Genetic and phylogenetic analyses of human T-cell lymphotropic virus type I variants from Melanesians with an without spastic myelopathy

Molecular variants of human T-cell lymphotropic virus type I (HTLV-I) have been isolated recently from lifelong residents of remote Melanesian populations, including a Solomon Islander with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) or HTLV-I myeloneuropathy. To clarify the genetic heterogeneity and molecular epidemiology of disease-associated strains of HTLV-I, we enzymatically amplified, then directly sequenced representative regions of thegag, pol, env, andpX genes of HTLV-I strains from Melanesians with and without TSP/HAM, and aligned and compared these sequences with those of HTLV-I strains from patients with TSP/HAM or adult T-cell leukemia/lymphoma and from asymptomatic carriers from widely separated and culturally disparate populations. Overall, the HTLV-I variant from the Solomon Islander with TSP/HAM, like HTLV-I strains from asymptomatically infected Melanesians, diverged by approx 7% from cosmopolitan HTLV-I strain. No disease-specific viral sequences were found. Gene phylogenies, as determined by the unweighted pair-goup method of assortment and by the maximum parsimony method, indicated that the Melanesian and cosmopolitan strains of HTLV-I have evolved along separate geographically dependent lineages, one comprised of HTLV-I strains from Papua New Guinea and the Solomon Islands, and the other composed of virus strains from Japan, India, the Caribbean, Polynesia, the Americas, and Africa. The total absence of nonhuman primates in Papua New Guinea and the Solomon Islands precludes any possibility that the Melanesian HTLV-I strains have evolved recently from the simian homolog of HTLV-I.     

Lack of tax diversity for tropical spastic paraparesis/human T-cell lymphotropic virus type 1 (HTLV-I) associated myelopathy development in HTLV-I-infected subjects in São Paulo, Brazil

The product of human T-cell lymphotropic virus type 1 (HTLV-1) tax gene has a transactivating effect of the viral and cellular gene expression. Genetic variations in this gene have been correlated with differences in clinical outcomes. Based upon its diversity, two closely related substrains, namely tax A and tax B, have been described. The tax A substrain has been found at a higher frequency among human T-cell leukemia virus type 1 (TSP/HAM) patients than among healthy HTLV-I-infected asymptomatic subjects in Japan. In this study, we determined the distribution of tax substrains in HTLV-I-infected subjects in the city of S?o Paulo, Brazil. Using the ACCII restriction enzyme site, we detected only tax A substrain from 48 TSP/HAM patients and 28 healthy HTLV-I carriers. The sequenced tax genes from nine TSP/HAM patients and five asymptomatic HTLV-I carriers showed a similar pattern of mutation, which characterizes tax A. Our results indicate that HTLV-I tax subtypes have no significant influences on TSP/HAM disease progression. Furthermore, monophyletic introduction of HTLV-I to Brazil probably occurred during the African slave trade many years ago.     

Genetic control and dynamics of the cellular immune response to the human T-cell leukaemia virus, HTLV-I.

About 1% of people infected with the human T-cell leukaemia virus, type 1 (HTLV-I) develop a disabling chronic inflammatory disease of the central nervous system known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Patients with HAM/TSP have a vigorous immune response to HTLV-I, and it has been widely suggested that this immune response, particularly the HTLV-I-specific cytotoxic T-lymphocyte (CTL) response, causes the tissue damage that is seen in HAM/TSP. In this paper we summarize recent evidence that a strong CTL response to HTLV-I does in fact protect against HAM/TSP by reducing the proviral load of HTLV-I. We conclude that HTLV-I is persistently replicating at a high level, despite the relative constancy of its genome sequence. These results imply that antiretroviral drugs could reduce the risk of HAM/TSP by reducing the viral load, and that an effective anti-HTLV-I vaccine should elicit a strong CTL response to the virus. The dynamic nature of the infection also has implications for the epidemiology and the evolution of HTLV-I.     

Tax mutation associated with tropical spastic paraparesis/human T-cell leukemia virus type I-associated myelopathy.

