Detection and differentiation of antibodies to human T-cell lymphotropic virus types I and II by the immunofluorescence method.

We compared the sensitivities of the prototype human T-cell lymphotropic virus type I (HTLV-I)- and HTLV-II-transformed cell lines, MT2 and Mo-T, with that of an HTLV-II-infected cell line, clone 19, established in our laboratory, in the immunofluorescence (IF) test for detection of antibody to HTLV-I and HTLV-II. In addition, IF antibody titers with the three antigens were determined, and the results were compared with HTLV-I and HTLV-II typing by polymerase chain reaction (PCR). The MT2 cell line was more sensitive than the two HTLV-II cell lines for detecting HTLV-I antibody by IF, and clone 19 was more sensitive than Mo-T or MT2 for measuring HTLV-II antibody. In the titration study, the antigen that gave the highest titer correlated completely with the HTLV type determined by PCR, indicating that the relatively simple IF titration method can be used for differentiating HTLV-I and HTLV-II antibody in sera and plasmas.     

Detection of human T-cell lymphotropic virus type 1 in plasma samples

Human T-cell lymphotropic virus type 1 (HTLV-1) is an RNA virus responsible for diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATL). Cell-to-cell contact and Tax-induced clonal expansion of infected cells are the main modes of virus replication, making virus detection during the viremic stage difficult. Consequently, the proviral load is the current virologic marker for disease monitoring, but the mechanisms of progression have not been established yet. Thus, this study investigated the presence of virus in plasma from asymptomatic HTLV-1 carriers and from HAM/TSP patients. Real-time PCR was performed on DNA from 150 plasma samples; 12 (8%) had detectable DNA amplification, including 6 (4%) asymptomatic HTLV-1 carriers and 14 (26%) HAM/TSP patients (p < 0.005). Of the 33 samples submitted for nested PCR, six (18%, p = 0.02) were positive for HTLV-1 RNA in the plasma. Additionally, 26 plasma samples were treated with DNAse enzyme to eliminate any DNA contamination before RNA extraction. Two of them (8%) showed amplification for HTLV-1 (p = 0.5). Therefore, this study described for the first time the detection of free HTLV-1 RNA in plasma from HTLV-1-infected subjects, regardless of their clinical status. Thus, HTLV-1 viral replication does occur in plasma, and other transmission pathways for HTLV-1 should be investigated further.     

Variations in Western blot banding patterns of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.

Serum samples from 27 patients infected with human T-cell lymphotropic virus type III (14 with acquired immune deficiency syndrome [AIDS] and 13 with AIDS-related complex) were examined for antibodies to viral proteins by the Western blot method and with four different commercial solid-phase enzyme-linked immunosorbent assays (ELISAs). Virus-specific bands on blots at molecular masses of 64, 55, 53, 41, 31, 24, and 17 kilodaltons were observed. Rank correlation matrices were calculated to relate the intensity of viral bands, stage of illness, and ELISA kit optical densities (ODs). Groups of bands tended to covary in intensity: p17, p24, and p55 (gag gene products); p53 and p64 (pol gene products); and p31 (pol/endonuclease gene product) and p41 (env gene product). Blots of sera from AIDS-related complex patients usually showed strong activity against all viral proteins, while those of sera from AIDS patients characteristically showed strong reactivity only at the pol/endonuclease and env bands. For one ELISA kit (Abbott Laboratories, North Chicago, Ill.), ODs correlated well with the env and pol band intensity scores, while ELISA ODs with other kits (from Litton Industries, Sunnyvale, Calif.; Electro-Nucleonics, Inc., Fairfield, N.J.; and E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.) correlated closely with gag band intensity scores. We conclude that human T-cell lymphotropic virus type III Western blot patterns are determined by (i) viral protein processing pathways and (ii) the stage of illness of the patient and may reflect (iii) the ELISA method used for serum screening.     

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.     

Detection of antibodies to trans-activator protein (p40taxI) of human T-cell lymphotropic virus type I by a synthetic peptide-based assay.

