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PubMed1993

[Localization of E(wa) in Drosophila melanogaster using rearrangement].

The white-apricot (wa) allele differs from the wild type white gene by the presence of the retrovirus-like transposable element copia within the transcription unit. The activity of wa is reduced in trans by a semidominant mutation in the gene Enhancer-of white-apricot (E(wa)). The map position of E(wa) is refined by analyzing recombination among three genes: px, E(wa) and sp. The recombinational distance between E(wa) and sp is 0.2 cM, which places E(wa) at 2-106.8. To localize E(wa) cytologically, four Y; 2 translocations, one 1;2 translocation and three 2R deficiency stocks were crossed to the suitable E(wa) stocks and the wa sons were scored for intensity of pigment and the speck at the axis of wings. The E(wa) was placed in the distal portion of 60B. The one E(wa) mutation and two sp mutations derived from our gamma-ray mutagenesis were used for examination of polytene chromosome. The former was a revertant which has a cytologically visible insertion of material from an unknown source at 60B5-13, most likely in the 60B8-9. It is likely that this insertion is within the E(wa) gene. The latter shows breakpoint at 60C1-2, where is likely the location of the sp gene. Combining the data of both cytological localization and recombinational mapping, the E(wa) was localized at 60B5-13, most likely in the 60B8-9, the left of sp but very close to it.

Animals↗
PubMed1992

Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction.

Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine.

Animals↗
PubMed1997

Immunologic and virologic characterization of the primary infiltrating cells in the aqueous humor of human T-cell leukemia virus type-1 uveitis. Accumulation of the human T-cell leukemia virus type-1-infected cells and constitutive expression of viral and interleukin-6 messenger ribonucleic acids.

PURPOSE: To characterize immunologically and virologically the infiltrating cells in the aqueous humor of patients with human T-cell leukemia virus type-1 (HTLV-1) uveitis (HU). METHODS: With their informed consent, patients had 0.1 ml of the aqueous humor in the anterior chamber collected with a needle under an operating microscope. An aliquot of the collected sample from patients without steroid therapy was examined by May-Giemsa staining and immunocytochemically. The presence of the HTLV-1-infected cells was investigated by polymerase chain reaction (PCR) using the gag and pol regions of the provirus genome. The population of the infected cells was compared by PCR testing the amplification of the virus genome from 60 cells, or determining the endpoint of successful amplification of the twofold dilution series of the samples, collected from the aqueous humor and peripheral blood mononuclear cells (PBMCs), which were obtained at the same time. Expression of viral and cytokine genes was studied by reverse transcriptase-PCR (RT-PCR). The interleukin-6 (IL-6) level in the aqueous humor of patients with HU and control subjects was measured by a high-sensitivity enzyme-linked immunosorbent assay kit. RESULTS: The number of the infiltrating cells ranged from 475 to 3563 (mean = 2111) per 0.1 ml of aqueous humor, and all the identifiable cells were lymphocytes. Most of them were CD3-positive T cells (mean = 78%), whereas CD4-positive cells constituted less than half (mean = 35.3%). HTLV-1 provirus was detected by PCR in the infiltrating cells of 36 of 38 patients with HU tested, whereas it was detected in 1 of 4 seropositive patients with other entities of uveitis. A higher population of the infected cells in the aqueous humor than in the PBMC was found in seven of nine patients with HU by two independent approaches. Expression of HTLV-1 env or pX genes or both was shown in all 12 patients with HU tested by RT-PCR. IL-6 messenger ribonucleic acid (mRNA) was detected by RT-PCR in 10 of these 12 patients, whereas those of interleukin-1 alpha, interleukin-2, interleukin-4, and tumor-necrosis factor-alpha were not, and that of interferon-gamma was detected in only 1 patient. The IL-6 level was elevated significantly in the aqueous humor of nine patients with HU compared with that of five control subjects (520.2 +/- 841 pg/ml versus 2.77 +/- 1.59 pg/ml, P < 0.01 by Mann-Whitney test). CONCLUSIONS: HU is characterized by lymphocytic infiltration with a predominance of T cells and by the presence and probable accumulation of HTLV-1-infected lymphocytes in the affected eye. Production of viral antigens and IL-6 by the infiltrating cells could be responsible for the development of HU.

