ABSTRACT Since its discovery as the etiological agent of AIDS in 1983, HIV-1 has been the focus of unrelenting research into an effective vaccine to control viral infection. Neutralizing antibodies constitute a correlate of immune protection for most available vaccines, but the induction of these antibodies against HIV-1 has become a major challenge. The HIV-1 envelope glycoprotein has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope to provide sterilizing immunity. The existence of a group of monoclonal antibodies, such as IgG1b12, 2G12, 2F5 and 4E10, capable of neutralizing a broad range of primary isolates signals vulnerable areas on the envelope glycoprotein. Furthermore, passive transfer of these antibodies can completely protect against viral challenge in animal models. The epitopes recognized by these antibodies are being intensely pursued as vaccine targets, in the hope of inducing such specificities. This thesis encompasses a series of studies on characterizing the epitopes recognized by these broadly cross-reactive monoclonal antibodies in the context of subtype C viruses. HIV-1 subtype C is responsible for the vast majority of infections worldwide, however, until recently, little research has been done on these viruses in contrast to the well characterized subtype B strains. Chapter Two describes the characterization of paediatric subtype C viruses for their sensitivity to IgG1b12, 2G12, 2F5 and 4E10. This study was done because of a planned clinical trial of some of these antibodies as post-exposure prophylaxis to prevent mother-to-child HIV-1 subtype C transmission. Only the MAb 4E10 was able to neutralize all the viruses tested, while IgG1b12 was only partially effective. 2F5 and 2G12 did not neutralize any of the viruses. The conclusion was that only 4E10 and IgG1b12 would be suitable for use as prophylactic agents in a population where HIV-1 subtype C is prevalent. Given that subtype C viruses were found to be largely insensitive to 2G12 neutralization, the commonly absent glycan at iv position 295 was introduced into envelope glycoproteins from this clade. The The work presented in Chapter Three explores the requirements of the 2G12 epitope on the envelopes of subtype C viruses. However, this antibody binding site was not readily reconstituted, suggesting structural differences from other HIV-1 subtypes in which the 2G12 epitope is naturally expressed. Chapter Four describes the study of 4E10 resistant virus quasispecies isolated from a seven year old perinatally HIV-1 infected child, in whom anti-MPER antibodies were found. Determinants of 4E10 neutralization were mapped to the epitope of this antibody in the MPER, as well as to the cytoplasmic tail, in particular, to four amino acids in the LLP-2 region. The role of neutralizing antibodies in natural HIV-1 subtype C infection was examined in Chapter Five by following the development of autologous and heterologous neutralizing antibodies in 14 patients during the first year of infection. Potent but relatively strain-specific neutralizing antibody responses were detected within 3-12 months of infection. The magnitude of the responses was associated with shorter V1-to-V5 envelope length and fewer glycosylation sites, in particular in the V1-V2 region. Furthermore, anti-MPER and anti-CD4i neutralizing antibodies were detected in some individuals; however, they were not associated with neutralization breadth. Finally, in Chapter Six these results are analyzed collectively, in the context of the latest findings in the field, and suggestions for further research are discussed.

