Identification of human T-cell lymphotropic virus infection in a semi-isolated Afro-Brazilian quilombo located in the Marajó Island (Pará, Brazil)

Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5 LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5 LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.     

Complete nucleotide sequence of the new simian T-lymphotropic virus, STLV-PH969 from a Hamadryas baboon, and unusual features of its long terminal repeat.

A third type of primate T-lymphotropic virus, PTLV-L, with STLV-PH969 as a prototype, has recently been isolated from an African baboon (Papio hamadryas). Classification of this virus has been based on partial sequence analysis of cDNA from a virus-producing cell line, PH969. We obtained the complete nucleotide sequence of this virus with a proviral genome of 8,916 bp. All major genes, homologous in all human T-cell lymphotropic virus (HTLV)-related viruses, and their corresponding mRNAs, including appropriate splicing, were identified. One additional nonhomologous open reading frame in the proximal pX region is accessible for translation through alternative splicing. Sequence comparison shows that STLV-PH969 is equidistantly related to HTLV type 1 (HTLV-1) and HTLV-2. In all coding regions, the similarity tends to be the lowest between STLV-PH969 and HTLV-1. However, in the long terminal repeat (LTR) region, the lowest similarity was found between STLV-PH969 and HTLV-2. The U3-R and R-U5 boundaries of the STLV-PH969 LTR were experimentally determined at nucleotides 268 and 524, respectively. This 695-bp LTR is 60 and 73 bp shorter than the LTRs of HTLV-1 and HTLV-2, respectively, but its general organization is similar to the one found in the HTLV-bovine leukemia virus genus. In the long region between the polyadenylation signal and the poly(A) site, sequence similarity with the HTLV-1 Rex-responsive element (RexRE) core and secondary structure prediction suggest the presence of a RexRE. The presence of three 21-bp repeats is conserved within the U3 region of HTLV-1, HTLV-2, and BLV. Only two direct repeats with similarity to these Tax-responsive elements were found in the STLV-PH969 LTR, which might suggest differences in the Tax-mediated transactivation of this virus. We conclude that STLV-PH969 has all the genes and genomic regions to suggest a replication cycle comparable to that of HTLV-1 and HTLV-2.     

Molecular epidemiology of human T-lymphotropic virus type II infection in Amerindian and urban populations of the Amazon region of Brazil

Molecular characterization of human T-cell lymphotropic virus II (HTLV-II) isolates in North America and Europe has shown the existence of two principal subtypes of the virus, HTLV-IIa and HTLV-IIb. Subsequent studies on HTLV-II isolates from Brazil have suggested the existence of a unique variant phylogenetically related to HTLV-IIa but phenotypically similar to HTLV-IIb with respect to the transactivatory protein, Tax. This variant has been designated HTLV-IIc. To better clarify the variability and distribution of HTLV-II in Brazil, the viruses present in two population groups from the Amazon region were tested for the presence of HTLV-II using serological and molecular assays. The groups consisted of blood donors from three Amerindian communities and of HIV-1/HTLV-II coinfected patients residing in Belém, an urban area. Nucleotide sequences and phylogenetic analysis confirmed the presence of HTLV-IIc subtype among Amerindian populations and, for the first time, the presence of the same virus among urban groups in Belém. The isolated occurrence of the HTLV-IIc subtype among Amerindian populations in the Amazon region could be attributed to (1) the different migratory pathways and founder effect, or (2) the local origin of a proto-HTLV-II carried by Amerindian ancestors who migrated to the Amazon circa 11,000 to 13,000 years ago. These results suggest that not only is HTLV-IIc unique to this region, but that its presence in urban areas of Brazil has resulted from admixture processes during the colonization of the country.     

Genetic characterization of human T-cell lymphotropic virus type 1 in Mozambique: transcontinental lineages drive the HTLV-1 endemic

Background

Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It has been estimated that 10–20 million people are infected worldwide, but no successful treatment is available. Recently, the epidemiology of this virus was addressed in blood donors from Maputo, showing rates from 0.9 to 1.2%. However, the origin and impact of HTLV endemic in this population is unknown.

Objective

To assess the HTLV-1 molecular epidemiology in Mozambique and to investigate their relationship with HTLV-1 lineages circulating worldwide.

