HTLV-III in saliva of people with AIDS-related complex and healthy homosexual men at risk for AIDS.

Peripheral blood leukocytes and saliva from 20 individuals, including four with the acquired immune deficiency syndrome (AIDS), ten with AIDS-related complex (ARC), and six healthy homosexual males at risk for AIDS, were compared as sources of transmissible human T-cell leukemia (lymphotropic) virus type III (HTLV-III), the virus found to be the etiologic agent of AIDS. All of the AIDS and ARC patients and four of the six healthy homosexuals had evidence of prior exposure to HTLV-III as indicated by seropositivity for antibody to HTLV-III structural proteins. Infectious virus was isolated from the peripheral blood of one of the AIDS patients, four of the ARC patients, and two of the healthy homosexual males, consistent with previous reports. HTLV-III was also isolated from the saliva of four of the ARC patients and four of the healthy homosexuals. Virus was also observed by electron microscopy in material prepared by centrifugation of the saliva of one AIDS patient. Although AIDS does not appear to be transmitted by casual contact, the possibility that HTLV-III can be transmitted by saliva should be considered.

Advances in the isolation of HTLV-III from patients with AIDS and AIDS-related complex and from donors at risk.

During the last 2 yr more than 100 independent isolates of human T-cell leukemia virus type III have been obtained in this laboratory. Most isolates were from peripheral blood T-lymphocytes established in cell culture from acquired immunodeficiency syndrome (AIDS) and acquired immunodeficiency syndrome-related complex (ARC) patients and healthy donors at risk for AIDS. Several were also obtained from leukocytes from bone marrow, lymph node, and brain tissue and from body excretions, e.g., saliva and semen. In addition HTLV-III was found in cell-free plasma. The incidence (number of isolates per number of patients or donors tested) of virus isolation was approximately 80% for ARC patients, approximately 50% for AIDS patients, and approximately 30% for healthy individuals at risk for AIDS. Inclusion of hydrocortisone in cell culture medium greatly facilitated the isolation of virus from primary leukocytes from AIDS/ARC patients and also promoted its transmission to fresh leukocytes in vitro. Biological analysis of cells from infected patients or donors, as well as from normal peripheral blood mononuclear cells exposed to virus in vitro, demonstrated that OKT4/leu3a+ T-lymphocytes were preferentially infected and were subjected to a characteristic cytopathic effect. In addition to the well-defined individuals at risk for AIDS, heterosexual transmission of HTLV-III with its subsequent pathological manifestations was found. Virus was isolated from males with heterosexual promiscuity as their only recognized risk factor and from the spouses of these and other AIDS and ARC patients.

Clinical spectrum of HTLV-III in humans.

We have studied the clinical and laboratory manifestations of infection with human T-cell lymphotropic virus type III in various epidemiological cohorts. The spectrum of infection ranges from an asymptomatic but apparently contagious carrier state to severe immunodeficiency with opportunistic infections and neoplasms. Study of virus structure-function relationships and host response to viral infection in hosts with different clinical manifestations should provide strategies for therapeutics and vaccine development as well as enhance our understanding of the biology of human retroviruses.

HTLV-I antibody status in hemophilia patients treated with factor concentrates prepared from U.S. plasma sources and in hemophilia patients with AIDS.

Serum samples from 85 Austrian hemophilia patients treated with lyophilized factor concentrates prepared from U.S. plasma sources, 24 hemophilia patients from Georgia on a home therapy program with factor concentrates, and 10 U.S. hemophilia patients with acquired immunodeficiency syndrome (AIDS) were analyzed by two different methods for the presence of antibodies to the major internal antigen of human T-cell leukemia virus I (HTLV-I) p24. All but one, a Georgia sample, were negative. The absence of antibody to HTLV-I p24 in the serum of European hemophilia patients, of U.S. hemophilia patients with no symptoms of AIDS, and of U.S. hemophilia patients with AIDS is interpreted as an indication of the lack of ready transmissibility of HTLV-I in lyophilized factor concentrates.

Radioimmunoassay and enzyme-linked immunoassay of antibodies to the core protein (P24) of human T-lymphotropic virus (HTLV III).