Thesis (M.Sc. (Med.))--University of the Witwatersrand, Faculty of Health Sciences, 2013. / Human immune deficiency virus (HIV) infection contributes to child mortality rates in South Africa. Investigations of newer technologies for improving early infant diagnosis of HIV in the South African setting could reduce child mortality as life saving treatment can be accessed early in life. This study investigated three technologies: a fourth generation rapid HIV test and two viral load (VL) platforms. Determine Combo (DC) is a qualitative fourth generation rapid test that is able to detect HIV antibodies and p24 antigen simultaneously. The performance of DC was evaluated in the field on samples from pregnant and postpartum women; in the laboratory, on stored samples from children and with the addition of heat denaturation. In the maternal DC study 90 (8 .8%) of 1019 women tested HIV positive of whom 59 (17.1% prevalence) were pregnant and 31 % (4.6% prevalence) were postpartum. The sensitivity and specificity of the antibody component of DC on plasma was 100%(Confidence Interval (CI): 95.9- 100%) and 99.8%(CI: 99.2-99.9%) respectively. Three postpartum patients tested false positive for HIV antibodies (n=2) and p24 antigen (n=1). No true positive p24 antigen was detected DC was performed on stored samples from 182 (90%) HIV-exposed and 20 (10%) HIV-unexposed children aged from birth to six years. The DC HIV antibody component returned false negative results in 2 HIV-infected children; one clinically symptomatic and one asymptomatic aged 7 and 23 months respectively. The sensitivity of DC HIV antibody was 100% (CI :94.3-100%) in infants aged 6 months and younger with a specificity of 100% (CI:81.6-100%) for all ages. Of the 61 HIV infected infants tested , the DC p24 antigen was reactive in only one clinically symptomatic infant resulting in a sensitivity for detection of HIV infection of 1.7% (CI 0.3-8.9%). A heat denaturation technique designed to improve p24 antigen detection was applied to HIVinfected samples but failed to enhance p24 antigen detection on DC. HIV viral load (VL) molecular assays are used to confirm an HIV-infected diagnosis and for VL monitoring. In South Africa, plasma is the gold standard sample for VL monitoring in infants even though dried blood spots (OBS) are the preferred specimen type in resource-constrained settings and for early infant diagnosis. The use of OBS specimens for HIV VL monitoring would convenience resource limited settings. The OBS matrix therefore requires validation to determine accuracy (for establishing diagnosis) and precision (for VL monitoring) compared to plasma VL. This study investigated the accuracy and precision limits of OBS VL on the Roche Cobas AmpliPrep-Cobas TaqMan HIV-1 v2.0 assay (CAP/CTM) and the Abbott RealTime HIV-1 assay (m2000) platforms on samples from HIV-infected adults and children. The CAP/CTM was investigated on OBS containing 751J1 blood and the m2000 was investigated using one (50IJI) and two (2x501J1) OBS. Compared to plasma VL, OBS VL from adults and children were higher in the lower range «310g,<1000copies/ml) and lower values in the higher range (>510g, >185,000copies/ml) on the CAP/CTM in the study of OBS VL accuracy. Additionally, OBS VL values were >log1.0 higher in 42/100 (42%) of adult and 16/49 (33%) of measurements from children, which will have clinical significance. On the m2000 platform, the differences between plasma and OBS VL were lower in the range >5 log and higher in the range 2 log copies/ml (100 copies/ml) to 4 log copies/ml (10000 copies/ml). Compared to plasma VL, OBS VL values were >log1.0 higher in 20/82 (24%) adult and 7/43 (16%) of measurements from children. Both platforms demonstrated 100% specificity in testing stored OBS from HIV-uninfected infants who were diagnosed negative on HIV DNA PCR. Acceptable limits for plasma VL precision is a coefficient of variation (CV) <35% and standard deviation (SO) :50.19 log. Where plasma VL :5510g, OBS VL demonstrated poor precision with CV>40% in 8/10 patients and total SO>0.30 log in 4/10 patients on the CAP/CTM. The m2000 total SO was >210g between adult plasma and OBS VLs under the 4 log copies/ml cut-off, irrespective of the number of DBS used. DBS VLs were unreliable when using precision limits used on plasma VLs on both platforms. In conclusion the DC test does not offer any advantage over currently available rapid tests in diagnosing new infection in women and children. The two VL platforms can be used to establish an HIV status in treatment naive patients in view of the 100% specificity. HIV-infected patients on treatment with undetectable plasma VL will always have detectable DBS VL on CAP/CTM, but equally undetectable DBS VL on the m2000. With DBS, the CAP/CTM assay generates higher VL values in the lower VL range than on plasma likely due to amplification of proviral DNA. Both platforms display poor intra- and inter- assay precision, using plasma VL based criteria and the variances would potentially affect clinical decision making. The acceptable limits for plasma VL precision cannot be applied to DBS VL on either platform.

Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / The HIV-­‐1 pandemic continues unabated. Although treatments exist that can substantially alleviate the morbidity and mortality associated with HIV, there is still a need for improved anti-­‐HIV treatments that reduce toxicities and administration frequency and mediate sustained inhibition of viral replication. Given the high mutability and variability of the virus, a strategy that is garnering increasing focus is the targeting of host factors that the virus requires to replicate, so-­‐called HIV-­‐dependency factors (HDFs). It is hoped this will reduce the emergence of viral drug resistance. A number of genome-­‐wide screens have been performed to identify HDFs, although many remain to be validated, particularly in relevant cells lines. An objective of this thesis was to validate three host factors as HDFs, in both TZM-­‐bl reporter and T cell-­‐derived cell lines, and to examine their potential as anti-­‐HIV-­‐1 therapeutic targets through exploitation of the cellular gene silencing pathway, RNA interference (RNAi). These were HIV-­‐1 Tat specific factor 1 (HTATSF1), DEAD (Asp-­‐Glu-­‐Ala-­‐Asp) box polypeptide 3, X-­‐ linked (DDX3X) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1), selected because they had been previously implicated in HIV-­‐ 1 pathogenesis. The well-­‐characterised HDF, PC4 and SFRS1 interacting protein 1 (PSIP1)/lens epithelium-­‐derived growth factor (LEDGF)/p75, was included in the study as a positive control. Cassettes expressing short hairpin RNAs (shRNAs) targeting the four host proteins were generated, although shRNAs did not suppress endogenous ddx3x mRNA levels. The ability of shRNAs to inhibit HIV-­‐1 replication in the reporter cell line, TZM-­‐bl, was examined. These HeLa-­‐ derived cells are permissive for R5-­‐tropic HIV-­‐1 infection and contain an integrated luciferase gene driven by the viral promoter. shRNAs mediated a dose-­‐dependent inhibition of luciferase activity in cells infected with a HIV-­‐1 subtype B molecular clone and, although production of the viral protein p24 was unaltered, infectious particle production was decreased in cells treated with a shRNA suppressing HTATSF1. Little effect was observed with a shRNA targeting SMARCB1, suggesting that this may not function as an HDF under these conditions. No effect on infectious particle production was seen with the shRNA targeting PSIP1, which was a result of the long half-­‐ life of this protein, highlighting a limitation of using such reporter systems for HDF validation. Importantly, shRNAs were not associated with any cytotoxic effects in TZM-­‐bl cells. Whether HTATSF1 is a potential therapeutic target was interrogated further in the more relevant T cell-­‐derived SupT1 cell line. Lentiviruses were used to generate populations where >90% had one copy of the integrated shRNA expression cassette. Replication of the subtype B molecular clone p81A-­‐4 was significantly inhibited in the shH1-­‐expressing SupT1 cell line, which targets HTATSF1, for over 14 days post-­‐infection, although inhibition was not as pronounced asthat observed in the shP1-­‐expressing SupT1 cell line, which targets PSIP1. In contrast to a previous report, no change in the ratio of unspliced to singly-­‐ or multiply-­‐spliced HIV-­‐1 transcripts were detected in shH1-­‐expressing SupT1 cells, suggesting that HTATSF1 does not function as a splicing cofactor in this system. A slight rebound in p24 levels at 14 days post-­‐infection was accompanied by increased HTATSF1 expression and a decrease in the percentage of cells with transgene expression in the population. In addition, there was a slight decrease in shH1-­‐derived guide strand expression, but no change in transcription rates of the htatsf1 gene, suggesting that cells within the population with shH1 expression and HTATSF1 suppression may have a growth disadvantage. Thus, although this work demonstrates for the first time that HTATSF1 functions as an HDF in T cell-­‐derived SupT1 cells, it may not constitute a viable therapeutic target. A second objective of this thesis was to examine the feasibility of transcriptional gene silencing (TGS) of HDFs as an anti-­‐HIV strategy. TGS is a small RNA-­‐induced gene silencing pathway that operates through chromatin remodelling with the potential to mediate long-­‐term silencing of gene expression. Thus, its application may reduce the frequency of drug administration and associated toxicities. Short interfering RNAs (siRNAs) targeting the htatsf1 promoter were able to reduce target mRNA expression, which was accompanied by decreased htatsf1 transcription rates in HEK293T cells, suggesting silencing via a TGS mechanism. The htatsf1 silencing inhibited infectious HIV-­‐1 particle production from TZM-­‐bl cells. This work provides proof of principle that TGS induction at a HDF may inhibit HIV-­‐1 replication. siRNAs targeting the ddx3x promoter did not induce TGS. To examine whether gene susceptibility to TGS may be influenced by promoter architectures, 49 promoter features were examined for enrichment in genes at which small RNA-­‐induced TGS has been reported. Initially, the TGS group was compared to a random set of 2,000 promoters and then all other promoters in the genome. To control for gene activation, two further analyses were performed comparing the TGS group features to those from promoters active in the THP-­‐1 cell line and housekeeping genes. Whilst difficult to ascribe differences between the TGS group and the control groups to anything beyond a variation in the proportion of active genes within each group, there was enrichment for certain promoter features that are independent of activity; the TGS group was characterised by broad transcription start regions, high CpG content and a single expression profile. Moreover, the fraction of promoters with reported non-­‐coding RNA overlap was greater in the TGS group than the control groups. Thus, there is some evidence that a number of promoter features are associated with TGS susceptibility. It is hoped this novel analysis will facilitate selection of future TGS targets, including HDFs. In summary, the work presented in this thesis paves the way for development of improved anti-­‐HIV therapies involving HDF-­‐targeted TGS-­‐based gene therapies that mediate sustained inhibition of the virus.

