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p. 1-10 Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-06-25T17:58:22Z No. of bitstreams: 1 9999999999999999999ff.pdf: 959098 bytes, checksum: 50b03165d54f8297f87c9e137a48338f (MD5) Approved for entry into archive by Rodrigo Meirelles (rodrigomei@ufba.br) on 2013-11-26T13:17:19Z (GMT) No. of bitstreams: 1 9999999999999999999ff.pdf: 959098 bytes, checksum: 50b03165d54f8297f87c9e137a48338f (MD5) Made available in DSpace on 2013-11-26T13:17:19Z (GMT). No. of bitstreams: 1 9999999999999999999ff.pdf: 959098 bytes, checksum: 50b03165d54f8297f87c9e137a48338f (MD5) Previous issue date: 2013 Background: Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiological agent of tropical spastic paraparesis/ HTLV-associated myelopathy (HAM/TSP) that can be identified in around 0.25%–3.8% of the infected population. Disease progression can be monitored by the proviral load and may depend on genetic factors, however, it is not well understood why some HTLV-1 infected people develop the disease while others do not. The present study attempts to assess the molecular diversity of gp46 glycoprotein in HAM/TSP patients and Health Carrier (HC) individuals. Methods: Blood samples were collected from 10 individuals, and DNA was extracted from PBMCs to measure the HTLV-1 proviral load. The gp46 coding sequences were amplified PCR, cloned and sequenced. The molecular characterization was performed using bioinformatics tools. Results: The median HTLV-1 proviral load of HC (n = 5) and HAM/TSP (n = 5) patients was similar (average 316,227 copies/106 PBMCs). The gp46 molecular characterization of 146 clones (70 HC and 76 HAM/TSP) revealed an overall diversity, within HC and HAM/TSP clones, of 0.4% and 0.6%, respectively. Five frequent mutations were detected among groups (HAM/TSP and HC clone sequences). A single amino acid (aa) substitution (S35L) was exclusive for the HC group, and three gp46 substitutions (F14S, N42H, G72S) were exclusive for the HAM/TSP group. The remaining frequent mutation (V247I) was present in both groups (p = 0.0014). The in silico protein analysis revealed that the mutated alleles F14S and N42H represent more hydrophilic and flexible protein domains that are likely to be less antigenic. The Receptor Binding Domain is quite variable in the HAM/TSP group. Two other domains (aa 53–75 and 175–209) that contain multiple linear T-cell epitopes showed genetic diversity in both HAM/TSP and HC groups. Further analysis revealed 27 and 13 T-cell epitopes for class I HLA alleles and class II HLA alleles, when analyzing the entire gp46. Conclusions: The most common gp46 mutations were not associated clinical status because they were found in only one individual, except for the V247I mutation, that was found at viral clones from HAM/TSP ad HC individuals. Because of this, we cannot associate any of the gp46 found mutations with the clinical profile. Salvador

p. 1-10 Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-06-25T17:58:22Z No. of bitstreams: 1 9999999999999999999ff.pdf: 959098 bytes, checksum: 50b03165d54f8297f87c9e137a48338f (MD5) Approved for entry into archive by Rodrigo Meirelles (rodrigomei@ufba.br) on 2013-11-26T13:17:19Z (GMT) No. of bitstreams: 1 9999999999999999999ff.pdf: 959098 bytes, checksum: 50b03165d54f8297f87c9e137a48338f (MD5) Made available in DSpace on 2013-11-26T13:17:19Z (GMT). No. of bitstreams: 1 9999999999999999999ff.pdf: 959098 bytes, checksum: 50b03165d54f8297f87c9e137a48338f (MD5) Previous issue date: 2013 Background: Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiological agent of tropical spastic paraparesis/ HTLV-associated myelopathy (HAM/TSP) that can be identified in around 0.25%–3.8% of the infected population. Disease progression can be monitored by the proviral load and may depend on genetic factors, however, it is not well understood why some HTLV-1 infected people develop the disease while others do not. The present study attempts to assess the molecular diversity of gp46 glycoprotein in HAM/TSP patients and Health Carrier (HC) individuals. Methods: Blood samples were collected from 10 individuals, and DNA was extracted from PBMCs to measure the HTLV-1 proviral load. The gp46 coding sequences were amplified PCR, cloned and sequenced. The molecular characterization was performed using bioinformatics tools. Results: The median HTLV-1 proviral load of HC (n = 5) and HAM/TSP (n = 5) patients was similar (average 316,227 copies/106 PBMCs). The gp46 molecular characterization of 146 clones (70 HC and 76 HAM/TSP) revealed an overall diversity, within HC and HAM/TSP clones, of 0.4% and 0.6%, respectively. Five frequent mutations were detected among groups (HAM/TSP and HC clone sequences). A single amino acid (aa) substitution (S35L) was exclusive for the HC group, and three gp46 substitutions (F14S, N42H, G72S) were exclusive for the HAM/TSP group. The remaining frequent mutation (V247I) was present in both groups (p = 0.0014). The in silico protein analysis revealed that the mutated alleles F14S and N42H represent more hydrophilic and flexible protein domains that are likely to be less antigenic. The Receptor Binding Domain is quite variable in the HAM/TSP group. Two other domains (aa 53–75 and 175–209) that contain multiple linear T-cell epitopes showed genetic diversity in both HAM/TSP and HC groups. Further analysis revealed 27 and 13 T-cell epitopes for class I HLA alleles and class II HLA alleles, when analyzing the entire gp46. Conclusions: The most common gp46 mutations were not associated clinical status because they were found in only one individual, except for the V247I mutation, that was found at viral clones from HAM/TSP ad HC individuals. Because of this, we cannot associate any of the gp46 found mutations with the clinical profile. Salvador

Centro de Pesquisas Gonçalo Moniz - FIOCRUZ, Bahia, Brasil Universidade Federal da Bahia, Brasil Escola Bahiana de Medicina e Saúde Pública, Bahia, Brasil Animal Models and Retroviral Vaccines Section, NCI/NIH, Bethesda, Maryland, Brasil Besides being extremely useful in measuring the level of HIV-1 diversity and prevalence in populations, the molecular analysis of genomic sequences provides crucial surveillance support and aids in the development of new therapies and effective vaccines. The present study focused on gag and env DNA and amino acid sequences that were generated from samples taken from 61 infected patients in the City of Salvador, Bahia, located in northeastern Brazil. In order to determine selective pressure and predict coreceptor usage, Bioinformatics tools were employed in phylogeny reconstruction. Fifty-six (91.8%) viruses were classified as belonging to subtype B, three (4.9%) from F1, and two (3.3%) from BF1 recombinants. Based on the characterization of the V3 region, the subtype B strains were represented by eight (18.2%) Brazilian variants (B'-GWGR), 20 (46.5%) European/EUA B variants (GPGR), and 15 (34.9%) GXGX variants. The mean time elapsed since diagnosis was 13 years among subtype B' and 9 years in subtype B. The mean dN/dS ratios from the GWGR, GPGR, and GXGX groups, when compared to an HXB2 reference, were 0.72, 0.77, and 0.67, respectively. Seventy-six percent of the viruses studied were predicted to use the CCR5 coreceptor for cell entry (R5 viruses), while 24% were predicted to use the CXCR4 or were classified as dual tropic viruses. The prevalence of subtypes B' and recombinant B/F1 was shown to be lower than findings from previous studies performed both in Brazil (B') and in Bahia (B/F1). The association between subtype B' and a lengthy period of time since diagnosis can be correlated with a slower disease progression in infected patients, when compared with those infected with subtype B.

Submitted by PMBqBM null (pmbqbm@ufba.br) on 2017-05-24T11:28:43Z No. of bitstreams: 1 Dissertação Juliana Sacramento Mota de Souza_PMBqBM-UFBA.pdf: 33818819 bytes, checksum: 7904ea127d9fe97a4dca2dc89cca3c2f (MD5) Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-07-06T13:24:46Z (GMT) No. of bitstreams: 1 Dissertação Juliana Sacramento Mota de Souza_PMBqBM-UFBA.pdf: 33818819 bytes, checksum: 7904ea127d9fe97a4dca2dc89cca3c2f (MD5) Made available in DSpace on 2017-07-06T13:24:46Z (GMT). No. of bitstreams: 1 Dissertação Juliana Sacramento Mota de Souza_PMBqBM-UFBA.pdf: 33818819 bytes, checksum: 7904ea127d9fe97a4dca2dc89cca3c2f (MD5) FAPESB O Vírus da imunodeficiência humana do tipo 1 (HIV-1) possui uma ampla variabilidade genética. Esta diversidade é representada pelos quatro grupos, pelos nove subgrupos do grupo M e também pelas formas recombinantes destes vírus. Dentre estes, os recombinantes BF tem se destacado pela alta dispersão global concomitante com o aumento em número e diversidade. Atualmente quatorze formas recombinantes circulantes (CRFs) BF e inúmeras formas recombinantes únicas BF (URFs) foram descritas. O objetivo deste trabalho foi realizar a caracterização molecular e geográfica de isolados virais recombinantes BF do HIV- 1. Para tal, sequências e informações de genomas completos de recombinantes BF do HIV foram coletadas dos bancos de dados NCBI e Los Alamos. Posteriormente foram realizadas a caracterização destas sequências, análises filogenéticas, de recombinação e da alça V3 da proteína glicoproteína 120 (gp120), além de busca por assinaturas nucleotídicas e predição do coreceptor de entrada viral. Foram encontradas 252 sequências de genoma completo (>7000 pb) de recombinantes BF do HIV oriundas de treze países diferentes, sendo a maioria delas provenientes de pacientes brasileiros (52,8%). Seis sequências previamente caraterizadas como recombinantes BF, foram reclassificadas como subtipo B puro. Algumas sequências formaram agrupamentos monofiléticos com as CRFs já descritas e, portanto, sugere-se a reclassificação das mesmas. Dentre as sequências avaliadas foram encontrados 114 padrões distintos de recombinação. A maioria das sequências apresenta recombinação na região pol (81%; 205/252), seguido das regiões gag (68%; 172/252) e env (54%; 137/252) sendo encontrado na região tat a menor taxa de recombinação (7%; 17/252). Enquanto em todos os outros genes acessórios e regulatórios predominou o genótipo B, em vif, o subtipo F prevaleceu (38%; 96/252). Além disso, foram identificados dois hotspots entre as sequências analisadas: um encontrado em 35 sequências (13,9%) na região entre as posições 5360 – 5390 do gene acessório vif e outro em torno da posição 9356 da região nef (11% das sequências). Foram identificadas assinaturas nucleotídicas conservadas exclusivas para cada CRF_BF descrita. As mutações mais frequentes foram encontradas na região da transcriptase reversa foram M41L (NRTIs) e K100N (NNRTIs). De maneira geral, as mutações encontradas nestas sequências conferem resistência a diversos fármacos sendo que as maiores taxas de resistência foram encontradas para os antirretrovirais Atazanavir, Saquinavir, Nevirapina, Zidovudina, Estavudina, Didanosina e Emtricitabina. A predição de uso de coreceptor viral mostrou estes recombinantes utilizam preferencialmente o coreceptor CCR5 (67,5%) sendo que a CRF42 utiliza exclusivamente este coreceptor, enquanto todas as sequências da CRF39 foram classificadas como R5X4/X4. Os tetrapeptídeos mais frequentes encontrados na alça V3 foram GPGR (49,4%), GPGQ (20%), APGR (6,7%), GPGK (5,1%), GWGR (4,3%) e GPGG (2%). Além disso, as sequências com o motivo GPGQ e APGR são preferencialmente classificadas como R5 e contem o subtipo F na região da alça V3. Pode-se observar que há uma grande diversidade dos padrões de recombinação evidenciando que a recombinação entre os subtipos B e F é frequente. Human immunodeficiency virus type 1 (HIV-1) has a wide genetic variability. This diversity is represented by the four groups, nine subgroups of group M and by the recombinant forms of these viruses. Among these, the recombinant BF has been distinguished by the high global dispersion concomitant with the increase in number and diversity. Currently fourteen BF Circulating Recombinant Forms (CRFs) and BF Unique Recombinant Forms (URFs) forms have been described. The objective of this work was to perform molecular and geographic characterization of HIV-1 recombinant BF viral isolates. To that end, complete genome sequences and information from recombinant HIV BFs were collected from the NCBI and Los Alamos databases. Subsequently, the characterization of these sequences, phylogenetic analysis, recombination and the V3 loop of the glycoprotein 120 protein (gp120) were carried out, as well as the search for nucleotide signatures and prediction of the viral entry coreceptor. A total of 252 complete genome sequences (> 7000 bp) of recombinant HIV BF from thirteen different countries were found, most of them coming from Brazilian patients (52.8%). Six sequences previously characterized as recombinant BF were reclassified as pure B subtype. Some sequences formed monophyletic clusters with the CRFs already described and, therefore, it is suggested to reclassify them. Among the sequences evaluated, 114 distinct patterns of recombination were found. Most of the sequences present recombination in the pol region (81%, 205/252), followed by the gag (68%, 172/252) and env (54%; 137/252) regions being found in the tat region at the lowest recombination rate (7%, 17/252). While in all other accessory and regulatory genes, genotype B predominated, in vif, the F subtype was dominant (38%, 96/252). In addition, two hotspots were identified among the sequences analyzed: one found in 35 sequences (13.9%) in the region between positions 5360-5390 of the vif accessory gene and another around position 9356 of the nef region (11% of sequences). Unique conserved nucleotide signatures were identified for each CRF_BF described. The most frequent mutations were found in the reverse transcriptase region were M41L (NRTIs) and K100N (NNRTIs). In general, the mutations found in these sequences confer resistance to several drugs, and the highest resistance rates were found for the antiretrovirals Atazanavir, Saquinavir, Nevirapine, Zidovudine, Stavudine, Didanosine, and Emtricitabine. The prediction of the use of viral coreceptor showed that these recombinants preferentially use the CCR5 coreceptor (67.5%) and CRF42 exclusively uses this coreceptor, while all CRF39 sequences were classified as R5X4 / X4. The most frequent tetrapeptides found in the V3 loop were GPGR (49.4%), GPGQ (20%), APGR (6.7%), GPGK (5.1%), GWGR (4.3%) and GPGG %). In addition, the sequences with the GPGQ motif and APGR are preferably classified as R5 and contain the F subtype in the V3 loop region. It can be observed that there is a great diversity of the recombination patterns evidencing that the recombination between the subtypes B and F is frequent.