Lack of correlation between human herpesvirus-6 infection and the course of human immunodeficiency virus infection.

Human herpesvirus-6 (HHV-6) and human immunodeficiency virus (HIV) are both tropic for CD4+ lymphocytes. To determine whether HHV-6 infection affects the susceptibility to or the course of HIV infection, HHV-6 titers were measured by an anticomplement immunofluorescence assay in serum of three groups of homosexual or bisexual men: (1) those with AIDS (n = 78), (2) those with HIV-associated lymphadenopathy (LAS; n = 81), and (3) those who were HIV-seronegative (n = 55). Early and late serum samples were available for 45 men with LAS (median interval 49 months). Men with early LAS did not differ from HIV-seronegative men in either the percentage that were HHV-6-seropositive or in the distribution of titers. There was a significantly lower percentage of seropositives in AIDS patients than in the other two groups (P less than .01). LAS patients who progressed to AIDS did not differ in percentage seropositivity or distribution of titers from nonprogressors. HHV-6 titers tended to decrease over time. HHV-6 titers late in LAS were similar to those in AIDS patients. These findings suggest that it is unlikely that previous exposure to HHV-6 either predisposes to or affects the course of HIV infection.

Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed paraffin-embedded cell cultures.

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.

Demonstration of human immunodeficiency virus by colorimetric in situ hybridization: a rapid technique for formalin-fixed paraffin-embedded material.

A colorimetric method of in situ hybridization has been developed for the rapid detection of human immunodeficiency virus (HIV) in formalin-fixed paraffin-embedded material. Following optimization of digestion conditions, biotin-labeled DNA probes are detected with an alkaline phosphatase conjugate. The method is verified using fixed paraffin-embedded cell blocks of HIV-infected and uninfected lymphocyte cell cultures. Hybridization specifically detects both viral RNA and proviral DNA. Formalin fixation for intervals up to 21 d did not significantly hamper the signal under the appropriate digestion conditions; however, Trump's fixation for even 12 h greatly reduced the intensity of the hybridization. This technique for in situ hybridization is amenable to automation, provides results within 6 h, and results in good morphologic preservation. A key feature of the technique is the use of human placental DNA as an endogenous positive control to optimize the empirically determined conditions for protein digestion.

Micromethod for assaying reverse transcriptase of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.

A micromethod for assaying the reverse transcriptase enzyme of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus in cocultures of clinical specimens for viral isolation was developed and compared with the macromethod in use. Ultracentrifuged, pelleted, and solubilized viral culture supernatants were transferred into either tubes (macromethod) or microtiter plates (micromethod) and incubated with tritiated enzyme substrate. Trichloroacetic acid-precipitated DNA was collected on individual filter papers with a Millipore filtration manifold (macromethod) or on filter sheets using a semiautomated cell harvester (micromethod). Filters were then placed in scintillation fluid and counted on a beta scintillation counter. Results of the micromethod significantly correlated to those of the macromethod, with a linear relationship between the two. The cutoffs for positivity based on the mean + 2 standard deviations for a set of known negative specimens (n = 19) was 4,973 cpm for the micromethod compared with 5,336 for the macromethod. The intrarun and interrun variations were comparable for both methods. There was a 67% increase in the maximal daily number of specimens which could be run (100 versus 60) as well as a reduction in reagent use. In summary, the micromethod utilizing a semiautomated cell harvester is comparable to the existing macromethod in accuracy and is an improvement due to savings in time and reagents.

Transfusion-associated acquired immunodeficiency syndrome in the United States.

By Aug 15, 1985, one hundred ninety-four cases of possible transfusion-associated acquired immunodeficiency syndrome (AIDS) had been reported to the Centers for Disease Control. Cases received their transfusions in 30 states. Infants account for 10% of the cases, suggesting an increased susceptibility to developing AIDS. Investigations one to six years after the transfusions have identified high-risk donors to 47 cases. Of 47 high-risk donors tested, 40 had a reactive serology for human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody, including five with no risk for AIDS by history. The HTLV-III/LAV was isolated from 23 of 26 seroreactive high-risk donors, most of whom remained asymptomatic. Blood components that transmitted HTLV-III/LAV included red cells, platelets, plasma, and whole blood. The time from transfusion to diagnosis of AIDS ranged from four to 84 months. The risk of developing AIDS after a blood transfusion has been low and will be lowered further by using both self-deferral and antibody screening.

