Evidence for exposure to HTLV-III in Uganda before 1973.

Fifty of 75 serum samples collected in the West Nile district of Uganda between August 1972 and July 1973 contained antibodies reactive with human T-cell leukemia (lymphotropic) virus type 3 (HTLV-III; mean titer, 601), while 12 of 75 samples were positive in a similar test for HTLV type 1 (HTLV-1) antibodies (mean titer, 236). The samples were screened by enzyme-linked immunosorbent assay and positive results were confirmed by a newly developed unlabeled antibody-peroxidase procedure with enhanced sensitivity for detection of antibody binding to immunoblots of HTLV-III antigen, demonstrating antibodies to proteins with molecular weights of 24,000, 41,000, and 76,000 in nearly all positive samples. Analysis of titration data indicated enhanced titers of antibody against HTLV-III and HTLV-I when coinfection occurred. The high prevalence and relatively low titers [compared to serum from patients with acquired immune deficiency syndrome (AIDS)] of antibodies recognizing HTLV-III proteins in sera from this population at a time that may predate or coincide with the appearance or spread of the AIDS agent (HTLV-III) suggest that the virus detected may have been a predecessor of HTLV-III or is HTLV-III itself but existing in a population acclimated to its presence. It further suggests an African origin of HTLV-III.

Transmission of HTLV-III infection from human plasma to chimpanzees: an animal model for AIDS.

Two of three chimpanzees given plasma from patients with acquired immune deficiency syndrome (AIDS) or pre-AIDS showed serum antibodies to type III human T-cell leukemia virus (HTLV-III) 10 to 12 weeks after transfusion. One animal also developed lymphadenopathy, transient depression of the ratio of T4 to T8 lymphocytes, and impaired blastogenic responses. No opportunistic infections occurred. Adenopathy persisted for 32 weeks, and antibody to HTLV-III persisted for at least 48 weeks. This transmission of HTLV-III by lymphocyte-poor plasma confirms the potential risk of such plasma or plasma derivatives to recipients. The susceptibility of the chimpanzee to HTLV-III infection and the ability to simulate the human lymphadenopathy syndrome in this animal makes it a valuable model for further study of AIDS.

Lack of expression of type C hamster virus after neoplastic transformation of hamster embryo fibroblasts by benzo(a)pyrene.

Syrian hamster embryo fibroblasts transformed in vitro with benzo(a)pyrene were analyzed for the presence of type C viral components, including extra- and intracellular reverse transcriptase activity, intracellular type C hamster virus-related RNA, and cellular hamster virus group-specific antigen. No evidence could be obtained for the presence of any of these components, although they were easily detectable in hamster fibroblasts producing either B-34 virus (a hamster virus pseudotype of Harvey murine sarcoma virus which contains an excess of helper type C hamster virus) or Harvey virus itself. In addition, intracellular viral RNA could not be detected in normal hamster embryo fibroblasts, in hamster fibroblasts transformed with simian virus 40, or in newborn hamster kidney and liver. Thus the detectable expression of the indigenous hamster type C virus is not required to maintain the transformed phenotype of these cells.

Antibodies to human herpes virus-6 in patients with acute lymphocytic leukemia.

Human herpesvirus-6 (HHV-6), a ubiquitous virus that causes exanthem subitum and occasional cases of infectious mononucleosis, hepatitis and other viral syndromes, has also been associated with acute lymphocytic leukemia (ALL) in children. To further investigate this association, we obtained sera from 50 patients with ALL and 50 age-sex matched controls. Antibodies to HHV-6 were determined using ELISA and indirect immunofluorescent antibody (IFA) tests. No significant difference between antibody titers in the cases and controls was observed. Since seroepidemiologic studies have demonstrated higher HHV-6 antibody titers in young children than in adults, this serologic study suggests that the previous association reported for HHV-6 and ALL was a result of the age of the population rather than a relationship between the virus and the disease.

Cross-sectional study of immunologic abnormalities in intravenous drug abusers on methadone maintenance in New York City.

One hundred and ninety-nine patients with a history of intravenous drug abuse, and enrolled on the St Luke's-Roosevelt Hospital Center Methadone Program, had baseline evaluations performed from September 1984 to April 1987. The study was designed to examine immunologic parameters associated with HIV seropositivity and those predictive of progression to AIDS-related complex (ARC) and AIDS. Sixty-four patients (32%) had antibodies to HIV by enzyme-linked immunosorbent assay (ELISA), with confirmation by Western blot and none of these patients had ARC or AIDS at the time of initial evaluation. The mean values for white blood-cell count, absolute lymphocyte count, proportion and absolute CD4, and CD4/CD8 ratio were decreased significantly in the HIV-seropositive group compared with the HIV-seronegative group. On the other hand, levels of circulating beta 2-microglobulin, SCD8, SIL-2R, and HIV p24 antigen were significantly elevated in the HIV-seropositive group compared with the HIV-seronegative group.

