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- Molecular epidemiology of HIV-1 circulating among injecting drug users in the great LisbonPublication . Sousa, Carina; Videira e Castro, Sandra; Pádua, Elizabeth; Parreira, Ricardo; Esteves, Aida; Piedade, JoãoMolecular epidemiology of HIV-1 circulating among injecting drug users in the Greater Lisbon Carina Sousa1; Sandra Videira e Castro1; Elizabeth Pádua3; Ricardo Parreira1,2; Aida Esteves1,2; João Piedade1,2 1Grupo de Virologia, UEI de Microbiologia Médica; 2Unidade de Parasitologia e Microbiologia Médicas (UPMM) Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa Rua da Junqueira nº100, 1349-008 Lisboa, Portugal 3Laboratório de Referência da Sida, Instituto Nacional de Saúde Dr. Ricardo Jorge Avenida Padre Cruz, 1649-016 Lisboa, Portugal Background and Objectives HIV/AIDS is recognized as a public health problem partly because of the high degree of viral genetic diversity that poses a major challenge for diagnosis, prevention by vaccination and treatment. Human immunodeficiency virus type 1 (HIV-1) genetic variants are classified in 4 phylogenetic groups: M, N, O and P. Group M, responsible for the great majority of HIV-1 infections worldwide, can be further subdivided into 9 phylogenetic subtypes (A–D, F–H, J and K) and, at least, 6 sub-subtypes (A1–A4 and F1–F2). Genetic recombination events, during infection with multiple subtypes, continuously give rise to mosaic viruses (unique recombinant forms, URFs), some of which can gain epidemic proportions (circulating recombinant forms, CRFs). According to their cellular coreceptor (CCR5 or CXCR4), HIV-1 variants can be also classified as R5-tropic, X4-tropic or dual/mixed (R5X4). Besides recombination, the main causes that contribute to HIV-1 impressive genetic heterogeneity are the lack of proof-reading ability by reverse transcriptase, the rapid viral turnover in vivo and host selective immune and drug pressures. These mechanisms, taken together, lead to the rapid formation, at the individual level, of distinct viral micropopulations, genetically related to each other, known globally as quasispecies, providing evidence for the ongoing evolution of HIV-1. In order to achieve an update picture of the molecular epidemiology of HIV-1 strains circulating among intravenous drug users (IDUs) from the Greater Lisbon, the aims of this study were: • to assess the genetic diversity of pol protease (PR), reverse transcriptase (RT), integrase (IN) and env C2V3C3 coding sequences; • to estimate the frequency of coreceptor usage (CCR5 and/or CXCR4). Results and Discussion RNA extracted from plasma taken from 61 HIV-1 seropositive IDUs, collected between 1998 and 2009, was amplified by nested PCR, after in vitro reverse transcription, to originate PR, RT, IN and C2V3C3 amplicons of 460, 650, 906 and 565 bp, respectively. A total of 158 DNA sequences was obtained, from 49 samples successfully amplified for, at least, one of the regions, by sequencing of PCR products or plasmid clones. Following sequence editing using BioEdit v.7.0.9.0 (Hall, 1999), subtype characterization was performed by manual phylogenetic analysis with MEGA4 (Tamura et al., 2007) and recombinant analysis was carried out by bootscanning using SimPlot v.3.5.1 (Lole et al., 1999). This study revealed a significant degree of HIV-1 diversity, suggesting the existence of a heterogeneous epidemic among the IDUs studied. On the whole, HIV-1 non-B subtypes were identified in 58.9% (93/158) of the sequences obtained: PR in this population comprehend mainly subtypes B and G, with a clear indication of the presence of a significant proportion of recombinants, mostly comprising unique mosaic structures of these two subtypes, CRF02_AG and CRF14_BG. Finally, 35 putative amino acid V3 sequences were obtained and coreceptor usage prediction (genotypic) was accomplished using the 11/25 (Fouchier et al., 1992) and the V3 net charge empiric rules (De Jong et al., 1992) and also the programs Fortinbras PSSM [matrix: subtype B X4/R5 (Poveda2009)] (at http://fortinbras.us/cgi-bin/fssm/fssm.pl) and geno2pheno[coreceptor] v.1.2. (at http://coreceptor.bioinf.mpi-inf.mpg.de/index.php), with a significance level of 20%. Considering the discrepant results obtained for some of the sequences, we considered as X4/R5X4-tropic all the sequences with coincident previsions with at least two of the algorithms used. Therefore, 30 sequences were identified as R5-tropic and five as X4 or dual/mixed-tropic. Overall, the prediction discrepancies were more frequent in non-B subtypes and in sequences with shorter V3 loops (34 amino acids), which may reflect a