Human T-cell lymphotropic viruses designated HTLV III or LAV are considered to represent the causative agent(s) of the acquired immunodeficiency syndrome (AIDS). Individuals who have been infected with these viruses may generally be identified on the basis of a positive serological test for antibodies against the protein components of these viruses. Purified viruses or viral proteins have been utilized for developing such tests. Since AIDS may be transmitted by blood transfusion and by blood products, screening of donors for antibodies to HTLV III/LAV has become a necessity. Such screening may be facilitated by the application of assays based on the use of crude virus-infected tissue culture media avoiding elaborate, expensive and potentially hazardous virus purification steps. Serum specimens were mixed with an appropriate dilution of an HTLV III-infected tissue culture-derived fraction, obtained by precipitation with polyethylene glycol 6000 and treatment with Tween 80 and tri-n-butylphosphate (to disrupt virus particles), and incubated with polystyrene beads coated with antibodies to HTLV III/LAV (anti-HTLV III). Subsequently, washed beads were incubated with either 125I- or beta-lactamase-labeled anti-HTLV III. The radioactivity or enzymatic activity associated with the beads was proportionate to the quantity of HTLV III antigen originally added to the beads. The presence of anti-HTLV III in serum specimens resulted in decreased antigen binding and thus in decreased radioactivity or diminished beta-lactamase activity associated with the beads. The test was specific for antibodies to the approximately equal to 24 kDa core protein of HTLV III. The prevalence of these antibodies (given in parentheses) in distinct populations was as follows: random blood donors (0.33%); hemophiliacs (36.4%); random homosexual males (25.1%); homosexual males preselected on the basis of positive markers for infection with hepatitis B virus (50%); and those with persistent lymphadenopathy (70%).

Detection of antibodies to human T-lymphotropic virus type 1 (HTLV-1).

Sera from 39,898 blood donors were tested for HTLV-1 antibodies using two enzyme immunoassays (EIA). Sera testing initially reactive (IR) were retested in duplicate by both EIAs. Sera testing repeatedly reactive (RR) were further tested by two Western blots (WB) and by two radioimmunoprecipitation assays (RIPA). There were 176 (0.44%) EIA IR and 68 (0.17%) RR results. On WBs, 10 of the 68 EIA RR sera demonstrated reactivity to HTLV-1 gag gene-encoded core protein p24, with or without reactivity to other core proteins (p19, p28, p53/55). These ten sera were the only ones demonstrating reactivity on RIPAs to other HTLV-1 gene products - env gene-encoded glycoproteins gp46, gp61/68, or tat gene-encoded HTLV-1 transcriptional activator p40x. These ten sera were interpreted as positive for HTLV-1 antibodies. Of the remaining 58 EIA RR sera, 21 were negative by WBs and RIPAs, 37 sera demonstrated reactivity to various combinations of p19, p28, and p53/55, but not to p24 on WBs. These 37 sera were interpreted as "indeterminate", because they were negative by RIPAs. We conclude that: 1) EIA testing and WB/RIPA verification identified 10 (0.025%) HTLV-1 infected individuals among 39,898 low-risk blood donors; 2) anti-p24 may be a more sensitive and specific indicator of HTLV-1 infection than antibodies to p19, p28, or p53/55; and 3) presently, both WB and RIPA are needed to verify HTLV-1 EIA reactivity.

Stability and inactivation of HTLV-III/LAV under clinical and laboratory environments.

The stability of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) under environmental conditions encountered in a clinical or laboratory setting and its inactivation by commonly used chemical disinfectants were investigated. Under our experimental conditions utilizing a highly concentrated viral preparation, virus with an initial infectious titer of approximately 7 log10 tissue culture infectious dose (TCID50) per milliliter can be recovered for more than a week from an aqueous environment held at room temperature (23 to 27 degrees C) or at 36 to 37 degrees C. Virus recovery is reduced at a rate of approximately 1 log10TCID50 per 20 minutes when held at 54 to 56 degrees C. Dried and held at room temperature, HTLV-III/LAV retains infectivity for more than three days with a reduction of approximately 1 log10TCID50 per nine hours. Viral infectivity is undetectable and reduced more than 7 log10TCID50 within one minute with 0.5% sodium hypochlorite, 70% alcohol, or 0.5% nonidet-P40, and within ten minutes with 0.08% quaternary ammonium chloride or with a 1:1 mixture of acetone-alcohol. These results help provide a rational basis to prevent the accidental spread of HTLV-III/LAV in the laboratory or clinical setting.

Human T-lymphotropic retroviruses.

The first human retroviruses have been discovered during the past six years. They cause two diseases which involve disturbances of the growth of the T4 lymphocyte, a remarkably specific target cell type. This cell, which is central to the regulation of the immune system, is induced by human T-lymphotropic virus type I (HTLV-I) to excessive proliferation (leukaemia) and by HTLV-III to premature death (acquired immune deficiency syndrome, AIDS). Both also seem to be indirectly involved in several other disorders. The genetic structures of these retroviruses and the mechanisms by which they usurp host-cell functions are novel among retroviruses.