Methods

Blood donors and HIV patients were screened for HTLV antibodies by using enzyme immunoassay, followed by Western Blot. PCR and sequencing of HTLV-1 LTR region were applied and genetic HTLV-1 subtypes were assigned by the neighbor-joining method. The mean genetic distance of Mozambican HTLV-1 lineages among the genetic clusters were determined. Human mitochondrial (mt) DNA analysis was performed and individuals classified in mtDNA haplogroups.

Results

LTR HTLV-1 analysis demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype. Mozambican HTLV-1 sequences had a high inter-strain genetic distance, reflecting in three major clusters. One cluster is associated with the South Africa sequences, one is related with Middle East and India strains and the third is a specific Mozambican cluster. Interestingly, 83.3% of HIV/HTLV-1 co-infection was observed in the Mozambican cluster. The human mtDNA haplotypes revealed that all belong to the African macrohaplogroup L with frequencies representatives of the country.

Conclusions

The Mozambican HTLV-1 genetic diversity detected in this study reveals that although the strains belong to the most prevalent and worldwide distributed Transcontinental subgroup of the Cosmopolitan subtype, there is a high HTLV diversity that could be correlated with at least 3 different HTLV-1 introductions in the country. The significant rate of HTLV-1a/HIV-1C co-infection, particularly in the Mozambican cluster, has important implications for the controls programs of both viruses.     

Retroviral interference on STAT activation in individuals coinfected with human T cell leukemia virus type 2 and HIV-1

Human T cell leukemia virus (HTLV) type-2 is a human retrovirus whose infection has not been tightly linked to human diseases. However, the fairly high prevalence of this infection among HIV-1-positive individuals indicates the importance of better understanding the potential interference of HTLV-2 infection on HIV-1 infection and AIDS. We previously demonstrated that one signature of PBMC freshly derived from HIV-1-infected individuals is the constitutive activation of a C-terminal truncated STAT5 (STAT5Delta). Therefore, we analyzed the potential activation of STATs in HTLV-2 monoinfected and HTLV-2/HIV-1 dually infected individuals. We observed that PBMC of HTLV-2-infected individuals do not show STAT activation unless they are cultivated ex vivo, in the absence of any mitogenic stimuli, for at least 8 h. The emergence of STAT activation, namely of STAT1, in culture was mostly related to the secretion of IFN-gamma. Of note, this phenomenon is not only a characteristic feature of HTLV-2-infected individuals but also occurred with PBMC of HIV-1(+) individuals. Surprisingly, HTLV-2/HIV-1 coinfection resulted in low/absent STAT activation in vivo that paralleled a diminished secretion of IFN-gamma after ex vivo cultivation. Our findings indicate that both HTLV-2 and HIV-1 infection prime T lymphocytes for STAT1 activation, but they also highlight an interference exerted by HTLV-2 on HIV-1-induced STAT1 activation. Although the nature of such a phenomenon is unclear at the present, these findings support the hypothesis that HTLV-2 may interfere with HIV-1 infection at multiple levels.     

5'-long terminal repeat-selective CpG methylation of latent human T-cell leukemia virus type 1 provirus in vitro and in vivo

CpG methylation of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) has been implicated in proviral latency, but there is presently little information available regarding the pattern of LTR methylation and its effect on viral gene expression. To gain insight into the mechanisms of HTLV-1 latency, we have studied methylation of individual CpG sites in the U3-R region of the integrated proviral LTR by using bisulfite genomic sequencing methods. Surprisingly, our results reveal selective hypermethylation of the 5' LTR and accompanying hypomethylation of the 3' LTR in both latently infected cell lines and adult T-cell leukemia (ATL) cells having a complete provirus. Moreover, we observed a lack of CpG methylation in the LTRs of 5'-defective proviruses recovered from ATL samples, which is consistent with the selective hypomethylation of the 3' LTR. Thus, the integrated HTLV-1 provirus in these carriers appears to be hypermethylated in the 5' LTR and hypomethylated in the 3' LTR. These results, together with the observation that proviral gene expression is reactivated by 5-azacytidine in latently infected cell lines, indicate that selective hypermethylation of the HTLV-1 5' LTR is common both in vivo and in vitro. Thus, hypermethylation of the 5' LTR appears to be an important mechanism by which HTLV-1 gene expression is repressed during viral latency.     

