Spatial visualization of progressive states of maturing lentivirus.

Progressive states of maturing lentivirus: maedia visna virus (MVV) and human immunodeficiency virus (HIV), respectively, have been visualized by 2-D electron microscopy and by 3-D electron microscopic tomography. A major fraction of MVV and a low percentage of HIV appear as immature particles 4 to 5 days post virus infection. Upon budding the gag-precursor material is densely packed inside the external envelope. After virus release the major portion of precursors is assembled within an approximately 25 nm thick layer directly attached to the envelope. Structural maturation of the core is different for the two viruses. Pleomorphic cores are observed in mature MVV in contrast to structurally defined cores of HIV. The latter are principally cone-shaped, spanning the entire diameter of the virion with a 40 to 60 nm wide free end and an approximately 20 nm narrow end attached to the envelope with a core-envelope-link.

Immunoglobulin class and subclass-specific monoclonal antibody sandwich ELISA for the detection of antibodies against coxsackieviruses B, types 1-5.

Immunoglobulin subclass-specific ELISAs were developed for human IgG1, IgG2, IgG3, IgG4, IgAtotal, and IgM directed against Coxsackie B (CB) virus types 1, 2, 3, 4, and 5. In all the assays the solid phase was coated with immunoglobulin class/subclass-specific monoclonal antibodies, followed by an incubation with the serum specimens. Incubation with one of the CB viruses, as well as an incubation with biotinylated serotype-specific monoclonal antibodies to the same virus type provided the virus specificity. Finally, there were incubations with peroxidase labeled Extravidin and the substrate-chromogen system. This ELISA method eliminated the competition between the immunoglobulin classes and subclasses. IgG3 and/or IgG1 were seen most frequently of the IgG subclasses, but IgG2 and IgG4 were also present infrequently. The viral specificity of the antibody subclass assays seems to be predominantly at the enterovirus group level, but this remains to be evaluated in a larger study. IgA and IgM were seen almost exclusively in specimens from patients with acute enteroviral infections, except in the assays with the crude CB5 antigen. This indicates the possible suitability of the IgA and IgM assays as diagnostic tests for enteroviral infections. A larger study is necessary to confirm this finding.

Comparison of immunoblots with neutralizing and complement fixing antibodies in experimental and natural cases of visna-maedi.

In this study the humoral antibody response in visna-maedi virus disease in sheep during long-term infection was analyzed utilizing immunoblot assays, neutralization tests and complement fixation tests. In immunoblot assays antibodies to several virus specific protein bands were detected, both against the viral envelope glycoproteins and internal proteins of the virus. The immunoblot reaction pattern resembled that found in HIV-1 infection in humans, consistent with reported similar molecular weight of the major proteins of these two viruses. The immunoblot band pattern was compared with the pattern of complement fixing and neutralizing antibodies through the preclinical and clinical course in natural and experimental cases of visna-maedi. Of six immunoblot bands identified as virus specific, the antibody response against three gag products and the major env glycoprotein appeared early in infection, at a similar time as the complement fixing antibodies. The response against two proteins, one presumably the transmembrane protein and the other possibly a gag precursor, was delayed.

Antibodies against varicella-zoster virus and herpes simplex virus deoxythymidine kinase in heterologous infections.

Antibody responses to varicella-zoster virus (VZV) deoxythymidine kinase (dTK) and herpes simplex virus (HSV) dTK in homologous and heterologous infections were studied. Antibodies blocking the enzymatic activity of VZV-dTK appeared late after varicella and decreased more or less in parallel with the decreasing complement fixing [CF] titre. In herpes zoster, on the other hand, antibodies to VZV-dTK appeared soon after infection. Antibodies against HSV dTKs appeared long after primary infection, but they were subsequently present in all other HSV-CF positive sera. In recurrent HSV, all acute sera were already HSV-dTK antibody positive, and three of nine persons showed an increase in titer between their acute and convalescent sera. Blocking antibodies to VZV-dTK appeared rapidly in specimens from three of 18 individuals positive by an immunofluorescence VZV-immunity test during HSV infection, whereas all other specimens remained devoid of blocking antibodies against VZV-dTK. A rise in antibody titre against HSV-dTK during VZV infections was observed in serum specimens from three of 13 HSV-CF positive patients, whereas an antibody response against HSV-dTK was not found in HSV-CF negative individuals in connection with VZV infections. The relevance of the sporadic increase in the titres of antibodies against heterologous viral dTKs is discussed.