Despite the improved survival of human immunodeficiency virus (HIV) infected individuals with the introduction of highly active anti-retroviral therapy (HAART) in the South African public sector in 2004, new challenges have been brought to the fore. These include drug-related toxicities, particular those of stavudine, which remains in common use within developing countries. A prospective analysis of 9040 HIV-1-infected adults initiated on HAART from 2004 to 2007 at the Themba Lethu Clinic, Helen Joseph Hospital in Johannesburg, confirmed the ability to roll out a successful HAART programme in a resource limited environment with a high retention rate of 70%. Nearly 30% of patients switched to non-stavudine based regimens due to side effects - predominantly peripheral neuropathy, symptomatic hyperlactataemia and lipoatrophy. In an attempt to look for safer options, a prospective randomized controlled trial comparing standard and low dose stavudine with tenofovir was undertaken in 2009. Sixty patients were randomized 1:1:1 to either standard (30-40 mg), low (20-30 mg) dose stavudine or tenofovir (300 mg) each combined with lamivudine and efavirenz. Adipocyte mitochondrial DNA (mtDNA) levels, gene expression, anthropometry, markers of inflammation, lipid and glucose metabolism were assessed at various time intervals. Results demonstrate early mitochondrial depletion among black South African patients receiving low and standard doses of stavudine, with preservation of gene expression levels, except for NRF1 and MTCYB, when compared to patients on tenofovir. Mitochondrial toxicities occurred in both the stavudine arms. Immunological and virological outcomes were similar for all three arms. Both drugs caused lipid changes, but tenofovir had a more favourable effect on anthropometry and adipokines. Both stavudine regimens increased fasting insulin and C-peptide levels, with the higher stavudine dose also causing increased fasting glucose and HOMA levels. This study demonstrates an early association between mitochondrial depletion and stavudine therapy in the black South African population and shows that tenofovir has a minimal effect on mitochondrial numbers. Only two of eight adipocyte genes were significantly affected by stavudine therapy when compared with tenofovir, but this was only seen with the standard dose. This study highlights the occurrence of significant metabolic abnormalities with both drugs. Therefore, awareness of the potential increased cardiovascular risk should be of concern with tenofovir and stavudine, although toxicity is lower in the low dose compared to the standard dose stavudine regimen with no attenuation of anti-retroviral effectivity.