Prevalence and genetic characterisation of HTLV-1 and 2 dual infections in patients with pulmonary tuberculosis in Central-West Brazil

Human T-cell lymphotropic virus (HTLV) may impact the clinical course of tuberculosis (TB). Both infections are highly endemic in Brazil. The aim of this study was to assess the prevalence of HTLV-1/2 in TB patients in Central-West Brazil and to perform a genetic characterisation of the respective isolates. Of the 402 patients, six (1.49%) were positive for anti-HTLV and five (1.24%; 95% confidence interval: 0.46-3.05) were infected with HTLV-1/2. Genetic characterisation demonstrated that the four HTLV-1 isolates belonged to the Transcontinental subgroup A of the Cosmopolitan subtype a and that the HTLV-2 isolate belonged to subtype a (HTLV-2a/c). The prevalence of HTLV infection observed in this study is higher than that observed in local blood donors and the HTLV-1 and 2 subtypes identified are consistent with those circulating in Brazil.     

Mutational analysis of human T-cell leukemia virus type 2 Tax.

A mutational analysis of human T-cell leukemia virus type 2 (HTLV-2) Tax (Tax-2) was performed to identify regions within Tax-2 important for activation of promoters through the CREB/ATF or NF-kappaB/Rel signaling pathway. Tax-2 mutations within the putative zinc-binding region as well as mutations at the carboxy terminus disrupted CREB/ATF transactivation. A single mutation within the central proline-rich region of Tax-2 disrupted the transactivation of the NF-kappaB/Rel pathway. Surprisingly, this mutation, which is thought to be in a separate activation domain, was suppressed by mutations within or around the putative zinc-binding region, suggesting an interaction between these two regions. These analyses indicate that the functional regions or domains important for transactivation through the CREB/ATF or NF-kappaB/Rel signaling pathway are similar, but not identical, in Tax-1 and Tax-2. Identification of these distinct Tax-2 mutants should facilitate comparative biological studies of HTLV-1 and HTLV-2 and ultimately lead to the determination of the functional importance of Tax trans-acting capacities in T-lymphocyte transformation by HTLV.     

Alternatively spliced mRNA of the pX region of human T lymphotropic virus type I proviral genome.

The pX region of human T lymphotropic virus type I (HTLV-I) is believed to be expressed as a consequence of a 2 step splicing. It is conceivable, however, that a donor site of the 1st splicing and an acceptor site of the 2nd splicing results in the production of an alternatively spliced mRNA which is capable of coding p21X-III. This possibility was examined by amplifying cDNA derived from HTLV-I+ cells between the 5' LTR and pX region. Bands of 2 different sizes were consistently observed. Sequencing of the longer band corresponded to a cDNA derived from a double-spliced pX mRNA as previously reported. The shorter band was derived from a single-spliced mRNA. HTLV-I+ cell lines had both mRNAs to a varying degree. Expression of p40tax and p21X-III seem to be well correlated with a double-spliced and a single-spliced mRNA, respectively.     

LTR point mutations in the Tax-responsive elements of HTLV-1 isolates from HIV/HTLV-1-coinfected patients

ABSTRACT: BACKGROUND: In Virology Journal 2011, 8:535, Neto et al. described point mutations into Tax-responsive elements (TRE) of the LTR region of HTLV-1 isolates from asymptomatic carriers from Sao Paulo, Brazil, and hypothesized that the presence of the G232A mutation in the TRE-1 increase viral proliferation and consequently the proviral load (PvL), while the A184G mutation in the TRE-2 do not have such effect. FINDINGS: We performed the real-time PCR assay (pol) and sequenced LTR region of HTLV-1 isolates from 24 HIV/HTLV-1-coinfected patients without HTLV-1-associated diseases from the same geographic area. These sequences were classified as belonging to the transcontinental subgroup A of the Cosmopolitan subtype a. The frequency of G232A mutation (16/24, 66.7%) was high as much as 61.8% reported by Neto's in HTLV-1 asymptomatic carriers with high PvL. High frequency (13/24, 54.2%) of double mutations G232A and A184G was also detected in HIV/HTLV-1-coinfected patients. We did not quantify PvL, but comparative analyses of the cycle threshold (Ct) median values of the group of isolates presenting the mutated-types sequences (Ct 33.5, n = 16) versus the group of isolates with the wild-type sequences (Ct 32, n = 8) showed no statistical difference (p = 0.4220). CONCLUSION: The frequencies of mutated-type sequences in the TRE-1 and TRE-2 motifs were high in HIV/HTLV-1-coinfected patients from Sao Paulo, Brazil. If these LTR point mutations have predictive value for the development of HTLV-1-associated diseases or they correspond to the subtype of virus that circulate in this geographic area has to be determined.     

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.     

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avan?ado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.     

Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection.