Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (> or = 8 proviral copies)/1x10(6) non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.     

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.     

Diagnosis of infection with human T-lymphotropic virus type II (HTLV-II) in Norwegian HIV-infected individuals

Sera from 298 HIV-infected individuals from Southern Norway were examined for antibodies against HTLV. 30 sera (10.1%) were HTLV-II positive and 1(0.3%) HTLV-I positive. 25 of the HTLV-II infected subjects were intravenous drug abusers (IVDAs), giving a prevalence of HTLV-II infection of 24.5% in this group. Examination of blood samples by polymerase chain reaction followed by restriction enzyme analysis or sequencing confirmed the serological diagnosis. To evaluate current screening and verification HTLV tests, 44 sera were examined using a gelatin particle agglutination test, 5 different enzyme-linked immunoassays (ELISA) and 4 Western blots (WB). While earlier ELISAs and WBs were inadequate, a recent ELISA and WB including recombinant envelope glycoproteins from both viruses permitted serological diagnosis and distinction between HTLV-I and HTLV-II. Thus, HTLV-II now spreads among IVDAs in a North-European country. Health authorities in other countries should estimate the magnitude of the problem to decide upon measures to avoid transmission through blood transfusion.     

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.     

Molecular detection and isolation of human T-cell lymphotropic virus type I (HTLV-I) from patients with HAM/TSP in São Paulo,Brazil

Background: Infection with HTLV-I is etiologically linked with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However some patients with chronic progressive paraparesis resembling HAM/TSP have been shown to be infected with HTLV-II.Objective: To clarify the role of each of these human retroviruses in the etiology of HAM/TSP in São Paulo, Brazil.Study design: A detailed serological and molecular analysis of HTLV-I/II infection was performed in a cohort of 19 patients with HAM/TSP attending a neurological clinic.Results: Plasma samples analyzed for anti-HTLV-I/II antibodies using a Western blot assay, comprising HTLV-I (rgp46^I)- and HTLV-II (rgp46^II)-specific recombinant env epitopes, demonstrated reactivity to rgp46^I and hence were typed as seropositive for HTLV-I. Presence of HTLV genomic sequences in peripheral blood mononuclear cells (PBMC) was sought after by PCR using consensus primers SK 110 and SK 111 for the pol region of HTLV proviral DNA followed by hybridization with type-specific probes—SK 112 (HTLV-I) and SK 188 (HTLV-II). Southern blots from all individuals hybridized with SK 112 but not with SK 188, further confirming HTLV-I infection. Cocultivation of PBMC from eight of these patients with activated lymphocytes from normal individuals resulted in active viral production, detected as presence of soluble p24^gag antigen in culture supernatants. Investigation of risk factors for HTLV-I infection in these individuals revealed that five out of 19 patients studied (26.3%) had received blood transfusions previous to disease onset.Conclusions: We demonstrate HTLV-I as the only viral type involved in the etiology of HAM/TSP in a cohort from São Paulo, Brazil, and emphasize that prevention measures, including widespread routine screening of blood donations for HTLV should be conducted in Brazil.     

Isolation,characterization, and transmission of human T-lymphotropic virus types I and II in culture

Highly sensitive coculture methods were developed both for isolation of human T-lymphotropic virus types I and II (HTLV-1 and HTLV-II) from infected individuals and for productive infection of lymphoid cells. Mitogen-activated peripheral blood mononuclear cells (PBMC) from 13 HTLV-I- and 20 HTLV-II-positive specimens were cocultured with an equal number of mitogen-activated PBMC from HTLV-seronegative individuals, and culture supernatants were tested for the presence of soluble p24^gag antigens at weekly intervals for 4 weeks. Eleven of 13 (85%) HTLV-I and 14 of 20 (70%) HTLV-II cultures were positive for p24 antigens. None of the 17 HTLV-seroindeterminate or six HTLV-seronegative specimens were positive for the presence of p24 antigen. The isolation rates for HTLV-I and HTLV-II by an alternative whole-blood lysis procedure were comparable to those obtained by standard PBMC cultures. Furthermore, cocultivation of PHA-stimulated PBMC from healthy donors with lethally irradiated HTLV-I- and HTLV-II-infected cell lines (SP and Mo-T, respectively) resulted in productive viral infection, as reflected by the appearance of p24^gag antigens concomitant with specific genomic amplification of HTLV proviral DNA after 3 weeks of cocultivation. Thus, the cocultivation technique provides a highly sensitive and specific procedure both for HTLV isolation and for infection of target cells.     

Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24.

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.     