The lack of an animal model that recapitulates the prominent features of HIV-1 infection in humans limits the search for preventative and curative strategies against HIV-1. As stated by the National Institutes of Health (NIH), developing highly pathogenic simian-human immunodeficiency viruses (SHIVs) that can establish persistent infections and AIDS progression in rhesus macaques (RMs) remains vital for advancing the field. The HIV-1 envelope (Env) serves as a major target in vaccine studies. SHIVs - which are chimeras of the simian immunodeficiency virus (SIV) backbone and a humanized Env, are utilized for preclinical evaluation of vaccines and therapeutics aimed at targeting the Env glycoprotein. However, SHIVs presently available poorly infect RM due to weak binding interactions between Env and rhesus CD4 (rhCD4). Position 375 (Env375) lies within the rhCD4 binding pocket of HIV-1 Env. Accordingly, substituting the wild-type (WT) amino acid in Env375 for bulky hydrophobic and/or basic amino acids may strengthen Env-rhCD4 interactions. These mutations should increase the pathogenicity of our original SHIV challenge stocks (SHIV162p3 and SHIVAE16) and allow for the development of an animal model that closely mirrors HIV-1 acquisition and chronic AIDS infection in humans. OBJECTIVES: To develop an animal model that recapitulates HIV-1 infection and AIDS progression in humans. MATERIALS AND METHODS: SHIV design involved insertion of human env sequences into a modified SIV backbone (provided by Dr. George Shaw, University of Pennsylvania). Site-directed mutagenesis was employed to introduce amino acid substitutions at Env375 for the following residues: serine, histidine, methionine, tryptophan, tyrosine, and phenylalanine. These constructs were then utilized for transfection of human embryonic kidney cells (293T) with viral supernatants collected 72 hours post-transfection. 293T viral supernatants were then used to infect human and rhesus PBMCs with the resulting supernatant harvested every three days and subjected to ELISA to monitor viral growth. Viruses were further characterized by RT-PCR for quantification and TCID50 assays to determine infectious dose. Resulting SHIVs were then used for challenge studies in rhesus macaques. Sixteen rhesus macaques were divided into groups of four that received the following SHIV challenge stocks: (1) original SHIV162p3, (2) modified SHIV162p3, (3) original SHIVAE16, and (4) modified SHIVAE16. Following infection, animal plasma and sera were collected and subjected to post-challenge analyses such as cellular assays and measurements of plasma viral RNA levels. RESULTS: Several analyses were conducted to study the pathogenicity of the modified SHIV162p3 and SHIVAE16 relative to the original stocks. Regarding SHIV infectivity, several versions of the modified SHIVs exhibited greater in vitro infectivity titers than the original stocks. Additionally, preliminary viral load analyses conducted in vivo indicate that all sixteen RMs challenged with original and modified SHIVs were infected at comparable levels. Furthermore, CD4+ T cell counts were measured and all sixteen animals exhibited declines in CD4+ T cell percentages. CONCLUSION: In sum, the modified SHIVs may improve animal models by closely recapitulating the events of HIV-1 infection in humans and serve better for future studies of preventative and curative treatments against HIV-1. / 2019-07-11T00:00:00Z

Resumo: Em 1997 o Departamento de DST/AIS e Hepatites Virais estruturou em todo Brasil uma rede de laboratórios para realizar o exame de Carga Viral Plasmática do HIV (CV) denominada Rede Nacional de Carga Viral. O exame quantifica o RNA do HIV no plasma do paciente infectado utilizando a metodologia do branched-DNA, um ensaio de amplificação do sinal luminescente, o qual utiliza uma plataforma de detecção. Atualmente, esta rede é reconhecida internacionalmente e realiza 520.000 exames/ano. No entanto, vários fatores podem influenciar o resultado do exame como integridade do RNA, volume de amostra disponível, método e plataforma utilizados. Assim, o Departamento de DST/AIDS e Hepatites Virais implantou um protocolo pré-analítico para ser utilizado em todo o território nacional. Entretanto, as regiões brasileiras são muito diferentes e alguns laboratórios não conseguem seguir este protocolo. O objetivo deste estudo foi (a) avaliar as diferentes condições de transporte e armazenamento das amostras utilizadas no teste de CV, (b) validar a utilização de volumes iniciais de plasma inferiores ao preconizado, (c) comparar plataformas de detecção e (d) metodologias disponíveis para a execução do exame. Os resultados mostraram que as amostras podem ser processadas em até 8 h sem perda ou degradação do RNA, volumes iniciais inferiores ao preconizado podem ser utilizado com perda de sensibilidade e, as duas plataformas disponíveis no Brasil são equivalentes para a execução do teste. Apesar de existirem outras metodologias para a realização do teste, os