Comparison of five ELISA assays for IgG antibody against coxsackievirus B1.

Enterovirus type and group specificities of five different IgG ELISA methods were compared, using neutralization titration tests as an indicator of the presence or absence of antibodies to coxsackie B (CB) viruses. One of the ELISA assays was a "standard" IgG assay, where the solid phase was coated directly with the purified virus, followed by incubations with human serum, biotinylated anti-human-IgG, streptavidin-peroxidase, and the substrate/chromogen. In a modified standard assay, blocking of common epitopes was attempted by incubating the CB1 virus antigen on the solid phase with a rabbit antiserum to CB5 before the human serum was added. In another modification the serum dilution buffer contained heat-denatured heterologous enteroviruses in an attempt to consume human antibodies reacting with common epitopes. In one assay the purified CB1 virus was captured by purified horse anti-CB1 IgG on the solid phase, before incubation with human serum. In the last of the five assays the serum specimen was incubated with CB1 virus (in the liquid phase) before the virus or virus-antibody complex was captured with purified horse anti-CB1-IgG. Reactions against common antigens dominated in the first three assays. The antigen-capture assay appeared to be at least predominantly type specific. Our data indicate that the liquid-phase assay may be type specific, but more studies are needed. The method of virus purification was critical for the type specificity of the antigen-capture and liquid-phase assays.

Subclass restriction of human enterovirus antibodies.

We studied antibodies to enteroviruses in four groups of serum specimens: those from healthy adults, cord blood specimens, serum specimens known to contain immunoglobulin M (IgM) to coxsackie B (CB) viruses by radioimmunoassay, and serum specimens from children with symptomatic enteroviral infections. Enzyme-linked immunosorbent assays (ELISAs) were developed to detect the IgG class- and subclass (IgG1, IgG2, IgG3, and IgG4)-specific responses to CB3. The CB3 virus ELISA was not type specific. There was very poor correlation between CB3 virus neutralizing titer and IgG anti-CB3 virus ELISA results, indicating that antibodies to heterologous picornaviruses cross-react with CB3 virus in the assay. All serum specimens tested except one were IgG positive for CB3 virus. All 32 cord serum specimens were positive for IgG1 and IgG3. No enterovirus-specific IgG2 or IgG4 was detected in any serum specimen tested. Most serum specimens from the IgM-positive group, healthy adults, and children with enterovirus infections were positive for IgG1 and IgG3. Class and subclass antibody titers remained constant over time. IgG antibodies to enteroviruses appear to be restricted to the IgG1 and IgG3 subclasses. This pattern is similar to results obtained by other investigators evaluating IgG subclass antibodies to protein antigens.

Indirect and reverse radioimmunoassays and their apparent specificities in the detection of antibodies to enteroviruses in human sera.

Indirect radioimmunoassays (RIAs) of IgM and IgG antibodies to enteroviruses have been developed, using coxsackieviruses B1 and B3, and echoviruses 11 and 30. The titres of IgM and IgG were assayed in paired sera from patients infected with one of these viruses or coxsackieviruses A7, A9, A16, B2, B4, B5 or echoviruses 4, 17, or 25. Both IgM and IgG were found in almost all serum pairs with each of the four viruses used as an antigen, and there were no certain differences between titres obtained with homologous and heterologous antigens. The convalescent phase specimens contained significantly higher titres compared with the acute phase specimens, the difference being most pronounced for IgG. Of the specimens from patients with nonenterovirus infections, a relatively high percentage contained IgM and IgG against enterovirus antigen. However, no increases in titres were seen between acute and convalescent specimens. When specimens from younger patients, aged 2 days to 22 months, without evidence of enterovirus infections, were assayed with enterovirus antigen, the frequency of IgM titres was seen to increase with age. Almost all specimens from newborns were negative, whereas the specimens from 12- to 22-month-old children showed a high frequency of IgM titres. In specimens from patients aged 2 days to 8 months, the ratio between IgM and IgG titres increased with age, probably due to a loss of maternal IgG. The IgG titres in specimens from 8.5- to 22-month-old children were similar to the titres of specimens from the patients with nonenterovirus infections. A reverse IgM assay was also developed, using the same viruses and serum specimens as for the indirect assays. In contrast to the indirect IgM assay, the reverse IgM assay was apparently type specific, provided that the amount of labeled virus was carefully standardized. The reverse IgM RIA detected and identified antibody responses better than the neutralization test. Attempts to develop a reverse IgG assay were promising concerning the specificity, but the sensitivity was low.

