Comparative evaluation of an immunofluorescent antibody test, enzyme immunoassay and western blot for the detection of HIV-1 antibody.

Screening blood and blood products for human immunodeficiency virus type 1 (HIV-1) antibody is predominantly performed by enzyme immunoassay (EIA), and results must be confirmed by the more immunospecific Western blot (WB) assay. This study evaluated an HIV immunofluorescent antibody (IFA) test relative to WB assay for use in confirming EIA designated HIV-1 antibody-positive sera. Specimens from seroconversion and CDC panels as well as clinical specimens obtained for routine EIA HIV-1 antibody screening were evaluated. Results with 209 specimens indicated that sensitivity and specificity of the Fluorognost-HIV assay were equivalent relative to WB. In addition, the Fluorognost-HIV IFA test was faster and easier to perform than the WB assay, and unlike the WB assay was not prone to indeterminate results.

Evaluation of a latex particle agglutination assay for the detection of cytomegalovirus antibody in patient serum.

The Virogen CMV Antibody Test is a simple and rapid latex agglutination assay for the detection of cytomegalovirus antibody in human serum and plasma. Evaluation of this assay with respect to enzyme immunoassay yielded a sensitivity of 98% with a specificity of 100%. In comparison to CMVScan, the Virogen CMV Antibody Test had a sensitivity of 98.4% and a specificity of 100%.

Kinetics of virus-specific IgG and IgM antibody response in patas monkeys experimentally infected with Delta herpesvirus by enzyme-linked immunosorbent assay.

The kinetics of virus-specific IgG and IgM antibody response in patas monkeys experimentally infected with Delta herpesvirus (DHV), was studied using enzyme-linked immunosorbent assay (ELISA). An indirect ELISA (IE) test was used for DHV IgG determination, whereas a "double sandwich" (triple antibody layer) ELISA (DSE) test was found to be very sensitive for DHV IgM detection. In DSE test DHV IgG appeared to interfere with IgM determination only in sera containing DHV IgG levels at least 100 times higher than IgM. IgM antibodies appeared 5-8 days after monkey infection, reached a peak titer in 5-7 days, and after 7 days started to drop in titer and disappeared within two months. IgG antibodies appeared 5 days later than IgM, reaching the plateau in the following 7 days and then remaining stable for at least 2 months. Experimental infection of the patas monkey with DHV appears to be a good animal model for human varicella. IE for IgG and DSE for IgM detection are sensitive ELISA tests for determination of the immune response to DHV infection in patas monkeys.

Serologic study by enzyme-linked immunosorbent assay of the IgM antibody response in the patas monkey following experimental simian varicella virus infection.

The time course of IgM and IgG antibody appearance in sera following experimental simian varicella virus infection in the patas monkey was studied. Serum IgM and IgG antibody to Delta herpes virus (DHV) were measured by a double indirect and indirect ELISA test, respectively. IgM antibody was detected 5-8 days postinfection (p.i.) and IgG antibody was present 10-14 days p.i., which was 5-7 days after IgM. Viremia preceded detectable IgM antibody by 2 days. Sucrose gradient separation of serum IgM and IgG was carried out to determine whether IgG competed significantly with IgM. Results of these studies indicated that competition by IgG with IgM antibody did not occur except when IgM had declined to a low level some 40 days p.i. Comparison of the double indirect ELISA test with a double indirect immunofluorescent antibody test for detection of IgM antibody to DHV antigen indicated the ELISA test was more sensitive.

Detection of antibody to BK virus by enzyme-linked immunosorbent assay compared to hemagglutination inhibition and immunofluorescent antibody staining.

The enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition test (HAI), and immunofluorescent antibody staining (IFAS) test were compared for their ability to detect IgG antibody to BK virus (BKV). Antibody titers to BKV antigen were determined for 35 sera by each of the three assays. The ELISA tests agreed with either HAI or IFAS with regard to BKV antibody status in all but one serum. On this basis the HAI accounted for four discordant determinations while IFAS was not in agreement in three cases. Geometric mean titers of ELISA and HAI were similar but greater than that of IFAS. The ELISA is particularly well suited for use in large-scale seroepidemiological studies since automated spectrophometric equipment can provide rapid, objective, numerical data.

Comparison of the Raji cell line fluorescent antibody to membrane antigen test and the enzyme-linked immunosorbent assay for determination of immunity to varicella-zoster virus.