Tumaco, Colombia, is an area with elevated rates of tropical spastic paraparesis/human T-cell leukemia virus type I (HTLV-I)-associated myelopathy (TSP/HAM). We have identified a mutation in nucleotide 7959 of the tax gene of 14 Tumaco HTLV-I isolates (14 positive of 14 tested) that was present in 5 of 14 (35%) TSP/HAM patients from Japan and in 8 of 11 (72%) TSP/HAM patients from other geographic locations. In contrast, this mutation was found in only 2 of 21 (9.5%) HTLV-I-infected subjects outside of Tumaco who did not have TSP/HAM. tax clones with nucleotide mutations including one at nucleotide 7959 showed a greater ability to transactivate the HTLV-I U3 promoter. However, this effect was not observed when two clones that differed only in nucleotide 7959 were compared. These results suggest that HTLV-I-infected individuals carrying isolates with this tax mutation are at higher risk for developing TSP/HAM.     

Complete nucleotide sequence of a highly divergent human T-cell leukemia (lymphotropic) virus type I (HTLV-I) variant from melanesia: genetic and phylogenetic relationship to HTLV-I strains from other geographical regions.

The high prevalences of antibodies against human T-cell leukemia (lymphotropic) virus type I (HTLV-I) reported for remote populations in Papua New Guinea and the Solomon Islands and for some aboriginal populations in Australia have been verified by virus isolation. Limited genetic analysis of the transmembrane portion (gp21) of the envelope gene of these viruses indicates the existence of highly divergent HTLV-I strains in Melanesia. Here, we report the complete nucleotide sequence of an HTLV-I isolate (designated HTLV-IMEL5) from the Solomon Islands. The overall nucleotide divergence of HTLV-IMEL5 from the prototype HTLV-IATK was approximately 8.5%. The degree of variability in the amino acid sequences of structural genes ranged between 3 and 11% and was higher (8.5 to 25%) for the regulatory (tax and rex) genes and the other genes encoded by the pX region. Since HTLV-IMEL5 was as distantly related to HTLV-II as to the other known HTLV-I strains, it could not have arisen from a reocmbinational event involving HTLV-II but rather might be an example of independent viral evolution in this remote population. These data provide important insights and raise new questions about the origin and global dissemination of HTLV-I.     

Modelling the Role of Tax Expression in HTLV-I Persistence in vivo

Human T-lymphotropic virus type I (HTLV-I) is a persistent human retrovirus characterized by life-long infection and risk of developing HAM/TSP, a progressive neurological and inflammatory disease, and adult T-cell leukemia (ATL). Chronically infected individuals often harbor high proviral loads despite maintaining a persistently activated immune response. Based on a new hypothesis for the persistence of HTLV-I infection, a three-dimensional compartmental model is constructed that describes the dynamic interactions among latently infected target cells, target-cell activation, and immune responses to HTLV-I, with an emphasis on understanding the role of Tax expression in the persistence of HTLV-I.     

Multistability in a Model for CTL Response to HTLV-I Infection and Its Implications to HAM/TSP Development and Prevention

Human T-cell leukaemia/lymphoma virus type I (HTLV-I) is a retrovirus that has been identified as the causative agent of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other illnesses. HTLV-I infects primarily CD4⁺ T cells and the transmission occurs through direct cell-to-cell contact. HAM/TSP patients harbor higher proviral loads in peripheral blood lymphocytes than asymptomatic carriers. Also, HAM/TSP patients exhibit a remarkably high number of circulating HTLV-I-specific CD8⁺ cytotoxic T lymphocytes (CTLs) in the peripheral blood. While CTLs have a protective role by killing the infected cells and lowering the proviral load, a high level of CTLs and their cytotoxicity are believed to be a main cause of the development of HAM/TSP. A mathematical model for HTLV-I infection of CD4⁺ T cells that incorporates the CD8⁺ cytotoxic T-cell (CTL) response is investigated. Our mathematical analysis reveals that the system can stabilize at a carrier steady-state with persistent viral infection but no CTL response, or at a HAM/TSP steady-state at which both the viral infection and CTL response are persistent. We also establish two threshold parameters R ₀ and R ₁, the basic reproduction numbers for viral persistence and for CTL response, respectively. We show that the parameter R ₁ can be used to distinguish asymptomatic carriers from HAM/TSP patients, and as an important control parameter for preventing the development of HAM/TSP.     

The presence of HTLV-I proviral DNA in the central nervous system of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis

Human T-lymphotropic virus type 1 (HTLV-I) is a pathogenic retrovirus associated with a chronic progressive myelopathy, termed HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), as well as adult T-cell leukemia (ATL). A chronic inflammatory process has been implicated in HAM/TSP by a pathological study, but the exact mechanism still remains unknown. To understand better the complex mechanism of disease induction by HTLV-I, I studied the spreading pattern of HTLV-I in both peripheral blood mononuclear cells (PBMNCs) and central nervous system (CNS) tissues in patients with HAM/TSP using a quantitative polymerase chain reaction (PCR) method. My results indicated the primary event to be the efficient replication of HTLV-I in vivo, whereas HTLV-I is likely to be present in the constituent cells of the CNS in addition to the infiltrating mononuclear cells.     

The influence of HLA class I alleles and heterozygosity on the outcome of human T cell lymphotropic virus type I infection

The inflammatory disease human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM/TSP) occurs in only 1-2% of HTLV-I-infected individuals and is associated with a high provirus load of HTLV-I. We hypothesize that a person's risk of developing HAM/TSP depends upon the efficiency of their immune response to the virus, which differs between individuals because of polymorphism in genes that influence this response. Previously we showed that the possession of HLA-A*02 was associated with a lower risk of HAM/TSP, and with a lower provirus load in healthy carriers of HTLV-I. However, HLA-A*02 did not account for all the observed difference in the risk of HAM/TSP. Here we present evidence, in the same study population in Japan, that HLA-Cw*08 was also associated with disease protection (probability value, two-tailed test = 0.002) and with a lower proviral load in healthy carriers. Possession of the A*02 and/or Cw*08 genes prevented 36% of potential HAM/TSP cases. In contrast, HLA-B*5401 was associated with a higher susceptibility to HAM/TSP (probability value, two-tailed test = 0.0003) and with a higher provirus load in HAM/TSP patients. At a given provirus load, B*5401 appeared to increase the risk of disease. The fraction of HAM/TSP cases attributable to B*5401 was 17%. Furthermore, individuals who were heterozygous at all three HLA class I loci have a lower HTLV-I provirus load than those who were homozygous at one or more loci. These results are consistent with the proposal that a strong class I-restricted CTL response to HTLV-I reduces the proviral load and hence the risk of disease.     

Increase in T-Cells Bearing CD25 in Bronchoalveolar Lavage Fluid from HAM/TSP Patients and HTLV-I Carriers

To determine the immunologic characteristics of T-cells in local pulmonary lesions of human T-cell lymphotropic virus type I (HTLV-I) carriers, we investigated lymphocyte surface markers in peripheral blood and bronchoalveolar lavage fluid (BALF) of 38 HTLV-I carriers, 8 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 44 HTLV-I seronegative patients with pulmonary diseases and 7 healthy volunteers using two-color flow cytometric analysis. In peripheral blood, activated T-cells, CD4+HLA-DR +, CD8 + HLA-DR + and CD3 + CD25 +, and CD4+CD29+ cells increased significantly in carriers and HAM/TSP patients compared with healthy volunteers and seronegative patients. In BALF, T-cells, especially CD25+ cells, increased significantly in carriers and HAM/TSP patients, compared with healthy volunteers and seronegative patients. These findings indicated that T-cells in the lungs, as well as in peripheral blood, are activated in carriers and HAM/TSP patients. Interestingly, there was dissociation between expression of CD3 + CD25+ cells in BALF and peripheral blood from these patients. These results suggest that T-cells activated probably by HTLV-I accumulate in the lungs in some carriers and HAM/TSP patients, and HTLV-I may be involved in the immunologic dysfunction in the lungs of these patients. However, we did not find any correlation between the degree of clinical features and the elevation of CD3 + CD25+ cells in BALF, or its characteristic features on chest roentgenograms.     

An altered peptide ligand antagonizes antigen-specific T cells of patients with human T lymphotropic virus type I-associated neurological disease

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease associated with HTLV-I infection, in which chronically activated, HTLV-I-specific CD8+ CTL have been suggested to be immunopathogenic. In HLA-A2 HAM/TSP patients, CD8+ HTLV-I-specific CTLs recognize an immunodominant peptide of the HTLV-I Tax protein, Tax11-19. We examined the functional outcome on activation of both cloned peripheral blood and cerebrospinal spinal fluid-derived CTL and bulk PBMC from HAM/TSP patients by altered peptide ligands (APL) derived from HTLV-I Tax11-19. In CTL clones generated from PBMC and CSF of HLA-A2 HAM/TSP patients, an APL substituted at position 5 significantly decreased CTL responses when compared with the native peptide. Moreover, these ligands were also shown to inhibit CTL responses to the native peptide in bulk PBMC of HLA-A2 HAM/TSP patients. These data suggest that a modification of an antigenic peptide at the central position can manipulate the T cell responses in bulk PBMC from different individuals with an inflammatory disease. Additionally, these results have implications for the potential use of APL-based immunotherapy in this T cell-mediated CNS disease.     

Human T cell lymphotropic virus type I (HTLV-I)-specific CD4+ T cells: immunodominance hierarchy and preferential infection with HTLV-I

CD4(+) T cells predominate in early lesions in the CNS in the inflammatory disease human lymphotropic T cell virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), but the pathogenesis of the disease remains unclear and the HTLV-I-specific CD4(+) T cell response has been little studied. We quantified the IFN-gamma-producing HTLV-I-specific CD4(+) T cells, in patients with HAM/TSP and in asymptomatic carriers with high proviral load, to test two hypotheses: that HAM/TSP patients and asymptomatic HTLV-I carriers with a similar proviral load differ in the immunodominance hierarchy or the total frequency of specific CD4(+) T cells, and that HTLV-I-specific CD4(+) T cells are preferentially infected with HTLV-I. The strongest CD4(+) T cell response in both HAM/TSP patients and asymptomatic carriers was specific to Env. This contrasts with the immunodominance of Tax in the HTLV-I-specific CD8(+) T cell response. The median frequency of HTLV-I-specific IFN-gamma(+) CD4(+) T cells was 25-fold greater in patients with HAM/TSP (p = 0.0023, Mann-Whitney) than in asymptomatic HTLV-I carriers with a similar proviral load. Furthermore, the frequency of CD4(+) T cells infected with HTLV-I (expressing Tax protein) was significantly greater (p = 0.0152, Mann-Whitney) among HTLV-I-specific cells than CMV-specific cells. These data were confirmed by quantitative PCR for HTLV-I DNA. We conclude that the high frequency of specific CD4(+) T cells was associated with the disease HAM/TSP, and did not simply reflect the higher proviral load that is usually found in HAM/TSP patients. Finally, we conclude that HTLV-I-specific CD4(+) T cells are preferentially infected with HTLV-I.     

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.     

Detection of HTLV-I in peripheral blood lymphocytes from patients with chronic HTLV-I-associated myelopathy/tropical spastic paraparesis and asymptomatic carriers by PCR-in situ hybridization

Less than 5% of people infected with human T-lymphotropic virus type I (HTLV-I) develop HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive neurologic disease. A number of factors have been implicated in the development of HAM/TSP including heterogeneity of viral sequences, host-genetic background, viral-specific cellular immune responses and viral load. This study examined the presence of HTLV-Itax DNA in peripheral blood lymphocytes (PBL) from 2 chronic HAM/TSP patients and 2 asymptomatic HTLV-I carriers by using PCR-in situ hybridization (PCR-ISH) for the in situ presence of proviral HTLV-Itax DNA. By this technique, rare PBL from these HTLV-I-infected individuals contained HTLV-I DNA. PCR-ISH did not detect any difference in the number of infected cells between HAM/TSP patients and asymptomatic carriers.     