Persistent infection with human T-lymphotropic virus type III/lymphadenopathy-associated virus in apparently healthy homosexual men.

PIP: A group of 14 apparently health homosexual men with serologic evidence of human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) infection were studied to determine the duration of their seropositivity, their immunologic status, and the frequency of isolation of HTLV-III/LAV from their peripheral blood. The men were selected from a larger sample of patients who attended a clinic for treatment of sexually transmitted diseases in San Francisco because they did not have acquired immunodeficiency syndrome (AIDS), signs or symptoms suggestive of the prodrome of AIDS, or laboratory evidence of anemia or leukopenia. 4 or more serum samples were available from previous clinic visits. The men ranged in age from 26-41 years, and had a median number of sexual partners in the last year of 23. The estimated duration of seropositivity ranged from 4-69 months (median, 33 months). 11 of the 14 had T-helper: T-suppressor cell ratios below 1 (the lower limit of normal), and low ratios were significantly correlated with duration of seropositivity. HTLV-III/LAV was isolated in peripheral blood samples from 8 of 12 men tested. Culture-positive and culture-negative men did not differ significantly in terms of age, presence of a palpable lymph node, T helper:T-suppressor cell ratio, or duration of seropositivity. These findings suggest that some seropositive men may remain asymptomatic for at least 5 years. However, the isolation of HTLV-III/LAV from the peripheral blood of most of these men indicates persistent infection may be common among asymptomatic seropositive men at risk for AIDS. It should be assumed that these men have the potential to transmit HTLV-III/LAV infection.^ieng

Lymphadenopathy associated virus infection of a blood donor--recipient pair with acquired immunodeficiency syndrome.

A retrovirus isolated from three patients with the acquired immunodeficiency syndrome (AIDS) in the United States was morphologically and antigenically identical to lymphadenopathy associated virus isolated in France. Two of these isolates were from a blood donor-recipient pair, each of whom developed AIDS. Lymphadenopathy associated virus was isolated from the blood donor's lymphocytes 12 months after his onset of AIDS symptoms and from the blood recipient's lymphocytes 1 month after her onset of AIDS symptoms. Two isolates from the blood donor-recipient pair and an isolate from an epidemiologically unrelated homosexual man were examined by competitive radioimmunoassay to determine their antigenic relatedness to each other and to other human retroviruses. The major core proteins (p25) of the isolates were antigenically identical and all three isolates were identical to prototype lymphadenopathy associated virus isolated in France.

Experimental allergic neuritis-like disease in rabbits after injection with influenza vaccines mixed with gangliosides and adjuvants.

An experimental allergic neuritis-like disease was induced in rabbits 3 to 8 weeks after injection with large doses of influenza vaccines mixed with gangliosides, cholesterol, and Freund complete adjuvant. The inclusion of gangliosides was essential to induce the experimental allergic neuritis-like disease. In trials with six different lots of vaccine, both swine influenza and non-swine influenza vaccines produced by four different manufacturers induced experimental allergic neuritis-like disease in 26 of 43 inoculated rabbits.

Recent experience with the complement fixation test in the laboratory diagnosis of rickettsial diseases in the United States.

