Seroepidemiology of human T-cell lymphotropic virus type I/II in northeastern Brazil

We investigated the prevalence of human T-lymphotropic virus I/II (HTLV-I/II) infection in Bahia, a state in Northeastern Brazil. Healthy individuals (n = 327) and patients (n = 337) with a variety of diseases were screened for antibodies to HTLV-I/II using an enzyme immunoassay and Western blot. The overall prevalence among healthy subjects was 1.8% (six of 327); among patients it was 18.4% (62 of 337). Patients with AIDS had the highest prevalence of HTLV-I/II infection, 22.7% (20/88), followed by randomly selected patients from an infectious disease hospital, 19.4% (25 of 129), and tuberculosis patients, 11.1% (10 of 90). Four of 14 patients with myelopathy and three of 16 patients with lymphoid leukemia or lymphoma were seropositive for HTLV-I/II. Sixty-three of 68 HTLV-I/II-positive specimens were then typed: 53 patients were HTLV-I positive, three patients were HTLV-II positive, and in seven patients the assay could not distinguish infection by HTLV-I or II. The finding among HIV-seropositive intravenous drug users in Bahia of coinfection with HTLV-I is contrasted with reports from other areas in which dual infection occurs with HTLV-II. Although high prevalence of HTLV-I infection was found in Bahia, the extent and clinical manifestations of HTLV-I/II infection in Brazil remains imprecisely defined, and further studies are needed.     

Simian T-cell lymphotropic virus type 1 from Mandrillus sphinx as a simian counterpart of human T-cell lymphotropic virus type 1 subtype D

A recent serological and molecular survey of a semifree-ranging colony of mandrills (Mandrillus sphinx) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR), env, and rex regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the env gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.     

Comparison of four enzyme immunoassays for detection of human T-cell lymphotropic virus type 2 antibodies

Four licensed enzyme immunoassay (EIA) kits for the measurement of antibody to human T-cell lymphotropic virus (HTLV) type 1, one from Organon Teknika Corp. (OTC), one from Cambridge Biotech Corp. (CBC), and two from Abbott Laboratories (the 1993 modification [Abb 93] and the 2.0 version licensed in 1995 [Abb 95]), were evaluated for sensitivity and specificity in the detection of HTLV type 2 antibody, and the results were compared with those previously obtained with earlier kit versions. The CBC, Abb 95, Abb 93, and OTC kits had sensitivities of 99.7, 97.6, 96.8, and 96.2%, respectively, compared with sensitivities of 89.1 and 60% for the Abbott and CBC (previously DuPont) kits, respectively, licensed in 1988. Thus, the abilities of commercial kits to detect HTLV antibody have improved. The relative specificities of the CBC, Abb 95, Abb 93, and OTC kits with negative blood donor specimens that had been reactive with the 1988 CBC EIA kit were 92.9, 64.5, 78.8, and 62.6%, respectively. Compared with those of the 1988 versions, the specificity of the Abbott EIA has decreased and the specificity of the CBC kit has been significantly improved.     

Immunocytochemical localization of the endogenous neuroexcitotoxin quinolinate in human peripheral blood monocytes/macrophages and the effect of human T-cell lymphotropic virus type I infection

Quinolinate (Quin), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that appears to act through the N-methyl-D-aspartate receptor system, was localized in cultured human peripheral blood monocytes/macrophages (PBMOs) by using a recently developed immunocytochemical method. Quin immunoreactivity (Quin-IR) was increased in gamma interferon (IFN-gamma)-stimulated monocytes/macrophages (MOs). In addition, the precursors, tryptophan and kynurenine, significantly increased Quin-IR. Infection of MOs by human T-cell lymphotropic virus type I (HTLV-I) in vitro substantially increased both the number of Quin-IR cells and the intensity of Quin-IR. At the peak of the Quin-IR response, about 40% of the cells were Quin-IR positive. In contrast, only about 2-5% of the cells were positive for HTLV-I, as detected by both immunofluorescence for the HTLV-I antigens and PCR techniques for the HTLV-I Tax gene. These results suggest that HTLV-I-induced Quin production in MOs occurs by an indirect mechanism, perhaps via cytokines produced by the infection but not directly by the virus infection per se. The significance of these findings to the neuropathology of HTLV-I infection is discussed.     

Progressive myelopathy caused by human T-cell lymphotropic virus type I (HTLV-I) in 3 patients in The Netherlands]

Liability to neural tube defects is increased by maternal dietary deficiency, and children with neural tube defects show a possibly related seasonal variation in date of birth. Since maternal dietary insufficiency may also increase liability to cleft lip, with or without cleft palate (CLP), we wondered if children with CLP would also show a seasonal variation in birth date. The multifactorial-threshold model predicts that any such effect would be more apparent in males than females, since CLP is more frequent in males. Month of birth was obtained from records of 598 children with CLP seen at The Montreal Children's Hospital between 1950 and 1996. Children with syndromes or associated malformations were excluded. There was a significant tendency for children with CLP to be born more often in the summer than in winter. The difference was greater in males than in females. The seasonal fluctuation in month of birth of children with CLP is consistent with the presence of an environmental factor increasing liability, with a maximal effect in November-December. This might be related, at least in part, to a seasonal fluctuation in maternal nutrition. The data support the prediction that analyzing the data for the sexes separately would amplify the effects of variation in liability for a multifactorial threshold trait that has a different frequency in males and females. This approach could be useful in the study of other gene-environment interactions.     

