Fernanda Khouri Barreto

Inferences about the global scenario of human T-cell lymphotropic virus type 1 infection using data mining of viral sequences

Human T-cell lymphotropic virus type 1 (HTLV-1) is mainly associated with two diseases: tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) and adult T-cell leukaemia/lymphoma. This retrovirus infects five-10 million individuals throughout the world. Previously, we developed a database that annotates sequence data from GenBank and the present study aimed to describe the clinical, molecular and epidemiological scenarios of HTLV-1 infection through the stored sequences in this database. A total of 2,545 registered complete and partial sequences of HTLV-1 were collected and 1,967 (77.3%) of those sequences represented unique isolates. Among these isolates, 93% contained geographic origin information and only 39% were related to any clinical status. A total of 1,091 sequences contained information about the geographic origin and viral subtype and 93% of these sequences were identified as subtype “a”. Ethnicity data are very scarce. Regarding clinical status data, 29% of the sequences were generated from TSP/HAM and 67.8% from healthy carrier individuals. Although the data mining enabled some inferences about specific aspects of HTLV-1 infection to be made, due to the relative scarcity of data of available sequences, it was not possible to delineate a global scenario of HTLV-1 infection.

Molecular study of HBZ and gp21 human T cell leukemia virus type 1 proteins isolated from different clinical profile infected individuals.

Human T cell leukemia virus type 1 (HTLV-1) is associated with a neurological syndrome named tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) and the disease progression involves viral factors. The gp21 glycoprotein is involved in envelope trafficking and membrane targeting while the bZIP protein is indispensable for cell growth and proliferation. This study aimed to assess the molecular diversity of gp21 and HBZ proteins in TSP/HAM and healthy carriers. DNA samples from HTLV-1-infected individuals were submitted to PCR and sequencing, and the molecular analyses were performed using bioinformatics tools. From eight gp21-analyzed sequences one amino acid change (Y477H) was associated with the switch of a helix to coil structure at secondary structure prediction. From 10 HBZ analyzed sequences, two amino acid changes were identified (S9P and T95I) at the activation domain. One mutation (R112C) located at the nuclear localization signal was present in 66.7% and 25% of healthy carriers (HC) and TSP/HAM groups, respectively. This is the first report of mutations in the HBZ region. These polymorphisms might be important for viral fitness.

Molecular study of HBZ and gp21 Human T cell lymphotropic virus type 1 proteins isolated from different clinical profile infected individuals.

The HTLV-1 gp21 glycoprotein is involved in envelope trafficking and membrane targeting while bZIP protein is indispensable for cell growth and proliferation. This study aimed to assess the molecular diversity of gp21 and HBZ proteins, in TSP/HAM and healthy carriers. DNA samples from HTLV-1 infected individuals were submitted to PCR, sequencing, and the molecular analyses were performed using bioinformatics tools. From eight gp21 analyzed sequences one amino acid change (Y477H) was associated to the switch of helix to coil structure at secondary structure prediction. From ten HBZ analyzed sequences, two amino acid changes were identified (S9P and T95I) at the activation domain. One mutation (R112C) located at nuclear localization signal was present in 66.7% and 25% of HC and TSP/HAM groups, respectively. This is the first report of mutations in HBZ region. These polymorphisms might be important for viral fitness.

Molecular characterization of the human T cell lymphotropic virus type 2 long terminal repeat region: A discussion about possible influences at viral gene expression.

Abstract This study aimed to identify nucleotide signatures in the promoter region of human T cell lymphotropic virus type 2 (HTLV-2) isolated from infected individuals from Salvador, Brazil and in sequences from the GenBank database. DNA samples from HTLV-2-infected individuals were submitted to nested polymerase chain reaction (PCR) and sequencing, and molecular analyses were performed using bioinformatics tools. The phylogeny of HTLV-2 strains isolated from patients from Salvador reveals that all sequences were subtype c. One hundred and fifty-one sequences from GenBank were selected, among which 30 belong to subtype a, 88 to subtype b, 32 to subtype c, and one to subtype d. Subtype-specific signatures were identified as well as mutations resulting in loss or gain of motifs important to transcription regulation. The subtypes a and b have two E box motifs, while subtypes c and d have only one. These polymorphisms may impact viral fitness and infection outcome and should be more closely investigated.

First detection of human T-lymphotropic virus in blood donors in Benin shows that testing is required to improve blood safety

Human T‐lymphotropic virus (HTLV) is a blood‐borne infection that can be transmitted via blood transfusion. Knowing that blood safety can improve blood transfusion to prevent dissemination of viral infections in medical facilities, there is no routine pre‐transfusion screening for HTLV in all blood banks in the Republic of Benin. This study aims to estimate the prevalence of HTLV infection in blood donors and describes the characteristics of positive donors.