Antibodies to human T-cell lymphotropic virus type I (HTLV-I) trans-activator protein (p40taxI) were determined in serum specimens from individuals infected with HTLV-I (n = 138) and HTLV-II (n = 19). Western blot (immunoblot) analysis using recombinant tax demonstrated the presence of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-I-associated myelopathy, 43% of those (20 of 46) with adult T-cell leukemia, and 61% of asymptomatic HTLV-I blood donors (40 of 66); only one of the HTLV-II specimens reacted with the recombinant tax protein. Synthetic peptides (Tax8(106-125), Tax22(316-335), Tax-23(331-350), and Tax-24(336-353) representing the immunodominant epitopes of¿ p40taxI detected anti-tax antibodies in 66 (48%), 50 (36%), 66 (48%), and 64 (46%) of 138 HTLV-I-positive specimens, respectively. An enzyme immunoassay using an equimolar ratio of these four peptides allowed sensitive detection of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-1-associated myelopathy, 52% of adult T-cell leukemia patients (24 of 46), and 62% of asymptomatic HTLV-1-infected donors (41 of 66). The synthetic peptide-based cocktail assay was HTLV-I specific, since none of the HTLV-II-infected specimens reacted with these peptides. Interestingly, the corresponding regions from the HTLV-II tax protein, Tax8II(106-125), and Tax-22II(312-331) did not react with either HTLV-II or HTLV-I specimens. Thus, a synthetic peptide-based assay composed of immunodominant epitopes located towards the amino terminus and the C terminus of p40taxI provides a reliable and sensitive assay for the detection of anti-tax antibodies in seroepidemiologic studies.     

Effect of using heat-inactivated serum with the Abbott human T-cell lymphotropic virus type III antibody test.

The Abbott enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.) for human T-cell lymphotropic virus type III (HTLV-III) antibody was evaluated to determine the effect of using heat-inactivated (56 degrees C for 30 min) serum as the sample. Each of 58 nonreactive serum samples gave a higher A492 value when tested after heat inactivation. Ten of the samples became reactive after heating. Heat-inactivated serum should not be used in the current Abbott HTLV-III antibody test, because this can cause false-positive results.     

Evaluation of a solid-phase immunoassay for the simultaneous detection of antibodies to human immunodeficiency virus type 1 and human T-cell lymphotropic virus type I.

We describe the evaluation of a solid-phase immunoassay developed for the simultaneous detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) in human serum. The immunoassay employs a mixture of HIV-1 and HTLV-I whole viral lysates immobilized in the wells of microtiter plates. Evaluation of genetically well-pedigreed specimens along with normal blood donor samples indicated that the performance characteristics of the test were equivalent to the sensitivity and specificity of individual tests licensed by the Food and Drug Administration for antibodies to HIV-1 and HTLV-I. Furthermore, the test was also able to detect the presence of cross-reacting antibodies in HTLV-II-infected individuals. The use of such a test would greatly reduce the continually mounting costs associated with screening transfusable products for infectious agents.     

Angiocentric T-cell lymphoma associated with human T-cell lymphotropic virus type I infection

We describe two patients who presented with vasculitic, ulcerative skin lesions that had the histologic features of lymphomatoid granulomatosis or angiocentric T-cell lymphoma. These patients were found to have antibodies to human T-cell lymphotropic virus type I.     

Salivary antibodies as a means of detecting human T cell lymphotropic virus type III/lymphadenopathy-associated virus infection.

Of 45 individuals seropositive for human T cell lymphotropic virus type III/lymphadenopathy-associated virus, 45 were found to have detectable salivary antibodies to viral antigens by a radioimmunoprecipitation assay. The results also showed that a Western blot assay for salivary antibodies may be possible. The feasibility of a diagnostic test for human T cell lymphotropic virus type III/lymphadenopathy-associated virus not requiring venipuncture is discussed.     

Detection and differentiation by sandwich enzyme-linked immunosorbent assay of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus- and acquired immunodeficiency syndrome-associated retroviruslike clinical isolates.