A prospective study was performed comparing the fluorescent antibody to membrane antigen (FAMA) test and the enzyme-linked immunosorbent assay (ELISA) for identifying susceptibility and seroconversion to varicella-zoster virus (VZV) infection. A total of 75 sera were collected from index cases and from sibling and parent contacts in 10 families. Varicella-zoster virus-infected human diploid embryonic fibroblasts and continuous lymphoblastoid cells (Raji cells) were compared as indicator cells in the FAMA test. Equivalent results were obtained with both types of cell. Results of the FAMA test and the ELISA were identical in two ways. (i) The same 11 individuals were initally defined as susceptible (seronegative), and 9 of them (82%) developed fourfold rises in antibody titers, clinical varicella, or both. (ii) Of 21 immune (seropositive) individuals, 4 developed fourfold antibody rises by FAMA tests, and 3 of these 4 responded by ELISA. Infection was asymptomatic in these individuals. The geometric mean titer by ELISA was significantly higher than by the FAMA test. The results indicated that the ELISA and the FAMA test have similar capacities to define susceptibility to varicella-zoster virus and that subclinical infection with varicella-zoster virus may be common.

Persistent Varicella-Zoster virus infection in a human rhabdomyosarcoma cell line and recovery of a plaque variant.

Varicella-zoster virus (VZV) has been found to persistently infect the human rhabdomyosarcoma cell line A204. Infectious center assays and fluorescent antibody staining demonstrated continuous production of infectious VZV and viral antigen. The level of infection determined by fluorescent antibody staining was variable, and usually only a small percentage of the cells were capable of producing plaques in permissive fibroblasts. The extent of infection was similar in cell cultures passaged at split ratios of 1:2 or 1:10 and grown at 33 or 37 degrees C. VZV recovered from A204 cells several months after establishment of the persistent infection had markedly increased syncytia-forming activity as compared with the parental VZV grown in human diploid fibroblast cells and the three monkey kidney-derived cell lines Vero, CV-1, and MA104. The expression of this altered phenotype continued after serial passage of the cell-associated virus in human diploid fibroblast and Vero cells. Consequently, we designated the reisolated VZV as plaque variant A. The buoyant densities of VZV plaque variant A and VZV DNAs in CsCl gradients were indistinguishable.

Effect of 5-iodo-5'-amino-2',5'-dideoxyuridine on varicella-zoster virus in vitro.

The antiviral effect of the nucleoside analog 5-iodo-5'-amino-2',5'-dideoxyuridine (AIU) was tested with three isolates of varicella-zoster virus (VZV). AIU concentrations of 10 to 800 muM (3.5 to 288 mug/ml) reduced the number of plaques produced by VZV-infected cells and cell-free VZV from approximately 30 to 95%. Smaller plaque size was also observed in the presence of AIU. AIU was less effective than arabinofuranosylthymine in reducing VZV-induced plaques since as little as 5 mug of arabinofuranosylthymine per ml completely blocked plaque formation by cell-free VZV. Toxicity assays with human diploid embryo fibroblast cells were also carried out. Drug concentrations as high as 800 muM were not toxic to human diploid embryo fibroblast cells as determined by radiolabeling of cell deoxyribonucleic acid, ribonucleic acid, and protein.

Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells.

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.

Characterization of human varicella-zoster virus DNA.

The DNA of varicella-zoster virus (VZV) has been characterized by sucrose gradient sedimentation, restriction enzyme cleavage with either EcoRI or HindIII site-specific endonucleases, and by isopycnic banding in caesium chloride. Comparisons of the DNAs from different clinical isolates have been made. DNAs from VZVs isolated from either varicella or herpes zoster are indistinguishable on the basis of size and restriction enzyme cleavage pattern. The buoyant density in caesium chloride of the DNA of VZV isolated from varicella was reproducibly slightly lower than that of the DNA of VZV isolated from herpes zoster.

Effect of ara-T on the replication of herpes simplex virus, varicella-zoster virus and cytomegalovirus.

The thymidine analogue, 1-beta-arabinofuranosylthymine (ara-T), has previously been found to selectively inhibit herpes simplex virus (HSV) replication. At a relatively non-toxic concentration (50 microgram/ml), ara-T reduced HSV yields by a factor of 10,000-100,000. Ara-T was also effective in inhibiting the replication of variecella-zoster virus (VZV) in vitro in human embryo fibroblasts, completely preventing VZV-specific cytopathic effects (CPE). Ara-T reduced the cell-free virus and plaque-forming cell (PFC) yields of VZV as well as of the simian varicella-like virus, Delta herpesvirus. In contrast to HSV and VZV, cytomegalovirus (CMV) replication was relatively resistant to ara-T. Neither CPE nor the incorporation of 3H-thymidine into acid-insoluble material in CMV-infected cells was markedly affected. Interpretation of these results with regard to virus-induced deoxypyrimidine kinase is discussed.