resultados podem ser diferentes mostrando a necessidade da utilização da mesma metodologia em todo Brasil / Abstract: The Department of the DST/AIDS and Viral Hepatitis implemented since 1997 a laboratory network in all Brazil to perform the HIV plasma viral load (PVL) test named Viral Load National Network as part of the attendance to infected patient. This exam quantify the HIV plasma RNA in infected patient using Branched-DNA methodology, a signal amplification nucleic acid probe assay, which use a detection platform. Nowadays this network has international appreciation and to execute 520.000 tests/year. However, several factors can alter the result of the test as RNA integrity, available sample volume, used method and detection platform. Then an optimized pre-analytic protocol was implanted by Department of the DST/Aids to be used in all national territory. However the Brazil regions are many different and some laboratories don't get lead this protocol. The goal of this study was (a) to evaluate the different transport and storage conditions of the samples used to the PVL test, (b) to valid the use of the lower plasma initial input in the exam, (c) to compare the detection platforms and (d) methods available to execution of the test. The results showed blood sample can be process in until 8h after collection without RNA loss or degradation, lower initial input can be used with loss of sensibility and the two detection platforms available in Brazil are equivalent. In spite of others methods are available to execution of test, the results can be distinct showing the importance of the all laboratories in Brazil used the same method / Orientador: Maria Inês de Moura Campos Pardini / Coorientador: Rejane Maria Tommasini Grotto / Banca: Emílio Carlos Curcelli / Banca: Paulo Inácio da Costa / Mestre

Viral protein R (Vpr) is an evolutionarily conserved but poorly understood protein encoded by all primate lentiviruses, including the lineages that gave rise to both human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), the causative agents of AIDS in humans. In this work, I sought to define the contribution of primate lentiviral Vpr to viral replication and evasion from cell-intrinsic antiviral defenses. I found that HIV-1 infection of human dendritic cells (MDDCs) is substantially attenuated upon infection with Vpr-deficient (HIV-1/ΔVpr) virus compared to wild-type (WT) infection. This replication defect to HIV-1/ΔVpr is evident in a single round of infection, results in reduced levels of viral transcription, and is relieved upon complementation by virion-associated Vpr. The block to transcription is alleviated through Vpr-engagement with the Cul4A/DCAF/DDB1 (DCAFCRL4) ubiquitin ligase complex and a yet-to-be identified host factor, hypothesized to induce the DNA damage response (DDR) in infected cells. MDDCs are critical immune cells that are poised to detect invading viruses through a variety of cell-intrinsic antiviral sensors, resulting in the production of type I interferon (IFN) and restriction of virus replication. Surprisingly, infection of MDDCs with Vpr-deficient lentiviruses (HIV-2 or SIVmac) resulted in production of type I IFN indicating that this pathway is targeted by Vpr. I determined that signaling cascades that induce NF-κB-dependent type I IFN production are triggered in response to lentiviral integration, an obligatory process in lentivirus life cycle that results in host DNA lesions and subsequent repair by cellular DNA repair machinery. I also demonstrated that mutations in SIVmac Vpr that ablate the ability to initiate DDR are unable to counteract the antiviral type I IFN response. Together, our work suggests the existence of a novel host factor that detects lentiviral integration in MDDCs to trigger an innate immune response that blocks virus dissemination. I hypothesize that Vpr by overcoming this cell intrinsic block to integration would be a critical viral adaptation to facilitate cross-species transmission that resulted in the HIV pandemic. / 2018-11-01T00:00:00Z

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No. of bitstreams: 2 Dissertação- Luciana Oliveira Bento - 2016.pdf: 3533613 bytes, checksum: f67fe73c6af12700e4cba39192ea7c95 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-24 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The prevalence of isolates of HIV-1 with resistance mutations to antiretroviral drugs should be monitored continuously and in different population groups from geographical regions Brazilian, since Brazil offers universal access to treatment for all people living with HIV and AIDS. Because of the scarcity of related studies in cities of the interior of Brazil, this study aimed to identify the HIV-1 genetic diversity and to evaluate the profile and the prevalence of HIV-1 isolates with mutations in non-antiretroviral (ARV) exposed patients attended at the Specialized Service in STD/AIDS of the Jataí city, southwestern of Goiás state. From January 2015 to