Counterimmunoelectrophoresis for detection of human serum antibody to HTLV-III.

We examined the usefulness of a counterimmunoelectrophoresis (CIE) technique for detecting antibodies to HTLV-III using sera that previously had been assessed for antibodies to HTLV-III by the standard enzyme-linked immunosorbent assay (ELISA). We selected a subset of 53 sera from patients with the acquired immune deficiency syndrome (AIDS) or the generalized lymphadenopathy syndrome (GLS) in which 81.1% were initially ELISA-positive, and 96.2% were positive by Western blot technique. In our standard HTLV-III CIE technique, 58.5% were positive and repeat testing increased the yield to 67.9%. Varying several parameters of the standard CIE assay did not improve sensitivity. We also studied 20 ELISA-negative and 10 ELISA-borderline sera from normal controls; all were negative by CIE. These results indicate that CIE may be used for detection of human serum antibodies to HTLV-III, but that the present assay was less sensitive than the HTLV-III ELISA.

Subgroup G HIV type 1 isolates from Nigeria.

PIP: Nucleotide sequences were obtained for portions of the envelope and gag genes of 4 HIV-1 isolates from Nigeria. The gag gene sequences clustered with previously described gag sequences from Gabon, Zaire, and Taiwan, which form the G clade of HIV-1. This documented for the first time the presence of subgroup G viruses in Nigeria. The env gene sequences were most closely related to env sequences reported from 2 isolates previously classified as gag subgroup G and, with those 2 env sequences, defined the subgroup G env genotype. Virus isolates were obtained by cocultivation of peripheral blood mononuclear cells (PBMCs) from Nigerian AIDS patients (isolates JV1018 and JP88) or healthy prostitutes (G3 and G9) with normal uninfected PBMCs (JV1083, JP88, and G3) or GEM-SS, a T cell line (G9). Clones containing env and gag gene sequences were obtained by polymerase chain reaction amplification, using DNA from these cultures. The region of gag amplified by primers SK37 and SK39 containing BamHI and KpnI sites, respectively, in 5' tails, was cloned into the KpnI-BamHI site of pKSBluescript and sequenced. When the gag sequences from 2 of the Nigerian viruses were subtyped by weighted parsimony, both viruses grouped with others of the gag G subgroup. A similar analysis of env sequences from all 4 Nigerian isolates showed that they were most closely related to 2 env sequences that had been assigned to env subgroup G. The Nigerian env sequences clustered more closely with each other than they did with the other 2 subgroup G viruses. Consensus of the inferred amino acid sequences of the 4 env genes showed that the V3 loop closely resembled the A to F consensus. 8 positions in the Nigerian consensus sequence appeared to be unique. However, whether these are signature residues for Nigerian subgroup G isolates or for subgroup G isolates will require the characterization of more isolates.^ieng

Seroepidemiology of human T cell lymphotropic virus in the Republic of Panama.

The human T-lymphotropic virus (HTLV) and associated diseases, adult T cell leukemia and spastic paraparesis, appear to be endemic in southwestern Japan and the Caribbean. This cross-sectional population-based study was conducted to describe the seroepidemiology of HTLV in the Republic of Panama. HTLV antibody was measured by first generation and commercial ELISA tests and confirmed by competitive binding ELISA, a radioimmunoassay for anti-p 24, and Western blot. Of 3,231 subjects greater than or equal to 15 years of age, 135 (4.2%) had antibody detected in ELISA screening tests, but because only 20% were confirmed positive, HTLV seroprevalence varied from 0.2-2% throughout the Republic. Infection with HTLV clustered in Guaymi Indians living in Bocas del Toro province (9.9% prevalence rate). With the exception of Guaymi Indians, no major geographic, urban/rural, male/female or racial differences in antibody prevalence were observed; specifically, HTLV infection rates were not elevated in black Panamanians. Clustering of infection in an isolated Amerind population must be further investigated. The small proportion of screen-positive sera which confirmed positive illustrates the importance of strict uniform criteria for seropositivity.

A simple method for measurement of cell-substrate attachment forces: application to HIV-1 Tat.

In order to understand the importance of cell attachment to HIV-1 Tat, we quantified the strength of cell attachment to immobilized Tat in microtiter plate wells by the application of buoyant force. By replacing the attachment medium with dense medium, and subjecting the attached cells in the microtiter plates to centrifugal force in the conventional upright position, weakly binding and strongly binding cells could be discriminated (and separated) by varying the centrifugal speed. The strength of attachment of HT1080 cells to Tat was compared with that of the well-known extracellular matrix (ECM) proteins fibronectin and vitronectin. We observed that all three proteins mediated significant attachment of HT1080 cells both at 4 degrees C and 37 degrees C. However, unlike the ECM proteins, Tat was unable to engage in higher strength binding when the temperature was raised to 37 degrees C. The relatively weak binding of HT1080 cells to Tat (in the order of 3.0 mudynes/picomole of coated Tat) and lack of strengthening of binding to Tat at physiologic temperature suggests that this protein does not mimic adhesion molecule function. We anticipate that the methodology developed and described here will be useful in a wide variety of cell-matrix and cell-cell interaction studies.