Retrovirus-like particles in salivary glands, prostate and testes of AIDS patients.

AIDS associated retrovirus-like particles were identified in the salivary gland, prostate and/or testicle of two AIDS patients. These findings further suggest that saliva and semen may transmit the infection to susceptible individuals.

PIP: This article presents electronmicroscopy evidence of retrovirus-like particles with bar shaped cores in salivary and prostate glands as well as testicles of 2 acquired immunodeficiency syndrome (AIDS) patients. The 1st case, a 38-year old black male homosexual, presented in 1982 with diarrhea, malabsorption, and weight loss. In the following 1 1/2 years, he experienced recurrent Candida esophagitis, cutaneous and pulmonary Kaposi's sarcoma, Pneumocystis carinii pneumonia, and cytomegalovirus. Autopsy in 1984 revealed residual Kaposi's sarcoma, disseminated cytomegalovirus, and M avium-intracellulare. The 2nd case, a 31-year old white male homosexual, presented in 1984 with Pneumocystis carinii penumonia and subsequently developed persistent fever, hepatomegaly, headaches, blurred vision, progressive liver function deterioration, and disseminated histoplasmosis infection. Autopsy in 1984 revealed an overwhelming disseminated histoplasmosis infection. Tissues taken at postmortem were examined by electron microscopy. Particles that conformed with the morphologic characteristics of AIDS retrovirus (a size of about 140 nm, a round shape with a double membrane, and an elongated core) were detected in the prostate gland of patient 2 and in the salivary glands and testes of both patients. This finding suggests that saliva and semen may be body fluids by which transmission of the AIDS virus occurs.

No evidence of LAV infection in the Republic of Liberia, West Africa, in the year 1973.

Sera collected 13 years ago from 592 residents of the Republic of Liberia have been tested for antibodies to LAV polypeptides. 7 sera were positive by ELISA using two commercially available test kits whereas immunoblotting did not confirm antibodies specific for LAV.

PIP: Liberian sera collected in 1973 from 592 residents of agricultural and iron ore mining companies were tested for LAV (HTLV-III), now known as HIV, by ELISA and western blotting, and no positives were found. The ELISA tests were kits from ELAVIA, Institute Pasteur, France, and VIRAMED, Electro Nucleonics, USA, and the immunoblot method was that of Towbin et al. The subjects included 430 men and non-pregnant women who had no positive findings, and 162 pregnant women at parturition, of whom 7 had positive ELISAs but negative confirmatory western blots. This population had high rates of onchocerciasis, hepatitis B, nematodes, Schistosomas, Marburg virus and Ebola virus.

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.

Infection with human T-lymphotropic virus type III/lymphadenopathy-associated virus: considerations for transmission in the child day care setting.

Many public health and social issues relate to infection with the human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV)-the virus that causes the acquired immunodeficiency syndrome. One such issue is the care and education of infected children, both in schools and in day care facilities. To date, transmission of HTLV-III/LAV in either the school or the day care setting has not been documented. However, because the virus has been isolated from a variety of body fluids, contact with such fluids from an infected child poses at least a theoretical risk of transmission. Past experience with transmission of hepatitis B virus provides a possible model to aid in understanding the epidemiology of HTLV-III/LAV infection. The risk of transmission of HTLV-III/LAV in day care settings is not known, and infection with this virus carries serious public health and clinical implications.

Acute HTLV-III infection. A case followed from onset to seroconversion.

An acute febrile illness with mouth ulcers and a skin rash was observed in a 37-year-old homosexual man. Acute human T-lymphotropic virus type III (HTLV-III) infection was suspected. The clinical features were documented prospectively, and striking changes in circulating T lymphocytes were demonstrated shortly after the onset of the illness. Seroconversion for HTLV-III antibody occurred between 46 and 78 days after the onset of the illness.

Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions.

The RNA-dependent DNA polymerase of the AIDS-associated retrovirus (ARV) gives highest activity with the synthetic template, poly(rA)oligo(dT) and prefers Mg2+ over Mn2+ as a divalent cation. It can use other template-primer combinations but with poly(rCm)oligo(dG), it prefers Mn2+ over Mg2+. Detection of ARV reverse transcriptase in culture fluids is substantially increased with an optimal KCl concentration and a special combination of EGTA and reducing agents. Our results indicate some distinguishing characteristics of the ARV reverse transcriptase and optimal conditions for its detection.

Genomic variability of selected LAV-related AIDS retroviruses.