Risk factors for human T cell lymphotropic virus type I among injecting drug users in northeast Brazil: possibly greater efficiency of male to female transmission

It was observed in the city of Salvador, State of Bahia, the highest seroprevalence of human T cell lymphotropic virus type 1 (HTLV-I) infection in Brazil as demonstrated by national wide blood bank surveys. In this paper, we report results of an investigation of drug use and sexual behavior associated with HTLV-I infection among male and female injecting drug users (IDUs) in Salvador. A cross sectional study was conducted in the Historical District of Salvador from 1994-1996 (Projeto Brasil-Salvador) and 216 asymptomatic IDUs were selected using the snowball contact technique. Blood samples were collected for serological assays. Sera were screened for human immunodeficiency virus (HIV-1/2) and HTLV-I/II antibodies by ELISA and confirmed by Western blot. The overall prevalence of HTLV-I/II was 35.2% (76/216). The seroprevalence of HTLV-I, HTLV-II and HIV-I was for males 22%, 11.3% and 44.1% and for females 46.2%, 10.3% and 74.4% respectively. HTLV-I was identified in 72.4% of HTLV positive IDUs. Variables which were significantly associated with HTLV-I infection among males included needle sharing practices, duration of injecting drug use, HIV-I seropositivity and syphilis. Among women, duration of injecting drug use and syphilis were strongly associated with HTLV-I infection. Multivariate analysis did not change the direction of these associations. Sexual intercourse might play a more important role in HTLV-I infection among women than in men.     

Human T-lymphotropic virus type I seroprevalence among Japanese Americans.

The epidemiology of human T-lymphotropic virus type I (HTLV-I) infection is not well defined in Japanese Americans. This impairs using approaches that could reduce viral transmission and monitor carriers for the disease. Using enzyme-linked immunosorbent assay and p21e recombinant Western blot testing, HTLV-I antibody was measured in unlinked samples from Japanese-American patients at 4 physicians'' offices in San Francisco, California. Of 442 patients, 4 (0.9%; 95% confidence interval 0.25%, 2.3%) were confirmed seropositive, all with an HTLV-I rather than an HTLV-II pattern on Western blot. Seroprevalence was highest among the issei or immigrant generation (3/230 or 1.3%) compared with the second-generation nisei (1/191 or 0.5%) or third-generation sansei (0 of 17). Prevalence did not differ by age or sex, although the number of positive subjects in each subgroup was small. Of 88 patients with familial origins in endemic areas of southern Japan, none were seropositive. In this sample of Japanese Americans, HTLV-I seroprevalence was lower than in residents of endemic southern Japan but higher than among American blood donors. The prevalence was most similar to that in nonendemic areas of Japan. The public health implications of HTLV-I infection among Japanese Americans are discussed.     

HTLV-II-specific antisera raised in rabbits immunized with a synthetic peptide of HTLV-II envelope protein.

In order to discriminate HTLV-II from HTLV-I, HTLV-II-specific polyclonal antibodies against a synthetic peptide of HTLV-II envelope sequence were raised in rabbits. We immunized two adult rabbits with a KLH-conjugated synthetic peptide corresponding to the amino acid sequence 171-196 of the HTLV-II envelope sequence, which is a specific region for HTLV-II as evaluated with an ELISA method. The resulting rabbit antisera to the synthetic peptide reacted with gp46 of HTLV-II lysates in Western blot analysis but not with that of HTLV-I. Flow cytometric analysis and immunohistochemical study revealed that these affinity purified antisera recognized some HTLV-II-producing cell lines examined, but not HTLV-I-producing cell lines or other cell lines uninfected by HTLV. These findings indicate that these antisera specifically recognized the envelope glycoprotein (gp46) of HTLV-II and suggest the specificity of this region in the immune response to HTLV-II. Such antisera are useful in distinguishing between HTLV-I and HTLV-II infection and in determining the presence of individual HTLV-II-infected cells both in vivo and in vitro, including non-lymphoid cells. They may also assist in the elucidation of the pathogenesis of HTLV-II.     

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.     

Mapping of homologous, amino-terminal neutralizing regions of human T-cell lymphotropic virus type I and II gp46 envelope glycoproteins.

