[Rapid spread of the HIV-1 circular recombinant CRF02-AG in Russia and neighboring countries].

The spread of the HIV-1circular recombinant CRF02-AG in countries of the former Soviet union (Commonwealth of Independent States, CIS) was studies using partial and full genome sequences. The full-genome sequence of the CRF02-AG recombinant circulating in Russia was obtained for the first time. A Global phylogenetic tree of CRF02-AG full-genome sequences was constructed. Three distinct groups of the sequences were detected as clustered by the geographical location (CIS, South Korea, and France), which is indicative of the single-virus introduction in each of the regions mentioned above. The CIS cluster exhibiting minimum genetic diversity was, therefore, relatively young. The phylogenetic analysis of the env gene sequences within the CIS cluster made it possible to clearly discriminate three branches: two of Russian and one of Uzbek origin. The low genetic diversity within the two Russian subclusters provides evidence of at least two recent independent introductions of the CRF02-AG recombinant from Central Asia into Russia. This work was performed within the framework of the 7th Federal Research Program (FP&), Project EURIPRED (European Research Infrastructures for Poverty Related Diseases), grant agreement No.312661.

HIV-1 genetic diversity in Galicia Spain: BG intersubtype recombinant viruses circulating among injecting drug users.

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.

Analysis of HIV type 1 protease and reverse transcriptase sequences from Venezuela for drug resistance-associated mutations and subtype classification: a UNAIDS study.

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.

Analysis of drug resistance-associated mutations in treatment-naïve individuals infected with different genetic forms of HIV-1 circulating in countries of the former Soviet Union.

There are few data on drug resistance-associated mutations in the former Soviet Union since, studies have usually been focused on the env or gag genes for subtype information. This study examines the prevalence and patterns of resistance-associated mutations to reverse transcriptase and protease inhibitors (RTI, PRI) in 278 HIV-1-infected treatment-naïve subjects from countries of Eastern Europe, and defines characteristic polymorphisms of RT and PR sequences in HIV-1 subtype A viruses. Blood samples were collected between 1997 and 2004. Plasma RNA was used for PR-RT amplification by reverse transcription coupled with nested PCR and sequencing. Phylogenetic analysis was done with neighbor-joining trees and bootscanning. Analysis of drug resistance mutations, with Stanford University HIV Drug Resistance Database's algorithm, resulted in an overall prevalence of 12.9% resistance to RTI and 3.9% to PRI. The most frequent substitutions in the RT region were at positions 62 and 236. V77I substitution in PR was found in 47.8% of samples. Polymorphisms in subtype A sequences were identified. This is the first study reporting the prevalence and patterns of both PRI and RTI resistance-associated mutations in naïve HIV-1 infected patients from the former Soviet Union. These data underline the importance of genotypic resistance testing of chronically HIV-1-infected patients before initiating treatment, in order to select the most suitable drug regimen.

Travel and the introduction of human immunodeficiency virus type 1 non-B subtype genetic forms into Western countries.

Both high mutation rates and recombination contribute to the genetic diversity of human immunodeficiency virus type 1 (HIV-1). Among viruses of the main group, which are responsible for the HIV-1 pandemic, 21 circulating genetic forms have been reported, 11 of which are recombinant between > or = 2 subtypes. In Western Europe and the Americas, the HIV-1 epidemic is largely dominated by B subtype viruses; however, infections with diverse non-B subtype genetic forms are increasingly being recognized. In Western Europe and North America, most of them have been identified in immigrants or travelers returning from areas with high HIV-1 prevalence, mainly from sub-Saharan Africa and Southeast Asia, where non-B subtype genetic forms predominate, but propagation within other groups has been reported in some Western countries. This may have implications for prophylactic and therapeutic strategies and, by bringing in contact different genetic forms, may favor the generation of novel recombinant viruses. Travelers from different categories--including immigrants, military personnel, seamen, tourists, expatriates, diplomats, and businessmen--may be at risk of transporting HIV non-B subtype genetic forms to Western countries.

Patterns of HIV-1 mRNA expression in transgenic mice are tissue-dependent.

To explore tissue-specific factors that may be important in HIV-1 transcriptional and post-transcriptional regulation, we examined a transgenic mouse model containing a mutant provirus deleted in the gag and pol region. The level of transgene expression was tissue-dependent. Skin, muscle, and tail consistently expressed the transgene abundantly; intestine, kidney, and thymus exhibited variable but generally low levels of expression; while liver expression was undetectable by Northern analysis. Individual mRNAs within the family of singly and multiply spliced messages were determined by reverse transcription (rt) of RNA samples from mouse tissues, polymerase chain reaction (PCR) amplification, and Southern hybridization with exon-specific probes. The exact percentage of Tat-coding mRNA that was multiply spliced was also determined by competitive rtPCR. When 2-, 4-, or 7-kb (full-length) mRNA species were calculated as a percentage of the total mRNA, two phenotypes of distribution were detected. Lymphoid tissue (thymus and spleen) and kidney had significantly greater amounts of unspliced message (P < 0.001) regardless of the level of expression. All other tissues expressed the multiply spliced messages encoding Tat, Rev, and Nef predominantly. Furthermore, utilization of the three major second exon splice acceptor sites for tat, rev, and nef was the same in transgenic mice as has been demonstrated in human cells but the splice acceptor site for the vpu/env was different in murine tissue. The marked tissue-dependent patterns of HIV mRNA expression suggest a potential mechanism for the organ-specific manifestations of AIDS.

Single- and multidrug resistance mutations to reverse transcriptase and protease inhibitors: human immunodeficiency virus type 1-infected patients from two geographical areas in Spain. Spanish Groups for Antiretroviral Resistance Studies.

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.

Detection and quantification of multiple drug resistance mutations in HIV-1 reverse transcriptase by an oligonucleotide ligation assay.

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.