ORFI genetic polymorphisms in geographically distinct HTLV-1 infections

The region known as pX in the 3’ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. The expression of ORF-I produces the protein p12 which can be cleavage resulting in the p8 protein. These proteins interfere with the ability of a biologically active of HTLV-1, influencing at the virus infectivity and persistence. Here, we evaluated whether natural mutations in HTLV-1 ORF-I can influence the proviral load and clinical manifestation of HAM/TSP. For that, we analyzed 1530 HTLV-1 ORF-I sequences from different clones of 156 patients with negative or positive diagnosis for HAM/TSP and demonstrated that some mutations may be associated with the outcome of HAM/TSP (C39R, L40F, P45L, S69G and R88K) or with proviral load (P34L and F61L). We further examined the presence of mutations in motifs of HBZ and observed that P45L mutation is located within the HBZ nuclear localization signal and was found more frequently between patients with HAM/TSP and high proviral load. This results suggest that some HTLV-1 ORF-I mutations may be associated with the development of HAM/TSP and the proviral load levels.

Molecular characterization of human T-cell leukemia virus type 1 gp46 glycoprotein from healthy carriers and tropical spastic paraparesis/HTLV-associated myelopathy infected individuals

The present study attempts to assess the molecular diversity of gp46 glycoprotein in HAM/TSP patients and Health Carrier (HC) individuals. 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. 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. We still cannot associate any of the gp46 found mutations with the clinical profile.

Inferences about global scenario of HTLV-1 infection using data mining of viral sequences

The human T-lymphotropic virus type 1 (HTLV-1) infects 15 to 20 million individuals throughout the world. Several HTLV-1 viral sequences are stored in HTLV-1 Molecular Epidemiology Database. The objective of this study was to describe the clinical, molecular and epidemiological scenarios of HTLV-1 infection through the stored sequences in the HTLV-1 Molecular Epidemiology Database. We have used the search algorithm implemented at the HTLV-1 database and the Excel to perform the descriptive analyses. There are currently 2,544 registered sequences with 2,057 (80.85%) of those sequences representing unique isolates. Among these sequences, 1,913 (92.99%) contained geographic information and only 797 (38.74%) are related to clinical status (TSP/HAM, 43%, ATLL, 19%, HC, 32.39%, and other diseases, 5.61%). 1,090 sequences contained information about geographic origin and viral subtype. Of these sequences 1,019 represent subtype a. The data about ethnicity are very scarce .Only 40 sequences have this information. 31 of them have information about clinical status, viral subtype, ethnicity and age at the same time. All of them were originated from women and 11 of them were younger than 40 years old. About the clinical status 9 of them were TSP/HAM and 21 HC. The HTLV-1 Molecular Epidemiology Database enabled some inferences about specific aspects on HTLV-1 infection, however due to the relative scarcity of data about the available sequences it was not possible to delineate a global scenario of HTLV-1 infection. Viral sequence data should be more offered because they can be used for planning public health policies.

Genetic diversity in HTLV-1 envelope (gp46), HBZ And LTR region from assymptomatic And TSP/HAM individuals

Most HTLV-1 carriers remain infected lifelong without developing any major clinical manifestation. It is known that several antibodies from the sera of HTLV-1-infected individuals can neutralize HTLV-1 transmission. The recently identified HBZ factor acts as a negative regulator of viral transactivation. To better investigate possible differences of gp46, among Health Care (HC) and TSP/HAM individuals, we have performed a point mutation characterization of this component in 146 clones from 10 HTLV-1 infected subjects. The generated sequences were aligned using CLUSTAL X, and edited manually using GENEDOC software to identify possible protein signatures. From the same patient samples, the analyses were performed in the PCR amplified products from HBZ and LTR sequences (n=10) to check the nucleotide sites changes and to perform a phylogenetic analysis, respectively. We had performed the Neighbor-joining and maximum likelihood phylogenetic analysis with PAUP* software. It was possible to identify, at least, 4 exclusive gp46 mutations among HC clones and 6 among TSP/HAM clones. The five common mutations were detected, with statistically significant difference, between HC and TSP/HAM. The overall genetic diversity was of 0.4% and 0.6% for HC and TSP/HAM clones, respectively. The potential protein domain analysis of gp46 showed, mostly, the presence of CK-2 and PKC phosphorylation, N-myristilation and N-glycosilation sites. In the HBZ sequences analysis it was possible to identify 12 mutations, and 11 of them was found in 100% of the generated sequences. The phylogenetic analysis of LTR region demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype.