Autonomous proliferation of HTLV-CD4+ T cell clones derived from human T cell leukemia virus type I (HTLV-I)-associated myelopathy patients

That HTLV-I infects CD4(+) T cells and enhances their cell growth has been shown as successful long-term in vitro proliferation in the presence of IL-2. It is known that T cells isolated from HAM patients possess strong ability for cell proliferation in vitro and mRNA of various cytokines are abundantly expressed in CNS tissues of HAM patients. Hence, the cytokine-induced proliferation could have an important role in pathogenesis and immune responses of HAM. In this study, we examined the relationship between cell proliferation and ability of in vitro cytokine production of CD4(+) T cell clones isolated from HAM patients. We started a culture from a single cell to isolate cell clones immediately after drawing blood from the patients using limiting dilution method, which could allow the cell to avoid in vitro HTLV-I infection after initiation of culture. Many cell clones were obtained and the rate of proliferation efficiency from a single cell was as high as 80%, especially in the 4 weeks' culture cells from HAM patients. These cells were classified as mainly Th0 phenotype that produce both IFN-gamma and IL-4 after CD3-stimulation. However, the frequency of proviral DNA in these cloned cells was significantly low. Our results indicate that the ability of cell proliferation in HAM patients is not restricted in HTLV-I-infected T cells. HTLV-Iuninfected CD4(+) T cells, mainly Th0 cells, also have a strong ability to respond to IL-2-stimulation, showing that unusual immune activation on T cells has been observed in HAM patients.     

Viral persistence of simian type D retrovirus (SRV-2/W) in naturally infected pigtailed macaques (Macaca nemestrina)

To elucidate sites of SRV-2/W persistence, tissue DNA from three groups of naturally infected Macaca nemestrina was analyzed for provirus: vertically transmitted, viremic, seronegative macaques; horizontally transmitted, viremic, seronegative macaques, and nonviremic seropositive macaques. In viremic animals infected vertically, provirus was found in many tissues, whereas in those infected horizontally, proviral DNA was limited. In V–Ab+ macaques, provirus was detected in bone marrow and/or ileocecal junction, confirming the presence of provirus in V –Ab+ animals.     

Multiple interspecies transmissions of human and simian T-cell leukemia/lymphoma virus type I sequences

Using two sets of nucleotide sequences of the human and simian T-cell leukemia/lymphoma virus type I (HTLV-I/STLV-I), one consisting of 522 bp of the env gene from 70 viral strains and the other a 140-bp segment from the pol gene of 52 viral strains, I estimated cladograms based on a statistical parsimony procedure that was developed specifically to estimate within-species gene trees. An extension of a nesting procedure is offered for sequence data that forms nested clades used in hypothesis testing. The nested clades were used to test three hypotheses relating to transmission of HTLV/STLV sequences: (1) Have cross-species transmissions occurred and, if so, how many? (2) In what direction have they occurred? (3) What are the geographic relationships of these transmission events? The analyses support a range of 11-16 cross-species transmissions throughout the history of these sequences. Additionally, outgroup weights were assigned to haplotypes using arguments from coalescence theory to infer directionality of transmission events. Conclusions on geographic origins of transmission events and particular viral strains are inconclusive due to small samples and inadequate sampling design. Finally, this approach is compared directly to results obtained from a traditional maximum parsimony approach and found to be superior at establishing relationships and identifying instances of transmission.      

Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison

We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease.     

Isolation of a novel simian T-cell lymphotropic virus from Pan paniscus that is distantly related to the human T-cell leukemia/lymphotropic virus types I and II.

An unusual serological profile against human T-cell leukemia/lymphotropic virus type I and II (HTLV-I and -II) proteins was reported in several human Pygmy tribes in Zaire and Cameroon with serum antibodies reactive with gp21 and p24. Here we describe a similar pattern of serum antibodies in a colony of captive pygmy chimpanzees and the isolation of a novel retrovirus, simian T-cell lymphotropic virus from Pan paniscus (STLVpan-p), from the peripheral blood mononuclear cells of several seropositive animals. Cocultures of peripheral blood mononuclear cells from three seropositive pygmy chimpanzees with human cord blood mononuclear cells led to the expression of an HTLV-I- and HTLV-II-related virus initially demonstrated by electron microscopy. Furthermore, several of these cocultures became immortalized T-cell lines expressing the CD4+ CD8+ DR+ phenotype of mature activated T cells. Southern blotting and DNA sequencing of a PCR fragment of viral DNA from these cell cultures demonstrated a distant evolutionary relationship of these viruses to HTLV-I and -II and distinct from the known STLV isolates. We designated this virus STLVpan-p. A genealogical analysis of the captive pygmy chimpanzees colony, originated from wild-caught animals, revealed a prevalence of seropositive offspring from infected mothers, as also observed with HTLVs. The presence in this old African Great Ape species of a virus which is genetically quite distinct from HTLV-I and -II could provide new insights in the phylogenesis of STLVs and HTLVs and be instrumental in the discovery of related human viruses.     

Dynamics of human T-cell lymphotropic virus I (HTLV-I) infection of CD4+ T-cells

Stilianakis and Seydel (Bull. Math. Biol., 1999) proposed an ODE model that describes the T-cell dynamics of human T-cell lymphotropic virus I (HTLV-I) infection and the development of adult T-cell leukemia (ATL). Their model consists of four components: uninfected healthy CD4+ T-cells, latently infected CD4+ T-cells, actively infected CD4+ T-cells, and ATL cells. Mathematical analysis that completely determines the global dynamics of this model has been done by Wang et al. (Math. Biosci., 2002). In this note, we first modify the parameters of the model to distinguish between contact and infectivity rates. Then we introduce a discrete time delay to the model to describe the time between emission of contagious particles by active CD4+ T-cells and infection of pure cells. Using the results in Culshaw and Ruan (Math. Biosci., 2000) in the analysis of time delay with respect to cell-free viral spread of HIV, we study the effect of time delay on the stability of the endemically infected equilibrium. Numerical simulations are presented to illustrate the results.     

Accelerated G(1) phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein.

Tax, the human T cell leukemia virus type I oncoprotein, plays a crucial role in viral transformation and the development of the virally associated disease adult T cell leukemia. Because oncogenesis involves alterations in cell growth, it is important to examine the effects of Tax on cell cycle progression. Using a synchronized cell system, we have found that Tax expression accelerates G(1) phase progression and S phase entry with concomitant DNA replication. This accelerated progression is accompanied by an earlier onset of cdk2 kinase activity. In contrast to the shortening of G(1) phase, the length of S phase is unaffected by Tax expression. As a result of a more rapid cell cycle progression, cells expressing Tax exhibit faster growth kinetics and display an altered cell cycle distribution. Additionally, the decreased time allowed for growth in the presence of Tax results in a decreased cell size. Tax-associated acceleration of cell cycle progression may play a role in the ability of this viral oncoprotein to mediate cellular transformation and promote the development of human T cell leukemia virus type I-associated diseases.     

Human T cell lymphotropic viruses (HTLV-I/II) in South America: Should it be a public health concern?

The presence of the human T cell lymphotropic virus (HTLV-I/II) in South America is well established. Its origin and spectrum in the continent still remain a matter of debate. There are signs now that HTLV-I/II was already present in the Amerindian population coming originally from Asia and that HTLV-I was also introduced with African slave trade and with immigration of individuals from endemic areas of Japan. South America has approximately 350 million inhabitants in its 13 countries. The presence of HTLV-I/II has been reported with impressive numbers in most of them and may be considered endemic in this continent. The distribution of HTLV I/II among native Amerindian populations has shown a geographic clustering of type I in the Andean highlands and Brazilian coast, while type II predominates in lowlands of South America. Although comparability between studies conducted among blood donors in different countries may be difficult, the data indicate that the viruses are also circulating among otherwise healthy individuals. Undoubtedly, HTLV-I/II infection and its related diseases should be considered a public health concern in South America and measures to prevent its spread should be emphasized.     

Phylogenetic associations of human and simian T-cell leukemia/lymphotropic virus type I strains: evidence for interspecies transmission.

Homologous env sequences from 17 human T-leukemia/lymphotropic virus type I (HTLV-I) strains from throughout the world and from 25 simian T-leukemia/lymphotropic virus type I (STLV-I) strains from 12 simian species in Asia and Africa were analyzed in a phylogenetic context as an approach to resolving the natural history of these related retroviruses. STLV-I exhibited greater overall sequence variation between strains (1 to 18% compared with 0 to 9% for HTLV-I), supporting the simian origin of the modern viruses in all species. Three HTLV-I phylogenetic clusters or clades (cosmopolitan, Zaire, and Melanesia) were resolved with phenetic, parsimony, and likelihood analytical procedures. Seven phylogenetic clusters of STLV-I were resolved with the most primitive (deeply rooted) divergence involving several STLV-I strains from Asian primate species. Combined analysis of HTLV-I and STLV-I revealed that neither STLV-I clusters nor HTLV-I clusters recapitulated host species specificity; rather, multiple clades from the same species were closer to clades from other species than to each other. We interpret these evolutionary associations as support for the occurrence of multiple discrete interspecies transmissions of ancestral viruses between primate species (including human) that led to recognizable phylogenetic clades that persist in modern species. Geographic concordance of divergent host species that harbor closely related viruses reinforces that physical feasibility for hypothesized interspecies virus transmission in the past and in the present.     

Evolutionary insights on the origin of human T-cell lymphoma/leukemia virus type I (HTLV-I) derived from sequence analysis of a new HTLV-I variant from Papua New Guinea.

Recent studies have established the presence of human T-cell lymphoma/leukemia virus type I (HTLV-I) in Melanesia. An HTLV-I strain, PNG-1, has now been isolated from a healthy member of the Hagahai, a remote, recently contacted group in Papua New Guinea. To further characterize PNG-1, we employed polymerase chain reaction amplification with subsequent cloning and sequencing of amplified products. Sequence analyses of amplified regions of pol, env, and pX genes of this variant indicate marked heterogeneity (approximately 7%) from prototype HTLV-I. Based on available sequence data, PNG-1 is distinct from all other known HTLV-I strains and diverges from the common ancestor of HTLV-I prior to prototype isolates. The data also suggest, therefore, that HTLV-I originated in the Indo-Malay region rather than Africa.     

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.     

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.     

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.