Adult↗
PubMed1993

Incidence of HTLV-I/II infection in seronegative high-risk individuals.

The frequency of human T-cell lymphotropic virus types I and II (HTLV-I/II) polymerase chain reaction (PCR) reactivity was studied in two groups of high-risk individuals in Amsterdam: hard drug users and heterosexual outpatients of the sexual transmitted diseases (STD) clinic. Both groups were seronegative as determined by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and radioimmuno-precipitation assay (RIPA). Detection of HTLV-I and HTLV-II in peripheral blood mononuclear cells (MNC) was performed by PCR, using primer sets indicative for the pol and tax genes. In the hard drug users group (n = 25) no evidence of HTLV-I/II infection was found whereas in the STD group (n = 21) one individual was identified with HTLV-II proviral DNA. Positive reactions in PCR were confirmed only for three seropositive controls after in vitro culture of MNC but not for the PCR-positive, seronegative individual. Virus production in vitro could not be detected by a sensitive HTLV-I antigen capture assay for viral p24gag proteins after in vitro T-cell stimulation of MNC, either from PCR-positive or PCR-seronegative individuals. This suggests again a low viral production rate. It is concluded that infection with HTLV-II can be detected among high-risk seronegative individuals.

Adult↗
PubMed1994

Genetic analysis and molecular phylogeny of simian T-cell lymphotropic virus type I: evidence for independent virus evolution in Asia and Africa.

Type C retroviruses, designated simian T-cell lymphotropic virus type I (STLV-I), have been isolated from several genera of Old World monkeys and apes, but not from New World monkeys and prosimians. To determine the genomic diversity and molecular evolution of STLV-I and to clarify their genetic relationship to human T-cell lymphotropic virus type I (HTLV-I), we enzymatically amplified, then directly sequenced selected regions of the gag, pol, env, and pX genes of STLV-I strains from Asia and Africa. STLV-I strains Si-2, Matsu, and JM86 from Japanese macaques, which exhibited sequence similarities ranging from 98.5 to 99.8% among themselves, diverged by 12.9 to 13.3% from STLV-I strain MM39-83 from a naturally infected rhesus macaque, by 9.7 to 11.2% from STLV-I strains from Africa, and by 8.8 to 11.2% from HTLV-I strains originating in Japan, India, Africa, the Caribbean, the Americas, Polynesia, and Melanesia. By contrast, the interspecies nucleotide sequence similarity among African STLV-I strains from green monkey, yellow baboon, sooty mangabey, and common chimpanzee was remarkably high, ranging from 96.9 to 97.4%, and these STLV-I strains diverged by only 2.2 to 2.8% from HTLV-I strain EL from equatorial Zaire. Phylogenetic trees constructed by using the neighbor-joining and maximum parsimony methods indicated that the Asian STLV-I strains diverged from the common ancestral virus prior to African STLV-I and cosmopolitan and Melanesian HTLV-I strains. Thus, our data are consistent with an archaic presence of STLV-I in Asia, probably predating macaque speciation, with subsequent independent virus evolution in Asia and Africa.

Africa↗
PubMed1995

In vivo cellular tropism of human T-lymphotropic virus type II is not restricted to CD8+ cells.