Twelve synthetic peptides containing hydrophilic amino acid sequences of human T-cell lymphotropic virus type I (HTLV-I) envelope glycoprotein were coupled to tetanus toxoid and used to raise epitope-specific antisera in goats and rabbits. Low neutralizing antibody titers (1:10 to 1:20) raised in rabbits to peptides SP-2 (envelope amino acids [aa] 86 to 107), SP-3 (aa 176 to 189), and SP-4A (aa 190 to 209) as well as to combined peptide SP-3/4A (aa 176 to 209) were detected in the vesicular stomatitis virus-HTLV-I pseudotype assay. Higher-titered neutralizing antibody responses to HTLV-I (1:10 to 1:640) were detected with pseudotype and syncytium inhibition assays in four goats immunized with a combined inoculum containing peptides SP-2, SP-3, and SP-4A linked to tetanus toxoid. These neutralizing anti-HTLV-I antibodies were type specific in that they did not inhibit HTLV-II syncytium formation. Neutralizing antibodies in sera from three goats could be absorbed with peptide SP-2 (aa 86 to 107) as well as truncated peptides containing envelope aa 90 to 98, but not with equimolar amounts of peptides lacking envelope aa 90 to 98. To map critical amino acids that contributed to HTLV-I neutralization within aa 88 to 98, peptides in which each amino acid was sequentially replaced by alanine were synthesized. The resulting 11 synthetic peptides with single alanine substitutions were then used to absorb three neutralizing goat antipeptide antisera. Both asparagines at positions 93 and 95 were required for adsorption of neutralizing anti-HTLV-I antibodies from all three sera. Peptide DP-90, containing the homologous region of HTLV-II envelope glycoprotein (aa 82 to 97), elicited antipeptide neutralizing antibodies to HTLV-II in goats that were type specific. In further adsorption experiments, it was determined that amino acid differences between homologous HTLV-I and HTLV-II envelope sequences at HTLV-I aa 95 (N to Q) and 97 (G to L) determined the type specificity of these neutralizing sites. Thus, the amino-terminal regions of HTLV-I and -II gp46 contain homologous, linear, neutralizing determinants that are type specific.     

Mapping of immunogenic regions of human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins with env-encoded synthetic peptides and a monoclonal antibody to gp46

Antigenic sites on human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins that are immunogenic in man were studied with envelope gene (env)-encoded synthetic peptides and a mAb to HTLV-I gp46 envelope glycoprotein. Antibodies in 78% of sera from HTLV-I seropositive subjects reacted with synthetic peptide 4A (amino acids 190 to 209) from a central region of HTLV-I gp46. Human anti-HTLV-I antibodies also bound to synthetic peptides 6 (29% of sera) and 7 (18% of sera) from a C-terminal region of gp46 (amino acids 296 to 312) and an N-terminal region of gp21 (amino acids 374 to 392), respectively. mAb 1C11 raised to affinity-purified HTLV-I gp46 reacted with gp46 external envelope glycoprotein and gp63 envelope precursor in immunoblot assay and also bound to the surface of HTLV-I+ cells lines HUT-102 and MT-2. Antibody 1C11 did not react with HTLV-II or HIV-infected cells or with a broad panel of normal human tissues or cell lines. In competitive RIA, anti-gp46 antibody 1C11 was inhibited from binding to gp46 either by antibodies from HTLV-I seropositive subjects or by HTLV-I env-encoded synthetic peptide 4A, indicating that 1C11 bound to or near a site on gp46 within amino acids 190 to 209 also recognized by antibodies from HTLV-I-seropositive individuals. When tested in syncytium inhibition assay, mAb 1C11 did not neutralize the infectivity of HTLV-I. Thus, HTLV-I infection in man is associated with a major antibody response to a region of gp46 within amino acids 190 to 209 that is on the surface of virus-infected cells.     

CD4-independent infection by human immunodeficiency virus type 1 after phenotypic mixing with human T-cell leukemia viruses.

Although human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome and related disorders, it has been suggested that viral cofactors may accelerate the progression of the disease. We present evidence that human T lymphoid cells productively coinfected by HIV type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) or HTLV-II generate a progeny of phenotypically mixed viral particles that allow the penetration of HIV-1 into previously nonsusceptible CD4- human cells, including mature CD8+ T lymphocytes, B lymphoid cells, epithelial cells, and skeletal muscle cells. The infection is independent of the major HIV-1 receptor, (i.e., the CD4 glycoprotein) since OKT4a, a neutralizing anti-CD4 monoclonal antibody, fails to block the penetration of HIV-1. Similarly, infection is not inhibited by monoclonal antibody M77, directed toward the neutralizing loop of the gp120 envelope glycoprotein of HIV-1. In contrast, pretreatment of the virus stock with HTLV-I-neutralizing human serum completely abolishes the penetration of phenotypically mixed HIV-1 into CD4- cells. These results suggest that HTLV-I or HTLV-II may increase the pathogenicity of HIV-1 by broadening the spectrum of its cellular tropism and, thus, favoring its spread within the organism of coinfected hosts.     

The Rex proteins of human T-cell leukemia virus type II differ by serine phosphorylation.

The Rex proteins of human T-cell leukemia virus types I and II (HTLV-I and HTLV-II) induce cytoplasmic expression of unspliced gag-pol mRNA and singly spliced env mRNA and are critical for virus replication. Two rex gene products, p27rex and p21rex of HTLV-I and p26rex and p24rex of HTLV-II, have been detected in HTLV-infected cells; however, the structural and biological relationship of the proteins has not been clearly elucidated. Endoproteinase digestion and phosphoamino acid analysis of HTLV-II Rex indicated that p24rex has the same amino acid backbone as p26rex and that the larger apparent molecular size of p26rex is attributable to serine phosphorylation.     

Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.