lower suitability of the matrices used in these cases 12 B, 1 F1, 17 G/G(14_BG), 5 CRF02_AG, 6 URFs; n=41; RT 17 B, 1 F1, 13 G/G(14_BG), 5 G(02_AG), 5 URFs; n=41; IN 13 B, 1 F1, 12 G/G(14_BG), 1 CRF02_AG, 14 URFs; n=41; and C2V3C3 [8 A, 23 B, 1 F1, 3 G; n=35]. It is also significant that 45.2% (19/42) of the PR/RT/IN/C2V3C3 concatenated sequences were derived from inter-genotype recombinants. Overall, the viral forms circulating Conclusions This study represents a contribution to a more complete understanding of the molecular epidemiology of the HIV/AIDS epidemic in IDUs of the Greater Lisbon. Our data confirm that this HIV-1 epidemic is dominated by B and G subtypes, and their recombinant forms. However, other subtypes (A, F1) and recombinant forms (CRF02_AG) were also found. This finding confirms our previous data and suggests that the HIV-1 epidemic in Portugal may be evolving to a unique epidemiological pattern, in which subtypes B, G and their recombinant forms prevail. The high prevalence of patients infected with R5-tropic viruses makes a CCR5 antagonist-based therapeutic regimen an option to be considered in this population, if necessary. However, the genotypic methods for coreceptor prediction still present limitations, especially when analysing non-B or genetically divergentstrains. Anyway, these methods are viable alternatives whenever phenotypic testing is not possible and they should be optimized in order to improve the sensibility and specificity of their predictions References Hall, Thomas. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, v.41, p.95-98. (1999). Tamura, Koichiro, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, v.24, p.1596-1599. (2007). Lole, Kavita, et al. Full-length human immunodeficiency virus type 1 genomes from subtype Cinfected seroconverters in India, with evidence of intersubtype recombination. Journal of Virology, v.73, p.152-160. (1999). Fouchier, Ron, et al. Phenotype-Associated Sequence Variation in the Third Variable Domain of the Human Immunodeficiency Virus Type 1 gpl20 Molecule. Journal of Virology, v.66, p.3183-3187. (1992). De Jong, Jean-Jacques, et al. Minimal Requirements for the Human Immunodeficiency Virus Type 1 V3 Domain To Support the Syncytium-Inducing Phenotype: Analysis by Single Amino Acid Substitution. Journal of Virology, v.66, p. 6777-6780. (1992).
- Genotypic resistance profiles to antiretroviral drugs in HIV-1 circulating among injecting drug users in the great LisbonPublication . Videira e Castro, Sandra; Sousa, Carina; Pádua, Elizabeth; Esteves, Aida; Parreira, Ricardo; Piedade, JoãoGenotypic resistance profiles to antiretroviral drugs in HIV-1 circulating among injecting drug users in the Greater Lisbon Sandra Videira e Castro1; Carina Sousa1; Elizabeth Pádua3; Aida Esteves1,2; Ricardo Parreira1,2; João Piedade1,2 1Grupo de Virologia, UEI de Microbiologia Médica; 2Unidade de Parasitologia e Microbiologia Médicas (UPMM) Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa Rua da Junqueira nº100, 1349-008 Lisboa, Portugal 3Laboratório de Referência da Sida, Instituto Nacional de Saúde Dr. Ricardo Jorge Avenida Padre Cruz, 1649-016 Lisboa, Portugal Background and Objectives The introduction of highly active antiretroviral therapy has significantly improved the ability to maintain human immunodeficiency virus type 1 (HIV-1) replication to very low levels for an extended period of time, providing a better quality of life to infected patients. The acquisition and transmission of HIV-1 drug resistance mutations is a major public health concern, so the search for better antiretroviral (ARV) therapeutic agents is a constant challenge. In recent years, in an effort to target other functions at the virus replication cycle beyond those carried out by the viral enzymes protease (PR), reverse transcriptase (RT) and integrase (IN), some novel agents have emerged, e.g. CCR5 coreceptor antagonists, as maraviroc (MVC), the first directed to a cellular target. Nevertheless, the discovery and licensing of new ARVs is followed by descriptions of the rapid emergence of resistant and multidrug-resistant viral variants (Hartman and Buckheit, 2012). Therefore, resistance information is useful in the clinical setting, as it can guide the choice of ARVs in therapy, also providing a repository of data on the extent of circulation of resistant viral strains (Vandamme et al., 2011). Monitoring of HIV-1 resistance to ARVs can be performed with genotypic tests. These assays are based on the direct nucleotide sequencing of target genes and the identification of mutations associated to resistance (and of other genetic polymorphisms) is