We have examined the in vivo and in vitro susceptibility of lymphocyte subpopulations to human T-lymphotropic virus type II (HTLV-II) to determine the cellular tropism for this virus. Monoclonal antibodies to T-cell subsets were used to separate highly purified CD4+ and CD8+ cells from peripheral blood lymphocytes of 35 individuals previously shown to be infected with HTLV-II. The purified T-cell subsets were analyzed for HTLV-II provirus (pol and tax gene sequences) by polymerase chain reaction (PCR) and cultured to determine virus expression by p24gag antigen detection. On the basis of PCR amplification in the pol and tax gene regions, both CD8+ subsets (89 to 91%) and CD4+ subsets (54 to 80%) from most infected subjects demonstrated HTLV-II provirus, irrespective of the viral genotype. Analysis of cultured lymphocytes demonstrated a higher spontaneous lymphocyte proliferation (17,986 +/- 4675 cpm) and p24gag antigen production (median 115 pg/ml; range 14-1360 pg/ml) in CD8+ cells compared to CD4+ cells (2333 +/- 826 cpm; p24gag antigen; 9 pg/ml; 2-250 pg/ml), suggesting a higher proviral load in CD8 cells. Limiting cell-dilution PCR analysis indicated that the CD8+ subset carried a higher HTLV-II provirus burden than the CD4+ subset. In vitro infection of purified CD4+ and CD8+ lymphocytes with irradiated HTLV-II cell lines resulted in productive infection of both subsets. Cell sorting and PCR analysis of lymphocyte subsets from 4 HTLV-II-infected subjects further demonstrated that in addition to CD4+ and CD8+ subsets, both CD45RO+ and CD45RO- and non-T-cells (CD14, CD16, and CD19) carried HTLV-II provirus. Taken together, these data suggest that HTLV-II possesses a broad tropism for peripheral blood mononuclear cells.

Antibodies, Monoclonal↗
PubMed1996

High frequencies of human T-lymphotropic virus type I (HTLV-I) infection and presence of HTLV-II proviral DNA in blood donors with anti-thyroid antibodies.

To investigate the relationship between human T-lymphotropic virus (HTLV) types I and II and the pathogenesis of autoimmune thyroid diseases, we examined serum anti-thyroid antibodies in 1019 blood donors with or without serum anti-HTLV-I antibody as well as proviral DNA for HTLV-II in leukocyte DNA by the polymerase chain reaction in 395 blood donors with or without anti-thyroid antibodies. The frequency of donors with anti-HTLV-I antibody who also showed anti-thyroid antibodies (7.9%) tended to be higher than that (6.3%) among donors who did not have the anti-HTLV-I antibody. The frequency of anti-thyroid antibodies in 125 young male donors aged 16-39 years with anti-HTLV-I antibody (4.8%) was significantly higher (P < 0.05) than that (0.6%) in 164 control donors without the antibody. In blood donors with anti-thyroid antibody, 25.0% of those with anti-HTLV-I antibody and 14.3% of those without the antibody had HTLV-II proviral DNA. In contrast, in donors without anti-thyroid antibody HTLV-II proviral DNA was detected in 2.3% of those with anti-HTLV-I antibody and in 0.6% of those without the antibody. Thus the detection rates in donors with anti-thyroid antibody were significantly higher (P < 0.001) than those in donors without the antibody, regardless of HTLV-I infection. These results suggest that HTLV-I infection and the presence of HTLV-II proviral DNA may be independently related to the pathogenesis of autoimmune thyroid diseases.

Adolescent↗
PubMed1995

Immune recognition of genetically diverse simian T-cell lymphotropic virus type I isolates.

Nucleotide sequence analysis of selected regions of the gag, pol, env and pX genes of simian T-cell lymphotropic virus type I (STLV-I) strains indicated that African isolates were more closely related to human T-cell lymphotropic virus type I (HTLV-I) than Asian isolates. Despite these recent comparative studies on nucleotide sequence homology between HTLV-I and STLV-I isolates, only limited information is available regarding the influence of genetic differences on antigen-antibody recognition of distinct STLV-I strains. In this study, we demonstrated that sera from STLV-I-infected yellow baboons (Papio cynocephalus) reacted strongly with env gp62/68 from HTLV-I-infected cell lines MT-2 and C10/MJ. In contrast, sera from Japanese macaques (Macaca fuscata) naturally infected with Asian STLV-I had weak reactivity to env gp62/68 of these prototypic HTLV-I strains. Pst-1 restriction enzyme analysis of proviral DNA indicated that the baboon virus isolates were more closely related to HTLV-I than the Japanese isolates. These results indicate that nucleotide sequence diversity, correlates with variations in proviral restriction enzyme sites and antibody recognition of viral envelope proteins. These differences in immunoreactivity may have important implications for serologic diagnosis, as well as epidemiological and vaccine studies of STLV-I infection.