Nucleic acid hybridization with RNA immobilized on filter paper.

RNA has been immobilized in a manner suitable for use in molecular hybridization experiments with dissolved RNA or DNA by a nonaqueous solid-phase reaction with carbonyldiimidazole and RNA "dry coated" on cellulose or, preferably, on previously activated phosphocellulose filters. Immobilization of RNA does not appear to alter its chemical character or cause it to acquire affinity for unspecific RNA or DNA. The versatility and efficiency of this method make it potentially attractive for use in routine analytical or preparative hybridization experiments, among other applications.

Evolutionary nature of human reverse transcriptase and of viral-related DNA synthesized in vitro by human leukemic cells.

The reverse transcriptase and endogenous DNA product synthesized by virus-like particles in the cytoplasm of human leukemic cells have been studied for their genetic relatedness to homologous components obtained from several animal RNA tumor viruses. The human reverse transcriptase activity was inhibited by antibodies prepared against reverse transcriptase from some animal RNA tumor viruses. The DNA molecules synthesized endogenously by the human cytoplasmic particle in the presence of actinomycin D, using the reverse transcriptase enzyme and RNA template residing in the particle, hybridized to 70S RNA purified from certain animal RNA tumor viruses. Both the human reverse transcriptase and DNA product are closely related to homologues from primate type-C viruses, more distantly related to those from murine type-C viruses, and essentially unrelated to similar structures from feline or avian type-C viruses. They are not related to type-B RNA tumor viruses. The results demonstrate that the components from the human leukemic cells are viral (type-C) and primate in nature.

Primate RNA tumor virus-like DNA synthesized endogenously by RNA-dependent DNA polymerase in virus-like particles from fresh human acute leukemic blood cells.

A particle of discrete biochemical composition was purified from fresh, unfrozen peripheral blood leukocytes of human patients with acute myeloblastic leukemia. This particle endogenously synthesized DNA by use of an RNA primer and template. About half of the DNA sequences synthesized in the presence of actinomycin D hybridized to RNA isolated from type-C sarcoma viruses of primates or mice; lower annealing values were obtained with RNA isolated from other sarcoma or leukemia viruses. The results confirm and extend previous results from molecular hybridization experiments related to the existence in human leukemia of components of RNA tumor viruses.

Occurrence of HTLV-I antibodies in Danish patients with cutaneous T-cell lymphoma.

10 of 68 CTCL (cutaneous T-cell lymphoma) patients without features of ATLL had antibodies against HTLV-I (human T-cell leukaemia virus, type I). The titre of antibody in these positive patients was generally much lower than that seen in cases of ATLL (adult T-cell leukaemia/lymphoma); geometric mean of 80 for CTCL vs. 8000 for Caribbean ATLL. The presence of HTLV-I antibody was unrelated to clinical remission, relapse, or stages of the disease, and some positives were detected in the earliest phases of mycosis fungoides. Among controls and normal donors between the ages of 40 and 65, only 1 of 36 and 3 of 113, respectively, had low titre antibodies to HTLV-I in their sera. Only 5 of 354 Danish normal donors of all ages had antibody, which was identical to the rate in over 2000 US normal donors. In negative control experiments, these antibodies were unreactive with bovine leukaemia virus. These data suggest that HTLV-I or a related retrovirus crossreactive with HTLV-I occurs in a low percentage of the Danish population and patients with CTCL have such antibodies at an increased rate, but less than the rate seen for ATLL (greater than 90%).

Production of virions with retrovirus morphology by human embryonal carcinoma cells in vitro.

Embryonal carcinoma (EC) cell lines established from human testicular germ-cell tumours produce, at low frequency, virions morphologically identical to type C retroviruses that have been observed by other workers in human placental tissues. The virus particles are formed while budding from the cell surface, and their numbers are increased by inducing the EC cells with 5-iodo-2'-deoxyuridine and dexamethasone. Assays for RNA-dependent DNA polymerase (reverse transcriptase) associated with purified virions suggested a low level of activity. In addition, another type of virus is occasionally produced by induced cells of three EC lines. These particles also form during the process of budding from the cell surface, but they have surface projections (spikes). Extracellular spiked virions frequently are pleomorphic, with a condensed, eccentric nucleoid, and thus morphologically resemble type B retroviruses. No virions of either type were detected with or without induction in cultures of differentiated EC cells or in cultures of yolk sac carcinoma or teratoma cells, both of which are considered malignant but differentiated derivatives of EC cells. The lack of virion production by these differentiated cells suggests developmental regulation of virus replication.