype or intersubtype recombinants with segments of subtype G. Nineteen (90.5%) were IDU. Fifteen (71.4%) were from the province of Pontevedra. An epidemiological link between these 21 individuals was known in only two patients (X-138 and X-279) who were sexual partners. A possible initial source of these G subtype viruses in Galicia could be through sexual contact of seamen with African women in African seaports, with a subsequent spread in Spain among IDU and their sexual partners. All 12 subtype G sequences (including G subtype clones of a dually infected individual) and four BG recombinant intersubtypes formed a monophyletic cluster supported by a high (93%) bootstrap value (when B subtype regions of the recombinants were excluded), indicating a common ancestry. The clinical and epidemiological data of these 16 patients are shown in Table 1. Thirteen (81.2%) were Spanish, and IDU was the risk exposure associated with HIV-1 infection. Twelve (75%) patients were treatment-naive, and four were under highly active antiretroviral therapy.Table 1: Epidemiological, clinical and virological data on HIV-1-infected patients with subtype G and GB recombinants in Spain (Galicia). The analysis of secondary mutations and polymorphisms showed that all subtype G viruses of the monophyletic group and one G subtype clone of the dually infected individual had the same RT and protease mutations (Table 1). Statistically significant differences were found in the frequency of these mutations between B and G subtype viruses (P < 0.001), being higher among G than among B subtype viruses. Two RT substitutions, A98S and R211S, were specifically detected in the viruses of the subtype G monophyletic group, but in none of the other non-B subtype or recombinant viruses included in the study. To our knowledge, none of these two mutations have been reported as characteristic of other subtype G strains, suggesting that they could be a signature of subtype G viruses circulating in this area of Spain. Two protease mutations, M46L, associated with resistance to indinavir [14], and 19P, were detected in three subtype G viruses and one G subtype clone of the dually infected individual. These four viruses were isolated from treatment-naive patients. To our knowledge, this is the first time these mutations have been detected in viruses from non-treated patients. The K20I substitution in protease has been published as a signature of subtype G [15]. We detected this mutation in subtype G but also in other non-B subtype viruses. To determine the clinical relevance of these findings, phenotypic resistance testing and biological characterization of G subtype viruses are now in progress. These data could reveal differences between B and G subtypes, and their impact on the use of antiretroviral drugs, allowing for a more reliable interpretation of resistance assays in the treatment of patients infected with non-B subtypes. Acknowledgements The authors would like to thank Dr José María Hernández Cochón, Conselleiro de Sanidade e Servicios Sociais and Dra Pilar Farjas Abadía Directora Xeral de Saúde Pública, Xunta de Galicia for their support in the development of the study in Galicia. The technical assistance of Isabel Herrero, Milagros Pinilla and Valentina García is gratefully acknowledged. *The Spanish Group for Antiretroviral Resistance Studies in Galicia: A. Agulla, A. Mariño, Hospital Arquitecto Marcide, Ferrol (La Coruña); S. López-Calvo, J.D. Pedreira, Hospital Juan Canalejo (La Coruña); A. Aguilera, E. Losada, A. Prieto, Complejo Hospitalario Universitario de Santiago (La Coruña); J. Corredoira, M.J. López-Alvarez, A. Rodríguez, Hospital Xeral-Calde (Lugo); J. García-Costa, R. Fernández-Rodríguez, Hospital Nuestra Señora del Cristal, (Orense); R. Rodríguez, Hospital Provincial Santa María Madre (Orense); C. Miralles, A. Ocampo, Hospital Xeral-Cíes, Vigo (Pontevedra); R. Ojea de Castro, Hospital Montecelo (Pontevedra); L.E. Morano, R. Pérez-Rodríguez, A. Rodríguez, J. Torres, Hospital Meixoeiro, Vigo (Pontevedra); J. Díz, R. Rodríguez-Real, Hospital Xeral Provincial (Pontevedra). Lucía Pérez-Alvareza Michael M. Thomsona María Luisa Villahermosaa Elena Vázquez de Pargaa Aurora Rodríguezb María Teresa Cuevasa Elena Delgadoa Nuria Manjóna Celia Mirallesb Leandro Medranoa Jose Antonio Taboadab Rafael Nájeraa and the Spanish Group for Antiretroviral Resistance Studies in Galicia*

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: 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.