Animals↗
PubMed1993

Reduction of HTLV-I-infected cells in blood by leukocyte filtration.

Leukocyte filtration was performed with HTLV-I-infected blood and with blood supplemented with cultured HTLV-I-transformed cells. Reduction of infectivity upon leukocyte filtration was determined by the polymerase chain reaction (PCR) using primers indicative for the HTLV-I-pol and tax genes. Two different commercially available filters were used: a column-shaped cellulose acetate and a flat-bed polyester filter. Both filters yielded reduction of at least 3 10logs for cultured HTLV-I-infected cells. When blood from HTLV-I-infected individuals was used for filtration, the number of infected cells was reduced by 1-3 10logs. Although filtered blood as yet cannot be regarded as safe, it is concluded that leukocyte filtration of HTLV-I-infected blood potentially contributes to reducing the spread of HTLV-I by blood transfusion.

Blood↗
PubMed1992

The presence of antibodies to purified p24gag protein of HTLV-I in sera of patients with systemic lupus erythematosus (SLE).

Sera of patients with systemic lupus erythematosus (SLE) were tested for their reactivity to HTLV-I by western blotting (WB). Seven (18%) of 40 SLE serum samples reacted to the p24gag protein of HTLV-I by WB using purified gag antigens. The specificity of anti-p24gag antibodies in the SLE sera was confirmed by competitive inhibition on WB. Two of the seven patients were shown to be HTLV-I carriers, because HTLV-I infected T cell lines were easily established from their peripheral blood mononuclear cells (PBMC). Except for these two carrier patients, the gag proteins were not detected in the lysates of PBMC by WB using anti-p24gag and anti-p19gag monoclonal antibodies. The gag and pX genes of HTLV-I were not detected by PCR in PBMC of the SLE patients, with the exception of the 2 HTLV-I carrier patients. These results show no direct involvement of HTLV-I in the etiology of SLE. However, the existence of a specific antibody to p24gag in the sera of some of the noncarrier SLE patients suggests a crossreactivity to either unknown viruses or some autoantigens.

Base Sequence↗
PubMedDate not supplied

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.

Central Nervous System↗
PubMed1992

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.

Actins↗
PubMed1991

Functional conversion from HIV-1 Rev to HTLV-1 Rex by mutation.

A nucleolar localizing rev gene mutant M10 of human immunodeficiency virus type 1 (HIV-1) lost a Rev function completely, instead, gained a Rex activity of human T cell leukemia virus type 1 (HTLV-1). The obtained compatibility between Rev M10 and Rex with their own nucleolar targeting signal (NOS) suggests a common molecular mechanism of their post-transcriptional regulation, despite no sequence similarities of both proteins and their responsive RNA elements, respectively.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a highly divergent HIV-2[GH-2]: generation of an infectious molecular clone and functional analysis of its rev-responsive element in response to primate retrovirus transactivators (Rev and Rex).

A highly divergent HIV-2 designated as HIV-2[GH-2] was obtained from an AIDS-related complex (ARC) patient in Ghana. A full-length molecular clone of this isolate was obtained and a biologically active clone was constructed. Its restriction pattern differed from that of prototype HIV-2[GH-1] in 25 of 35 restriction sites, but was strikingly similar to a previously characterized HIV-2 isolate from a Ghanaian (HIV-2ALT). The conserved integrase region (288-bp fragment) previously displayed 95% identity with that of ALT but 17-20% divergence from the HIV-2 prototype member, and a new distinct subgroup (HIV-2b) of HIV-2 consisting of GH-2 and ALT was postulated (Miura et al. 1991.) These isolates, however, were biologically distinguishable from each other by its replication capacity in a monocyte line, U937, in which GH-2 could not grow but ALT grew well. In addition, the nucleotide sequence of the LTR of this new isolate displays 21% divergence from that of prototype HIV-2[GH-1], but the core enhancer, Sp1 binding sites and TATA box were conserved. Although the 3' half of the env gene sequence which is deleted in HIV-2ALT clone showed 27% diversity from the prototype, functional differences in the rev-responsive element were not observed.