Genetic heterogeneity in human T-cell leukemia/lymphoma virus type II.

DNA from the peripheral blood mononuclear cells of 17 different individuals infected with human T-cell lymphoma/leukemia virus type II (HTLV-II) was successfully amplified by the polymerase chain reaction (PCR) with the primer pair SK110/SK111. This primer pair is conserved among the pol genes of all primate T-cell lymphoma viruses (PTLV) and flanks a 140-bp fragment of DNA which, when used in comparative analyses, reflects the relative degree of diversity among PTLV genomes. Cloning, sequencing, and phylogenetic comparisons of these amplified 140-bp pol fragments indicated that there are at least two distinct genetic substrains of HTLV-II in the Western Hemisphere. These data were confirmed for selected isolates by performing PCR, cloning, and sequencing with to 10 additional primer pair-probe sets specific for different regions throughout the PTLV genome. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-II, while the prototype MoT isolate defines the original substrain. There was greater diversity among HTLV-II New World strains than among HTLV-I New World strains. In fact, the heterogeneity among HTLV-II strains from the Western Hemisphere was similar to that observed in HTLV-I and simian T-cell lymphoma/leukemia virus type I isolates from around the world, including Japan, Africa, and Papua New Guinea. Given these geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV, these data suggest either that HTLV-II has existed for a long time in the indigenous Amerindian population or that HTLV-II isolates introduced into the New World were more heterogeneous than the HTLV-I strains introduced into the New World.     

HTLV-1: A real pathogen or a runaway guest of a diseased cell?

The human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus claimed to be aetiologically linked to the adult T-cell leukaemia/lymphoma (ATLL) and associated myelopathy/tropical spastic paraparesis (HAM/TSP) besides other minor pathologies. HTLV-1 infection is worldwide distributed, despite its heterogeneous prevalence. Environmental factors and host-genetic background are very likely to determine the epidemiological profile of HTLV-1 prevalence and related disease confinement in distinct human ethnic populations and geographical coordinates, which raises the question if the virus is a real pathogen or a runaway well-organized packed genome of a burden host cell near death process. New methodological approaches need to be proposed and applied in order to prove or discard the hypotheses emerged in the present review.     

Human T cell leukemia virus type I and neurologic disease: events in bone marrow, peripheral blood, and central nervous system during normal immune surveillance and neuroinflammation.

Human T cell lymphotropic/leukemia virus type I (HTLV-I) has been identified as the causative agent of both adult T cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the exact sequence of events that occur during the early stages of infection are not known in detail, the initial route of infection may predetermine, along with host, environmental, and viral factors, the subset of target cells and/or the primary immune response encountered by HTLV-I, and whether an HTLV-I-infected individual will remain asymptomatic, develop ATL, or progress to the neuroinflammatory disease, HAM/TSP. Although a large number of studies have indicated that CD4(+) T cells represent an important target for HTLV-I infection in the peripheral blood (PB), additional evidence has accumulated over the past several years demonstrating that HTLV-I can infect several additional cellular compartments in vivo, including CD8(+) T lymphocytes, PB monocytes, dendritic cells, B lymphocytes, and resident central nervous system (CNS) astrocytes. More importantly, extensive latent viral infection of the bone marrow, including cells likely to be hematopoietic progenitor cells, has been observed in individuals with HAM/TSP as well as some asymptomatic carriers, but to a much lesser extent in individuals with ATL. Furthermore, HTLV-I(+) CD34(+) hematopoietic progenitor cells can maintain the intact proviral genome and initiate viral gene expression during the differentiation process. Introduction of HTLV-I-infected bone marrow progenitor cells into the PB, followed by genomic activation and low level viral gene expression may lead to an increase in proviral DNA load in the PB, resulting in a progressive state of immune dysregulation including the generation of a detrimental cytotoxic Tax-specific CD8(+) T cell population, anti-HTLV-I antibodies, and neurotoxic cytokines involved in disruption of myelin-producing cells and neuronal degradation characteristic of HAM/TSP.