Reverse radioimmunoassays of IgM and IgG antibodies to Coxsackie B viruses in patients with acute myopericarditis.

A reverse radioimmunoassay (RIA) of antibodies to enteroviruses, previously developed for the detection of IgM antibodies to Coxsackie B1 (CB1) and B3 (CB3) and to Echo 11 (E11) and 30 (E30) viruses, was extended in the present study for the detection of IgM antibodies to Coxsackie B2 (CB2), B4 (CB4), and B5 (CB5) viruses and of IgG antibodies to CB1-CB5, E11, and E30 viruses. After standardisation of the assays and application to a collection of serum specimens from patients with proven enterovirus infections, specimens from patients with diagnosed or suspected acute myo- and/or pericarditis (myopericarditis group), and control specimens from patients with nonenterovirus infections, were studied, as well as from apparently healthy subjects. Of the patients with enterovirus infections, 29 of 30 (97%) were positive in the IgM RIA and 19 of 25 (76%) in the IgG RIA. In the myopericarditis group, 18 of 37 (49%) patients showed Coxsackie B (CB) virus-specific IgM titres and 9 of 37 (24%) CB virus-specific IgG titres. In the control specimens very few positive responses were detected. The RIAs appeared to be type specific or at least predominantly type specific, provided that the amount of labeled virus was carefully standardised. The sensitivity of the RIAs seemed to be rather high for IgM but low for IgG. In the neutralisation (NT) test no significant rise or fall in titre against CB viruses was demonstrated in the myopericarditis group. It is concluded that the reverse IgM RIA may be valuable for studies of the role of CB viruses in acute myo- and/or pericarditis.

Human serum antibodies to varicella-zoster virus thymidine kinase.

The conditions required for the production of varicella-zoster virus (VSV)-induced deoxythymidine kinase (dTk) have been studied. Extracts from Vero cells harvested 62 h after VZV infection were found to contain VZV-induced dTk activity, with a minimal contribution from the cellular dTk activity. VZV dTK was shown to have a broad substrate specificity phosphorylating both deoxythymidine, deoxycytidine, and iododeoxyuridine. Deoxythymidine triphosphate inhibition studies revealed an intermediate deoxythymidine triphosphate sensitivity when compared with that of the cellular cytosolar enzyme and the deoxythymidine triphosphate-insensitive herpes simplex virus dTk. An assay for VZV dTk-blocking antibodies was developed, with [125I]iododeoxyuridine as a substrate in the presence of a deoxythymidine triphosphate concentration which selectively blocked the dTK of host cell origin. A total of 79 serum samples were studied; these included serum pairs from patients with varicella or herpes zoster and single sera from immune and nonimmune adults. VZV dTk blocking antibodies were detected exclusively in sera from patients with herpes zoster. All serum pairs showing VZV dTK seroconversion also showed a parallel conversion of complement fixation titers. The VZV dTk antibodies were found to be of the immunoglobulin G class. The immunological specificity of VZV dTK was investigated, and no cross-reactivity with herpes simplex virus type 1 or 2 dTk was found.

False RIA IgM titres to herpes simplex virus and cytomegalovirus: factors causing them, and their absorption by protein A-Sepharose/IgG-protein A-Sepharose.

A method for the absorption of false radioimmunoassay (RIA) IgM titres against herpes simplex virus (HSV) and cytomegalovirus (CMV) is presented. The serum specimens were absorbed by a mixture of protein A-Sepharose and protein A-Sepharose saturated with normal human gamma globulin (PAS/IgG). The detection of rheumatoid factor of IgM class (IgM-RF) as well as antinuclear antibodies (ANA) of both IgM and IgG class by solid-phase RIA is also described, and their role in the false IgM results was studied. It was found that the PAS/IgG absorption removed 50-90% of both IgM-RF and total IgG. The reduction of IgM-ANA clustered at 50-90% or nothing, whereas the reduction of IgG-ANA was approximately 50%. The studies with HSV and CMV antigens indicated that the removal of false IgM titres was more effective than the removal of each of these four factors. It was concluded that the IgM-RF titres alone were not sufficiently high to explain the false IgM results, but the ANA activity probably contributed.

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus.

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.