made through the use of algorithms that allow the reporting of associations of mutation patterns with different levels of susceptibility to ARVs. Thus, the aims of this study were: • to identify PR, RT and IN mutations associated to different levels of resistance to currently licensed enzymatic inhibitors, in HIV-1 strains circulating among injecting drug users (IDUs) from the Greater Lisbon; • to describe, in the same population, natural genetic polymorphisms putatively associated with reduced susceptibility to MVC. Results and Discussion RNA extracted from plasma taken from 61 HIV-1 seropositive IDUs, collected from 1998 to 2009, was amplified by RT-nested PCR using the Titan One Tube RT-PCR kit (Roche Applied Science, Germany), followed by the illustra™ puReTaq Ready-To-Go PCR Beads (GE Healthcare, England) according to the manufacturers’ instructions. HIV-1 DNA sequences for PR (n=41), RT (n=41), IN (n=41) of pol gene and C2V3C3-gp120 (n=35) of env gene were generated by sequencing, in both directions, of PCR products or plasmid clones. PR, RT, IN and C2V3C3 nucleotide sequences were edited with BioEdit v.7.0.9.0 (Hall, 1999) to originate fragments of 297, 598, 813 and 516 bp, respectively. The analysis of mutations associated with decreased susceptibility to currently licensed ARV enzymatic inhibitors was performed using the HIVdb program, implemented through the Genotypic Resistance Interpretation Algorithm from the Stanford University HIV Drug Resistance Database (at http://hivdb.stanford.edu). The analysis for PR, RT, IN coding regions revealed a total of 15 resistance associated mutations, present in 50.0% (22/44) of the subjects, with a distribution of 1-3 mutations/sequence (4 minor mutations in PR; 3 high-level and one low-level resistance mutations and 2 polymorphisms in RT; 5 mutations in IN, one major and 4 minor). Half of the subjects infected with these strains (11/22) were drug-naïve, showing transmission of resistance mutations between infected individuals. However, only 26.7% (4/15) of these mutations confer a putative phenotypic profile of resistance and just to one class of inhibitors (RT or IN), being found in 9.1% (4/44) of the subjects. Conversely, the mutations associated with resistance to MVC were analyzed after translation of the C2V3C3 nucleotide sequences and subsequent comparison to a subtype B consensus. Based on published data, we observed a total of 12 different single mutations, as well as two mutational patterns, in the V3 loop, present in all the sequences studied, at variable numbers. The presence of the patterns 11S+26V and 20F+25D+26V, in 3 sequences, is of particular interest, since these patterns have unequivocally been associated with MVC resistance in vivo. The presence of these mutations in MVC-naïve patients is certainly the result of the high variability of theV3 region. Their impact at MVC treatment baseline is still unknown, but may have important clinical implications. Conclusions Overall, a very high frequency of genetic polymorphisms was found, both for pol (PR, RT, IN) and env (C2V3C3) coding regions. Although the majority of the polymorphisms described has only a marginal importance on ARV resistance, they may play a role on viral fitness and/or the evolution of resistance, once drug pressure is applied. On the other hand, even though a resistance profile for MVC is not defined yet, the presence of these mutations in MVC-naïve populations may have a significant impact in their clinical management in the future, especially considering the introduction of this drug in salvage therapy. Finally, the presence of resistance associated mutations in drug-naïve individuals shows that the transmission of these mutations is ongoing among IDUs in the Greater Lisbon area. The monitoring of ARV resistance is a fundamental issue to the control of the HIV/AIDS epidemic. The results reported will surely be of value for ARV therapy implementation and monitoring of infection in this IDU population, having also an impact on the clinical management of HIV-1 drug-experienced individuals, particularly in those who have experienced previous virologic failures. References Hartman, Tracy and Buckheit Jr., Robert. The continuing evolution of HIV-1 therapy: identification and development of novel antiretroviral agents targeting viral and cellular targets. Molecular Biology International, v.2012, 401965 (2012). Vandamme, Anne-Mieke, et al. European recommendations for the clinical use of HIV drug resistance testing: 2011 update. AIDS Reviews, v.13, p.77-108. (2011). Hall, Thomas. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, v.41, p.95-98. (1999).