Monoclonal antibodies can be used in sandwich enzyme-linked immunosorbent assays to measure viral antigens. Such an assay was developed to detect the core protein, p24, of human T-cell lymphotropic virus type III and lymphadenopathy-associated virus, etiologic agents of the acquired immunodeficiency syndrome (AIDS). Another AIDS-associated virus, AIDS-associated retrovirus type 2 (ARV-2) could not be detected in this assay because of the low affinity of one of the monoclonal antibodies to ARV-2 p24. Detection of ARV-2 was accomplished with a monoclonal antibody-rabbit polyclonal antibody sandwich enzyme-linked immunosorbent assay. These two assays were used to efficiently detect AIDS-related viruses in lymphocyte cell cultures and to distinguish strains of the viruses.     

Geographical clustering of human T-cell lymphotropic virus type 1 infection in Honduras.

Geographical clustering of human T-cell lymphotropic virus type 1 (HTLV-1) infection has been identified in the nonmestizo communities in several cities along the Atlantic coast of Honduras. Of the 2,651 serum samples tested, 122 samples were repeatedly reactive for HTLV-1 antibodies in two different enzyme immunoassays and 3 were indeterminate. These sera did not react in the HTLV-2-specific antibody tests. The presence of HTLV-1 antibodies was confirmed by HTLV-1 immunoblots or Western blots (immunoblots), and the infection was verified by the detection of HTLV-1-specific genetic sequences in the cellular DNA by PCR. Genomic DNA from the peripheral blood mononuclear cells was first tested with generic primers and probes that identified both HTLV-1 and HTLV-2. Next, all DNA samples that showed HTLV reactivity were tested by PCR with specific primers and probes that distinguished HTLV-1 sequences from those of HTLV-2. Our results indicate that only HTLV-1 infection was present in the blood of both mestizo and nonmestizo residents of 15 cities in the Republic of Honduras. The overall prevalence of HTLV-1 infection in the nonmestizo population was 8.1% (95% confidence limit, 6.6 to 9.7%). The mestizo population residing in the same geographical vicinities showed a HTLV-1 antibodies in 0.5% of serum samples tested (95% confidence limit, 0.6 to 1.7%), indicating a significantly greater prevalence of HTLV-1 infection in the nonmestizo population than in the mestizo ethnic groups living in Honduras (P = 0.0001). Since no HTLV-2 antibody reactivity or HTLV-2-specific genetic sequences were detected by PCR with different primers and probes, it was concluded that HTLV-2 infection was not present in the Honduran population groups we tested. Our study also suggested an endemic nature for this virus because there was no difference in the prevalence rate of HTLV-1 antibodies in the nonmestizo community living in the coastal towns of Honduras between 1989 and 1993. This is the first report of HTLV-1 cluster identification in Honduras, Central America.     

Transmission of human T-cell lymphotropic virus type 1 tax to rabbits by tax-only-positive human cells

The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40(tax). Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40(tax), while they lacked sequences and antibodies related to gag, pol, or env. Thus, it seemed important to determine whether the "tax-only" state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegative tax-only-positive MF patients or from healthy tax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted by tax-only-positive mononuclear cells.     

Quantitative estimation by a standardized enzyme-linked immunosorbent assay of human T-cell lymphotropic virus type I antibodies in adult T-cell leukemia and acquired immune deficiency syndrome.

Sera from patients with adult T-cell leukemia and asymptomatic carriers of human T-cell lymphotropic virus type I (HTLV-I) from widely separated areas of the world reacted strongly in a standardized quantitative enzyme-linked immunosorbent assay procedure with HTLV-I viral antigen prepared from a strain isolated in the United States. There was a sharp differentiation of the values seen in the patients as compared with a normal population. Of the 35 acquired immune deficiency syndrome patients with Kaposi's sarcoma, only 2 were positive for HTLV-I antibodies in this test, and the distribution of the negative assay values in the other acquired immune deficiency syndrome patient sera was similar to that seen in the normal sera. Sera which contained extremely high levels of antibodies to other unrelated viruses (rubella virus, cytomegalovirus, and herpes simplex virus) all showed negative anti-HTLV-I results, in a pattern similar to the normal sera. Sera from patients with several autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, thyroiditis) as well as those with infectious mononucleosis or myeloma all also showed the normal distribution of negative results, in spite of the presence of very high levels of the autoantibodies, etc., associated with their illnesses.     

Effect of heat and fresh human serum on the infectivity of human T-cell lymphotropic virus type III evaluated with new bioassay systems.

MT-4 cells, which are a human T-cell lymphotropic virus type I (HTLV-I)-positive cell line highly permissive to HTLV-III infection, were used to detect the biologically active virus. For quantitation of the virus, induction of HTLV-III-specific antigen(s) and inhibition of DNA synthesis in infected MT-4 cells were assessed by indirect immunofluorescence and by a proliferation assay measuring [3H]thymidine uptake, respectively. HTLV-III was fully inactivated by treatment at 56 degrees C for 30 min. It was not inactivated by treatment with fresh anti-HTLV-III-negative serum. Thus, these assay systems with MT-4 cells would be useful in further studies on acquired immune deficiency syndrome.     

Adenosine deaminase isoenzyme levels in patients with human T-cell lymphotropic virus type 1 and human immunodeficiency virus type 1 infections.

In serum, the enzyme adenosine deaminase (ADA) is known to be divided into two isoenzymes, ADA1 and ADA2, which have different molecular weights and kinetic properties. The present study investigated ADA isoenzyme levels in the sera of patients infected with retroviruses associated with adult T-cell leukemia (ATL), human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM), and AIDS, ADA isoenzyme activities were found to be significantly (P < 0.001) higher in the sera of patients with ATL, HAM, and AIDS than in the sera of healthy controls. In the case of the ADA subtypes in the sera of patients with ATL, ADA1 activity was significantly (P < 0.001) elevated in patients with the acute and lymphoma types of ATL compared with that in patients with the chronic and smoldering types of ATL. ADA2 activity was significantly elevated in the sera of patients with the acute, lymphoma, and chronic types of ATL (P < 0.001) compared with that in patients with smoldering ATL and HTLV-1 carriers. In the case of patients with human immunodeficiency virus type 1 (HIV-1) infection, ADA1 and ADA2 activities in the sera of patients with AIDS and HIV-1 antibody-positive individuals were significantly (P < 0.001) higher than those in the sera of HIV-1 antibody-negative individuals. A significant elevation in ADA2 activity was also seen in the sera of AIDS patients (P < 0.01) compared with that in the sera of HIV-1 antibody-positive individuals. These results suggest that the magnitude of elevation of ADA isoenzyme levels in serum correlates well with the clinical conditions of the patients with these diseases. Measurement of the activities of ADA isoenzymes may therefore provide an additional parameter for distinguishing the subtypes of ATL and may prove to be useful as prognostic and therapeutic monitors in diseases associated with HTLV-1 and HIV-1 infections.     

Genetic characterization and phylogeny of human T-cell lymphotropic virus type I from Chile

Infection with Human T-Cell Lymphotropic Virus type I (HTLV-I) have been associated with the development of the HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phylogenetic analyses of HTLV-I isolates have revealed that HTLV-I can be classified into three major groups: the Cosmopolitan, Central African and Melanesian. In the present study, we analyzed the tax, 5' ltr, gag, pol, and env sequences of proviruses of PBMC from ten HAM/TSP patients to investigate the phylogenetic characterization of HTLV-I in Chilean patients. HTLV-I provirus in PBMC from ten Chilean patients with HAM/TSP were amplified by PCR using primers of tax, 5' ltr, gag, pol, and env genes. Amplified products of the five genes were purified and nucleotide sequence was determined by the dideoxy termination procedure. DNA sequences were aligned with the CLUSTAL W program. The results of this study showed that the tax, 5' ltr, gag, pol, and env gene of the Chilean HTLV-I strains had a nucleotide homology ranged from 98.1 to 100%, 95 to 97%, 98.9 to 100%, 94 to 98%, and 94.2 to 98.5% respect to ATK-1 clone, respectively. According to molecular phylogeny with 5' ltr gene, the Chilean HTLV-I strains were grouped with each other suggesting one cluster included in Transcontinental subgroup.     