Four-layer radioimmunoassay for detection of adenovirus in stool.

A four-layer antispecies radioimmunoassay (RIA) was developed for the detection of adenovirus in stool specimens. Polystyrene beads were used as the solid phase, anti-adenovirus guinea pig immunoglobulin (1 microgram per bead) was used as the primary antibody, anti-adenovirus rabbit immunoglobulin (16 micrograms/ml) was used as the secondary antibody, and 125I-labeled sheep anti-rabbit immunoglobulin was used as the indicator antibody. A highly purified, crystallized adenovirus type 2 hexon antigen was used as the immunizing antigen for the production of hyperimmune sera. The sensitivity of the test was 1 ng of hexon protein per ml. Each of the 13 stool specimens positive for adenovirus by electron microscopy was positive for adenovirus by the RIA. Of 200 nonconcentrated stool specimens negative by electron microscopy, 14 additional specimens were positive by the RIA, increasing the detection rate from 6% by electron microscopy to 13% by the RIA. A confirmatory test was done on the RIA-positive, electron microscopy-negative specimens, and the test indicated a true specific result with each specimen. A confirmatory test was also done on each specimen with a low positive counts per minute value. The specificity of the RIA was further demonstrated by the fact that a positive result was found with only 3 of 295 specimens positive by the rotavirus RIA. In two of these three specimens, adenovirus and rotavirus were also detected simultaneously by electron microscopy, and the third specimen was from a patient with serological evidence for a dual infection. The adenovirus and rotavirus RIAs are now in a routine diagnostic laboratory, and in the 307 stool specimens tested during the first 5 months, the positive rate was 32% for rotavirus and 9.5% for adenovirus.

IgA antibody response in acute rubella determined by solid-phase radioimmunoassay.

A solid-phase radioimmunoassay (RIA) for detecting rubella virus IgA serum antibodies was developed. Purified rubella virus grown in roller cultures of Vero cells was adsorbed onto polystyrene beads. The coated beads were then incubated with dilutions of serum, and rubella IgA antibodies which attached to the virus antigen on the solid-phase were subsequently detected with 125I-labelled anti-human-alpha antibodies. The specificity of the iodinated anti-human immunoglobulins was confirmed by RIA analysis of fractions obtained by chromatography of an early convalescent serum on an agarose column. A complete separation of IgM, IgA, and IgG was observed. A total of 144 serial serum specimens from 31 adult patients with an acute rubella infection were tested for rubella IgA antibodies, and the results were compared with the RIA IgG and IgM titres reported earlier from the same specimens. The RIA IgA response was detected in each of the 31 patients and the IgA antibodies appeared almost simultaneously with the IgG and IgM antibodies. The maximum titres, which were lower than the IgG and IgM titres, were reached in about 1 week after the onset of rash. In 6 patients out of 31 the IgA antibody response was transient and persisted approximately two months, while in the remaining 25 patients the IgA antibodies persisted throughout the study period of more than 5 months. The results obtained indicate that the presence of rubella IgA antibodies in serum is not an indication for a recent rubella infection.

Solid-phase radioimmunoassay of serum immunoglobulin A antibodies to respiratory syncytial virus and adenovirus.

A solid-phase radioimmunoassay for detecting respiratory syncytial virus and adenovirus serum immunoglobulin A (IgA) antibodies was developed. An antigen consisting of purified adenovirus type 2 hexons or a crude lysate of respiratory syncytial virus-infected cells was first adsorbed onto polystyrene beads. The coated beads were then incubated with dilutions of serum, and IgA antibodies which attached to the solid-phase virus antigen were subsequently detected with 125I-labeled anti-human alpha antibodies. The anti-human alpha antibodies used were isolated by immunosorbent chromatography from rabbit antiserum produced by immunization with IgA purified from serum of an IgA myeloma patient. A total of 46 serum specimens from 13 patients with respiratory syncytial virus infections and 10 patients with adenovirus infections were tested. Complement fixation, homologous IgG and IgM radioimmunoassay, and heterologous IgA radioimmunoassay testing were also done. Specific values higher than 10,000 cpm were often reached with convalescent serum specimens, and positive-to-negative serum binding ratios of 50 or more were frequently obtained with lower serum dilutions. IgA titers of convalescent sera were from 1,000 to 16,000, and with few exceptions a fourfold or greater rise in the IgA titer was detected in the homologous IgA radioimmunoassay.