71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human T-cell lymphotropic virus type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and beta-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-beta-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

The humoral immune response to human T-cell lymphotropic virus type 1 envelope glycoprotein gp46 is directed primarily against conformational epitopes

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.     

A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.     

Substrates and inhibitors of human T-cell leukemia virus type I protease

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.     

Induction of tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic virus type 1 Tax

To examine the role of human T-lymphotropic virus type 1 (HTLV-1) Tax1 in the development of neurological disease, we studied the effects of extracellular Tax1 on gene expression in NT2-N cells, postmitotic cells that share morphologic, phenotypic, and functional features with mature human primary neurons. Treatment with soluble HTLV-1 Tax1 resulted in the induction of tumor necrosis factor alpha (TNF-alpha) gene expression, as detected by reverse-transcribed PCR and by enzyme-linked immunosorbent assay. TNF-alpha induction was completely blocked by clearance with anti-Tax1 monoclonal antibodies. Furthermore, cells treated with either a mock bacterial extract or with lipopolysaccharide produced no detectable TNF-alpha. Synthesis of TNF-alpha in response to soluble Tax1 occurred in a dose-dependent fashion between 0.25 and 75 nM and peaked within 6 h of treatment. Interestingly, culturing NT2-N cells in the presence of soluble Tax1 for as little as 5 min was sufficient to result in TNF-alpha production, indicating that the induction of TNF-alpha in NT2-N does not require Tax1 to be continually present in the culture medium. Treatment of the undifferentiated parental embryonal carcinoma cell line NT2 with soluble Tax1 did not result in TNF-alpha synthesis, suggesting that differentiation-dependent, neuron-specific factors may be required. These results provide the first experimental evidence that neuronal cells are sensitive to HTLV-1 Tax1 as an extracellular cytokine, with a potential role in the pathology of HTLV-1-associated/tropical spastic paraparesis.     

Human T cell lymphotropic virus type I does not increase human immunodeficiency virus viral load in vivo

Human T lymphotropic virus type I (HTLV-I) can increase human immunodeficiency virus (HIV) replication in vitro, and several studies suggest that HTLV-I accelerates the progression of HIV infection. To determine whether HTLV-I enhances HIV replication in vivo, a case-control study was done of serum HIV viral load, using polymerase chain reaction, in 23 subjects with HTLV-I/HIV coinfection and 92 control subjects with HIV single infection. The geometric mean serum RNA level was 11,482 copies/mL in the coinfected group and 13,804 in the single-infection group (P = .57), a result that did not change after adjustment for zidovudine use and CD4 cell count. Among subjects with advanced HIV infection, there was a trend toward higher viral load among singly infected subjects. HTLV-I did not appear to increase HIV plasma RNA levels in subjects with coinfection. These results do not provide a biologic basis for the hypothesis that HTLV-I accelerates the course of HIV infection.     

Defective human T-cell lymphotropic virus type I (HTLV-I) provirus in 10 Chilean seronegative patients with tropical spastic paraparesis or HTLV-I-associated myelopathy

We studied the presence of tax and ltr genes from human T-cell lymphotropic virus type I (HTLV-I) provirus in the peripheral blood mononuclear cells from 15 seronegative patients with tropical spastic paraparesis or HTLV-I-associated myelopathy by PCR. Only a region of the tax gene from 10 patients was amplified. The nucleotide homologies of six Chilean isolates to the ATK-1 clone ranged between 98.7 and 99.4%.     

Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome

Two principles are presented that describe directional confounds associated with baseline differences: Principle 1: Change scores are confounded with baseline whenever data are skewed. Principle 2: When baseline differences are real, ANCOVA has a directional bias that magnifies differences in one direction and masks those in the other direction. Both principles involve a directional bias that is related to the direction of baseline difference and the direction of the hypothesized difference in change. Ethical dilemmas arise if decisions (whether or not to transform, whether or not to use ANCOVA) are chosen in order to maximize power by capitalizing on the directional bias and Type 1 error.     

Accumulation of human T lymphotropic virus type I-infected T cells in the salivary glands of patients with human T lymphotropic virus type I-associated Sj?gren's syndrome

OBJECTIVE: To clarify the involvement of human T lymphotropic virus type I (HTLV-I) in the pathogenesis of Sjogren's syndrome (SS). METHODS: In HTLV-I-seropositive patients with SS, HTLV-I proviral DNA in the labial salivary glands (SG) was detected by polymerase chain reaction (PCR) amplification of the extracted cellular DNA, and the localization in the SG was examined by in situ PCR hybridization. RESULTS: The cellular DNA extracted from the SG contained full HTLV-I proviral DNA, which was present in the nucleus of the infiltrating T cells, but not in either the SG epithelial cells or the acinar cells. Furthermore, the viral loads in the SG were approximately 8 times to 9 x 10(3) times higher than those in the peripheral blood mononuclear cells. CONCLUSION: Accumulation of HTLV-I-infected T cells in the SG suggests that HTLV-I likely causes the self-reactive T cells to proliferate, which, as a result, induces SS.     

Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli

The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.