Sera from patients suspected of having rickettsial infections were tested in the complement fixation test with antigens prepared from the rickettsiae of Rocky Mountain spotted fever (SF), rickettsial pox (RP), murine typhus, epidemic typhus, and from Rickettsia canada (RC). Eight units of antigen were used in all cases and two units in man. Only those patients with antibody titers of 1:16 or higher were included in the study. Largely on the basis of comparative titers, the patients were divided into two groups: 102 with SF and 35 with infections by one of the members of the typhus group. The antibody titers were higher with SF antigen than RP antigen in 72% of the SF patients, and in only two SF patients was the RP titer higher, and then by only one tube (twofold dilution). There seemed little advantage in including the RP antigen in the battery of rickettsial antigens. Cross-reaction with at least one of the typhus antigens was observed in the sera from 64% of the SF patients. It was extensive enough to be confusing (within one tube) in 17% with eight units of antigen, but the differentiation was more distinct with two units of antigen. The cross-reaction with typhus antigens was as frequent in children with SF as it was in adults; thus, it is unlikely that these cross-reactions resulted from previous typhus vaccination. The serological differentiation between murine typhus and epidemic typhus was frequently difficult, but the epidemiological background was distinct. Five patients had higher titers to RC antigen, and four of these may possibly have had RC infections.

Mumps class-specific immunoglobulins in radioimmunoassay and conventional serology.

Antibodies against mumps virus have been studied by using immunoglobulin class-specific indicators labeled with (125)I in the radioimmunoassay (RIA) procedure. The immunoglobulins in paired acute and convalescent sera were allowed to react with mumps virus in a solid-phase RIA system. Class-specific immunoglobulin indicators (anti-immunoglobulin M [IgM] and anti-immunoglobulin G [IgG]) labeled with (125)I revealed that immunoglobulins of early antisera were preponderantly IgM, whereas immunoglobulins of late antisera were predominantly IgG. These indicators detected antibodies of the early (IgM) and late (IgG) phases of the immune response. These findings are consistent with the classical temporal order of appearance of 19s (IgM) and 7s (IgG) globulins. Specificity of these indicators for reacting with fractionated 7s and 19s globulins is also presented. Mumps virus RIA obtained with anti-IgG correlated well with conventional serological data obtained by neutralization and hemagglutination inhibition, but most strongly with complement-fixation data. In addition, antibody bound by solid phase was capable of distinguishing between related antigens of the myxovirus group.

Mumps class-specific immunoglobulins in radioimmunoassay and conventional serology.

In assessing the host cell range of bovine parvoviruses, these viruses were found to replicate optimally in actively dividing bovine fetal lung and spleen cells. Other primary bovine fetal cells supported growth to a lesser extent, but bovine line cells and line cells of other animal species tested did not. Minimal infectivity remained after passage of bovine parvovirus in cells from chicken embryos and guinea pig fetuses. During bovine parvovirus replication in bovine fetal lung and spleen cells, production kinetics of infectious virus and hemagglutinins were determined. An eclipse period of 16 h occurred, and viral release from cells was not detected until 30 h after inoculation of bovine fetal lung cells and 36 h after inoculation of bovine fetal spleen cells. Cell-associated virus titers were always higher than extracellular virus titers. Hemagglutinins were detected in parallel to infectious virus.

Solid-phase radioimmunoassay of total and influenza-specific immunoglobulin G.

An antigen-antibody system of polystyrene tubes coated with immunoglobulin antibody was used for quantitating immunoglobulins. A similar radioimmunoassay method was adapted for a viral antigen-antibody system. The viral system can be used for quantitating viruses and for measuring virus-specific antibodies by reacting with (125)iodine-labeled anti-immunoglobulin G (IgG). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay method were investigated. Comparison of direct and indirect methods of assaying for immunoglobulins or viral antibody indicates that the indirect method is more sensitive and can quantitate a minimum of 0.037 mug of IgG per ml. Results of solid-phase radioimmunoassay for influenza antibody correlate well with hemagglutinin antibody titers but not with complement-fixing antibody titers. Radioimmunoassay results for influenza antibody by solid phase are likewise in agreement with results by the carrier precipitate radioimmunoassay method. The simplicity, reproducibility, and versatility of the solid-phase procedure make it diagnostically useful.

Preparation of type 2 herpes simplex virus complement-fixing antigen.

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.