Risk of nosocomial infection with human T-cell lymphotropic virus III (HTLV-III)

Infection with human T-cell lymphotropic virus III (HTLV-III) is closely linked to the acquired immunodeficiency syndrome (AIDS). We evaluated the risk of nosocomial infection with HTLV-III by testing for antibodies to HTLV-III among hospital employees, including victims of needle-stick exposure, endoscopists, pathologists, and laboratory workers. Assays for antibody against the virus were performed by enzyme-linked immunosorbent assay and electrophoretic (Western blot) techniques. Although all 22 of our patients with AIDS and 6 of 7 with AIDS-related complex were found to have antibodies to HTLV-III when both assays were employed, none of the 85 employees with nosocomial exposure to specimens from patients with AIDS were positive for HTLV-III antibody. These studies must be regarded as preliminary, but they suggest that when current hospital isolation procedures are employed, the risk of nosocomial transmission of HTLV-III is low.     

Monoclonal antibodies and chemiluminescence immunoassay for detection of the surface protein of human T-cell lymphotropic virus.

Monoclonal antibodies (MAbs) raised against human T-cell lymphotropic virus type I (HTLV-I) recognized five distinct antigenic domains of viral env gene-encoded proteins. By using recombinant env proteins and synthetic peptides as mapping antigens, it was determined that the most immunogenic region represented a central portion of the retroviral surface protein (domain 2; amino acids 165 to 191). However, only a single MAb was able to react strongly with native viral proteins. This antibody (clone 6C2) was directed to an epitope within domain 4 (amino acids 210 to 306) of the retroviral env gene and reacted with envelope proteins in both HTLV-I and HTLV-II, as determined by immunoprecipitation, solid-phase binding, and immunoblotting. No reactivity against envelope components of other human retroviruses, including human immunodeficiency virus types 1 and 2, was present. Flow cytometry data demonstrated that MAb 6C2 reacted with cell lines chronically infected with HTLV-I or HTLV-II and also with surface antigens expressed on fresh adult T-cell leukemia cells, following up-regulation with interleukin-2. By a chemiluminescence immunoassay procedure, picogram amounts of viral surface protein could be detected in the unconcentrated supernatants of HTLV-infected cell lines and in diagnostic cultures. Levels of env and gag proteins released by cells into culture supernatants were not directly related to percent expression of cell surface viral-coat proteins. Further, the molar ratio of p19 to gp46 in conditioned media varied from strain to strain, possibly reflecting differences in viral assembly or packaging mechanisms. MAb 6C2 will be of value in characterizing the biochemical and immunological behavior of retroviral env gene proteins and in studying the interaction of HTLV-I and HTLV-II with their receptors.     

Analysis of human lymphotropic T-cell virus type II-like particle production by recombinant baculovirus-infected insect cells

The molecular processes involved in retrovirus assembly and budding formation remain poorly understood. The gag-pro-pol genes of human lymphotropic T-cell virus type II (HTLV-II) are translated into Gag, Gag-Pro, or Gag-Pro-Pol by frameshift events. In the present study, we investigated the roles of the gag, pro, and pol regions of HTLV-II in viral particle formation using recombinant baculoviruses. In this study we could successfully produce mature HTLV-II viral particles containing core structures using a construct expressing the entire gag-pro-pol region. We also investigated the role of the pol region in particle formation. Deletion of the pol region affects viral particle assembly or release very little, indicating that the gag-pro region is sufficient for viral particle formation and maturation. Expression of the Gag proteins alone or Gag proteins with inactivated viral proteases (Pro) resulted in the formation of viral particles; however, these particles did not contain core structures. These results suggest the intracellular expression of Gag with Pro of HTLV-II is essential for the production of mature virus particles, whereas that of Pol is not.