Differential effect of arabinofuranosylthymine of the replication of human herpesviruses.

The thymidine analog 1-beta-arabinofuranosylthymine (ara-T) has previously been found to selectively inhibit herpes simplex virus replication. At a relatively nontoxic conentration (50 microgram/ml), ara-T reduced herpes simplex virus yields by 4 to 5 log10. Ara-T was also effective in inhibiting the replication of varicellazoster virus (VZV) in vitro in human embryo fibroblasts, completely preventing VZV-specific cytopathic effects. The inhibition of VZV was reversible upon drug removal at 48 h after addition but was not reversible after 5 days of treatment. ara-T also reduced cell-free virus infectivity and the plaque-forming cell yield of VZV. Compared with the untreated controls, which demonstrated a 1-log10 increase over input plaque-forming cells at 24 h after infection, 50 microgram of ara-T per ml resulted in a 1-log10 decrease. In contrast to herpes simplex virus and VZV, cytomegalovirus replication was relatively resistant to ara-T. Neither cytopathic effects nor the incorporation of [3H]thymidine into acid-insoluble material in cytomegalovirus-infected cells was markedly affected. Analysis of the newly synthesized labeled DNA by CsCl buoyant density determinations indicated that the same relative proportions of cell and virus DNA were synthesized with or without added drug. Interpretation of these results with regard to virus-induced deoxypyrimidine kinase is discussed.

Adenovirus of ring-necked pheasants: purification and partial characterization of marble spleen disease virus.

A method for purification of marble spleen disease virus (MSDV) from the spleens of infected turkeys and pheasants is described. It combines chloroform or fluorocarbon extraction with subsequent purification by centrifugation on a cushion of cesium chloride (CsCl). Further purification of MSDV was accomplished with a CsCl equilibrium density gradient. A viral buoyant density of approximately 1.32 to 1.33 g/cm3 was determined. Negative-stain electron microscopy revealed that virus isopycnically banded by CsCl gradient had non-enveloped icosahedral capsids composed of 252 capsomeres. The direct colorimetric diphenylamine assay indicated that MSDV has deoxyribonucleic acid as its nucleic acid. The above evidence demonstrates that MSDV is an avian adenovirus, the first recognized in the ring-necked pheasant (Phasianus colchicus L.).

Demonstration of an avian adenovirus as the causative agent of marble spleen disease.

A purification procedure, using chloroform or fluorocarbon extraction and centrifugation on a cushion of cesium chloride (CsCL), was designed to isolate the causative virus of marble spleen disease. Virus was purified, inoculated into turkeys, and subsequently reisolated by purification from the spleen of inoculated turkeys, thus fulfilling Koch's postulates. Splenic antigen was detected by the agar gel precipitin test, and viral inclusions with viral particles were observed by light and electron microscopy. Results of further studies indicated splenic lymphoreticulum cell hyperplasia was a sensitive indicator of marble spleen disease virus (MSDV) infection. Direct fluorescent antibody staining revealed nuclear fluorescence in virus-infected splenic cells from turkeys inoculated with purified MSDV. With negative stain electron microscopy, MSDV was observed to be 90 nm across with an icosahedral capsid composed of 252 capsomeres. This morphologic feature was consistent with that of an avian adenovirus. Serologic evidence for classification of MSDV as an adenovirus was the cross reaction of MSDV antigen with antiserum to turkey adenovirus serotypes TA-1 and TA-2 in the agar gel precipitin test.

A novel approach to study the DNA of herpes zoster virus.

Herpes zoster virus DNA was isolated from infected human embryo lung cells because it is released into the "Hirt supernatant". The buoyant density of the virus DNA was found to be slightly higher than that of cellular DNA. The molecular weight of this DNA was estimated to be 92 x 10(6) daltons by cosedimentation with T4 DNA in neutral sucrose gradients. As with the DNA of other herpesviruses, the DNA of herpes zoster virus is fragmented when denatured under alkaline conditions and is therefore "alkali-labile". Studies of virus DNA using reassociation kinetics reveal that the kinetic complexity of the DNA correlates with the genome size as estimated by sucrose gradient sedimentation.

Experimentally transmitted marble spleen disease in pen-raised wild turkeys.

Pen-raised North American wild turkeys (Meleagris gallopavo L.) were experimentally infected with marble spleen disease (MSD) to determine their susceptibility to this disease. Gross and microscopic lesions were consistent with experimental MSD in pheasants and domestic turkeys: an enlarged mottled spleen, intranuclear inclusion bodies, and absence of pulmonary edema and hemorrhage. Detectable levels of viral antigen were not demonstrable in sera of turkeys using the agar gell precipitin test.