All AIDS retroviruses isolated from different patients have shown degrees of heterogeneity as defined by restriction fragment polymorphisms. Despite this variability, all these virus isolates share a number of structural features, including immunological cross-reactivity of virally encoded proteins. In this paper, we compare restriction patterns of integrated proviral DNA from viral isolates of patients belonging to different geographical groups, at risk or not for the disease. We confirm the existence of different clones in the same isolate of the Lymphadenopathy-associated virus (LAV) and of Human T lymphotropic virus (HTLV-III), which are identical. One of these forms is very similar to a Haitian isolate, as well as to an isolate from an early recognized New York case, suggesting a common origin for these viruses. More variation is apparent with Zairian viral isolates, and one of two clones found in a virus from a child who received an allogeneic bone marrow transplant. Greater disparity was also found in the restriction pattern of an isolate from an individual belonging to none of the so-called high-risk groups. We also show that this variation occurs mainly but not only in the env region of the genome, as previously described.

Noma in children with severe combined immunodeficiency.

Three Native American children with severe combined immunodeficiency developed noma, a necrotizing gingivostomatitis not previously reported in this country. The similarity between the clinical findings and those observed in monkeys with simian AIDS prompted us to evaluate our patients and their families for human retroviral infection. Antibodies to HTLV-I or HTLV-III/LAV proteins were not identified in patients nor in their family members. Standard bacterial and viral cultures similarly failed to identify a suspect pathogen.

HLA antibodies as a cause of false-positive reactions in screening enzyme immunoassays for antibodies to human T-lymphotropic virus type III.

15,680 volunteer blood donors were screened by enzyme-linked immunosorbent assay (EIA) for antibodies to human T-lymphotropic virus Type III (HTLV-III). On at least two of three determinations, 0.37 percent were found to be reactive. When the first 38 of these samples were sent for repeat EIA and Western blot, the EIA was positive in 14. In only three of these 14 samples was the EIA result confirmed by Western blot. Eight of these eleven false-positive samples were from women who were found to have HLA antibodies. In seven of these women, the HLA antibodies were directed to Class II antigens expressed on the cell used to grow HTLV-III in the preparation of screening EIA kits. We conclude that HLA antibodies are an important cause of false-positive reactions in the screening test for HTLV-III antibody.

Decision analysis of the HTLV-III screening test for blood donors.

Recent research has identified the human T-lymphotropic virus type III (HTLV-III) as a probable etiologic agent of the acquired immune deficiency syndrome (AIDS). This has prompted the U.S. Public Health Service to recommend that all blood used for transfusions or in the manufacture of blood products be screened. An enzyme-linked immunosorbent assay (ELISA) has been approved for use as a screening test. From the perspective of the low-risk blood donor, however, our analysis indicates that the expected utility of no-testing may exceed that of testing. This is primarily due to the risk of a false-positive test result. It follows that informed low-risk individuals may be hesitant to donate blood. We support the discarding of blood that tests positive on ELISA but, to decrease donor risk, a positive confirmatory test, such as the Western blot, should be considered as necessary before the testing outcome is treated as positive from the donor's perspective. Additionally, individuals should be given the option to donate blood without being told the testing outcome.

AIDS serology testing in low- and high-risk groups.

The performance characteristics of the acquired immunodeficiency syndrome (AIDS)-retrovirus serological tests including enzyme-linked immunosorbent assay (ELISA), Western blot, and immunofluorescence assay were defined in a clinical laboratory setting by testing 1,257 serum specimens from low- and high-risk groups for AIDS. The three prototype AIDS retroviruses (lymphadenopathy-associated virus, human T-lymphotropic virus III, and AIDS-associated retrovirus) were equally suitable as target antigen for these assays. Sera from six of 74 laboratory and health care personnel and 91 of 1,014 unselected blood donors were falsely positive by ELISA (positive to negative ratio [P/N], greater than or equal to 2) based on the lack of Western blot confirmation. Only two true-positives (two [0.2%] of 1,014 blood donors) were detected in these low-risk groups. In contrast, 106 of 108 specimens with ELISA P/N ratios of 2 or greater from the high-risk groups including asymptomatic homosexual men, hemophiliacs, AIDS-related complex patients, and AIDS patients were positive by Western blot and immunofluorescence assay. Four false-negative ELISA results based on positive immunofluorescence assay and Western blot were found in the AIDS patient group. Ten of 69 AIDS patients were negative by all three serological tests. The consequence of maintaining high sensitivity for the ELISA (P/N ratio, greater than or equal to 2) as a screening test was a loss of specificity. The number of false-positive results necessitated the use of a confirmation test with greater specificity.