Cloning, Molecular↗
PubMed1994

Prototypical HTLV-I/II infection is rare in LGL leukemia.

The etiology of LGL leukemia is not known; however, we recently detected HTLV-II in a patient with LGL leukemia. In this study, we found that sera from 6 of 28 patients with LGL leukemia were positive for HTLV-I/II using a whole virus ELISA; moreover, the ELISA-negative sera were near the positive cut-off value. Therefore, we performed additional studies on these sera using commercially available assays which can confirm and distinguish HTLV-I from HTLV-II infection. Serum from only one patient was confirmed positive using conventional criteria (HTLV-II+). Sera from 25 patients (89%) had indeterminate reactivity on Western blot assays. Of these, sera from 21 (84%) reacted to gag protein p24; 12 (48%) reacted with recombinant env protein p21e, and 10 (40%) reacted with both. We could not detect HTLV-I/II pol or pX gene sequences in these patients using polymerase chain reaction analyses, with the exception of the HTLV-II-infected patient described previously. These data show that most patients with LGL leukemia are not infected with prototypical HTLV-I or HTLV-II. The frequent reactivity of patient sera to HTLV-I/II gag protein p24 and to env protein p21e, however, suggests that a deleted or variant form of HTLV-I/II may be associated with LGL leukemia.

Blotting, Western↗
PubMed1996

Presence of antibodies to p21X and/or p27rex proteins in sera from human T-cell leukemia virus type I-infected individuals.

The human T-cell leukemia virus type I (HTLV-I) pX gene encodes three nonstructural proteins, p40tax, p27rex and p21X. So far, natural antibodies to p27rex and/or p21X have not been found in sera from HTLV-I-infected individuals, although antibodies to p40tax have been found. Recently, the viral transcripts specific for these proteins were detected in fresh peripheral blood mononuclear cells from HTLV-I-infected individuals by the polymerase chain reaction coupled to reverse transcription, showing the in vivo expression of these proteins. We detected antibodies to p21X and p27rex by an enzyme-linked immunosorbent assay (ELISA) system using a recombinantly produced p21X protein as a common antigen, because p21X is identical to the C-terminal portion of p27rex. The sensitivity of the ELISA was determined to be approximately 100 times greater than that of Western blotting. From the analyzed sera of 31 ATL patients, 30 asymptomatic carriers, 18 HAM patients and 100 healthy donors, three specimens from one ATL patient and two carriers were found to be positive for anti-p21X/p27rex antibodies. The specificity of the ELISA reaction was confirmed by the competitive ELISA test with the highly purified recombinant p21X protein. As of result, we first determined the presence of anti-p21X/p27rex antibodies in a small percentage (3.8%) of the sera from HTLV-I-infected individuals. Even sera from the ATL patients, whose fresh PBMCs contained the transcripts for these proteins, were not found to contain these antibodies, suggesting that the immune response to these proteins is low in HTLV-I-infected humans.

Animals↗
PubMed1998

Quantification of proviral DNA load in human T-cell leukaemia virus type I infections.

A nested PCR was designed using primers from the pol and tax genes of human T-cell leukaemia virus type I (HTLV-I). The assay reliably detected a single copy of HTLV-I proviral genome in DNA from 1 x 10(5) Peripheral blood mononuclear cells (PBMCs). Using serial dilutions of sample DNA, the assay was applied prospectively to study proviral load in patients with HTLV-associated disease and carriers. The median proviral load expressed as number of copies/100 PBMCs was found to be 14.0 copies in patients with HAM and 1.55 copies in initially asymptomatic carriers. The assay was used to test for low proviral load in subjects who may have HTLV-I infection, and to monitor response to therapy.

Cell Line↗