January 2016, 57 patients not exposed to ARVs were recruited and whole blood samples were collected. The protease (PR) and about 2/3 of the reverse transcriptase (RT) regions were amplified in 46 samples by "nested"-PCR and sequenced. Resistance mutations to ARVs were determined by Calibrated Population Resistance Tool from Stanford University and HIV-1 subtypes were identified by REGA and phylogenetic inference. In this study, the prevalence of HIV-1 resistant was more frequent among young male population, heterosexual, especially in the reproductive age group and brown race. Among 46 HIV-1 isolates sequenced, 5 had primary resistance mutations to ARVs, giving a prevalence of 10.9%. The mutations were detected both non-nucleoside RT inhibitors-NNRTIs (K103N, E138K/A and V179E) and for PR inhibitors-IP (M46L and T74S). As the K103N mutation confers resistance to high profile ARV composing the first line of treatment (EFV), it was introduced into the second line with IP for this patient. The E138K mutation confers resistance to an ARV not used in Brazil (RPV), allowing the introduction of the first line of treatment with the fixed-dose combination (formulation 3 in 1). Two isolates of HIV-1 were IP resistance mutations (M46L and T74S) but not yet started therapy. In this case, the introduction of the treatment with the formulation 3 to 1 is possible, since the first line has no IP in its formulation. HIV-1 subtype B was the prevalent isolates and three recombinants involving subtypes B and F1 were observed. The subtype C and recombinant forms were first reported in Goiás southwestern region. The moderate prevalence of primary resistance of HIV-1 isolates among patients from southwestern Goiás state and co-circulation of “pure” HIV-1 subtypes and recombinant forms, it is evident the importance of monitoring of newly diagnosed patients to optimize initial therapy, improving clinical management and control of transmission of HIV-1. / A prevalência de isolados do HIV-1 com mutações de resistência aos antirretrovirais deve ser monitorada continuamente nas diferentes regiões geográficas e grupos populacionais brasileiros, visto que o Brasil disponibiliza acesso universal ao tratamento para todas as pessoas vivendo com HIV e aids. Devido à escassez de estudos relacionados em cidades do interior do Brasil, este trabalho teve como objetivo identificar a diversidade genética do HIV-1 e avaliar o perfil e a prevalência de isolados do HIV-1 com mutações, em pacientes não expostos aos antirretrovirais (ARVs) atendidos no Serviço de Atendimento Especializado em DST/aids do município de Jataí, no sudoeste goiano. De janeiro de 2015 a janeiro de 2016, foram recrutados 57 pacientes não expostos aos ARVs e amostras de sangue total foram coletadas. Os genes da protease (PR) e cerca de 2/3 da transcriptase reversa (TR) foram amplificados em 46 amostras pela “nested”-PCR e sequenciados. As mutações de resistência aos ARVs foram determinadas mediante a ferramenta Calibrated Population Resistance Tool da Universidade de Stanford e os subtipos do HIV-1 foram identificados pela análise por REGA e inferência filogenética. Neste estudo, a prevalência da resistência aos antirretrovirais foi mais frequente na população jovem não exposta do sexo masculino, heterossexual, na cor parda, e especialmente nas faixas etárias de 30 a 34 anos, e de 40 a 49 anos de idade. Entre os 46 isolados de HIV-1 sequenciados, 5 apresentaram mutações de resistência primária aos ARVs, conferindo uma prevalência de 10,9%. Foram detectadas mutações tanto para inibidores da TR não nucleosídicos-NNRTI (K103N, E138K/A e V179E) quanto para inibidores da PR-IP (M46L e T74S). Como a mutação K103N confere alto perfil de resistência ao ARV que compõe o esquema de primeira linha de tratamento (EFV), foi introduzida a segunda linha com IP (LPV/r) para este paciente. A mutação E138K confere resistência a um ARV ainda não utilizado no Brasil (RPV), o que permitiu a introdução da primeira linha de tratamento constituída pela dose fixa combinada com TDF+3TC+EFV (formulação 3 em 1). Dois isolados do HIV-1 apresentaram mutações de resistência (M46L e T74S) que conferem resistência ao IP (NFV), mas ainda não iniciaram a terapia. Nesse caso, a introdução do tratamento com a formulação 3 em 1 será possível, já que a primeira linha não tem o IP (NFV) em sua formulação. O subtipo B do HIV-1 foi o prevalente e três isolados recombinantes foram observados, envolvendo os subtipos B e F1. O subtipo C e as formas recombinantes foram relatados pela primeira vez na região do sudoeste goiano. Com a identificação de uma prevalência moderada de isolados de HIV-1 com resistência primária entre pacientes do sudoeste goiano e a co-circulação de subtipos “puros” e mosaicos do HIV-1, fica evidente a importância do monitoramento dos pacientes recém-diagnosticados para a otimização da terapia inicial, melhorando a conduta clínica e o controle da transmissão do HIV-1.

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