Comparison of immunodiffusion and enzyme linked immunosorbent assay in the detection of abnormal antibodies in pigeon breeder's disease.

AIMS: To compare the sensitivity of two methods for the detection of serum antibodies to pigeon faecal antigens in patients with pigeon breeder's disease. METHODS: Serum samples stored at -20 degrees C from 50 patients with pigeon breeder's disease, 50 control samples from patients with other respiratory diseases, and 50 healthy blood donors were examined for the precipitating antibodies and IgG antibodies to antigens present in extract of pigeon droppings by immunodiffusion and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: Both antigen preparations of pigeon dropping extract were equally effective. A positive immunodiffusion reaction gave one or more precipitin lines and these antibodies were detected only in undiluted sera from 80% of the patients with pigeon breeder's disease. In the ELISA the sera were tested at a starting dilution of 1 in 100 because positive reactions were observed with sera from healthy blood donors at lower dilutions. All sera which gave optical density readings above 3 SD of the control value were considered to have IgG antibodies. These antibodies were detected in sera from all the patients with pigeon breeder's disease. The antibody titres were much higher in those patients who had precipitating antibodies (range 800-51,200) than those without (range 100-800). The antibodies were not detected in the sera of patients with respiratory diseases or healthy blood donors by either method. CONCLUSIONS: Antibodies to pigeon dropping antigens were detected by immunodiffusion and ELISA in sera from patients with pigeon breeder's disease but not in control sera. ELISA was a more sensitive method for detecting antibodies and therefore seems to have considerable potential as a routine technique in the serological diagnosis of pigeon breeder's disease.

Virus-specific antibodies to Epstein-Barr virus, varicella-zoster virus and rubella virus in renal transplant patients with cytomegalovirus infections.

Renal transplant patients with primary and recurrent cytomegalovirus (CMV) infection had higher antibody titres to Epstein-Barr virus viral capsid antigen (EBV-VCA-IgG) before and after transplantation than healthy blood donors. The geometric mean titres (GMT) of EBV-VCA-IgG were higher in renal transplant patients without CMV infection than in renal transplant patients with CMV infection. Four-fold or greater rises in EBV-VCA-IgG antibody were detected in six patients and a similar rise in antibody to EBV early antigen (EBV-EA-IgG) was detected in one other patient. IgM antibody to EBV-VCA (EBV-VCA-IgM) was detected in only three of these patients. EBV-EA-IgG was present in 39% patients and in 30% control subjects. IgG titres to varicella zoster virus (VZV-IgG) and rubella virus (rubella HI) were higher in patients without CMV infection compared to the patients with CMV infection. Raised titres were detected to VZV in five patients and to rubella virus in three patients. Reductions in antibody titre of four-fold or more were also detected in EBV-EA-IgG (one patient) and to rubella virus (one patient). Raised antibody titres to EBV, VZV, and rubella virus in renal transplant patients may indicate reactivation of these viruses without any symptoms.

Patients with cervical cancer produce an antibody response to an HSV-inducible tumour-specific cell polypeptide.

Anti-sera raised against HSV-2-infected cells (WI) and the sera of animals bearing tumours (TBS) to HSV-2 transformed cells contain antibodies to a set of tumour-specific cell-coded polypeptides. The specificity of these polypeptides for tumour cells is monitored by the ability of [35S]-L-methionine labelled proteins to be immunoprecipitated by these anti-sera, in contrast to control cells from which the polypeptides are not precipitated. The polypeptides which share an epitope and are co-precipitated are of MWs 90,000 (a doublet), 40,000 and 32,000. The upper 90,000-MW polypeptide (U90) is induced by HSV-2 infection. This communication deals with the 40,000-MW polypeptide which was shown to be immunoprecipitated by TBS and a monoclonal antibody (MAb) raised to the DNA-binding proteins of HSV-2-infected cells. Immunological and biochemical studies reveal that the 40,000-MW protein which is immunoprecipitated comprises more than one polypeptide, and that the proteins may need to interact to produce the peptide pattern specific for the tumour form of the immunoprecipitated 40,000-MW protein. WI antisera and TBS both recognise antigens specific for tumour cells in sections of cervical-carcinoma tissue. Sera from patients with cancer of the cervix contain antibodies to a cell-coded polypeptide of MW 40,000, which by peptide analysis is indistinguishable from the 40,000-MW polypeptide induced by HSV-2 infection and immunoprecipitated by WI and TBS.

A study of HPV 1, 2 and 4 antibody prevalence in patients presenting for treatment with cutaneous warts to general practitioners in N. Ireland.

Three hundred and seventy-six patients attending their general practitioner with cutaneous warts at five health centres in Northern Ireland were screened for human papilloma virus (HPV) types 1 and 2 IgM antibody using an indirect immunofluorescence test. Eight-eight (23.4%) patients were positive for HPV type 1 IgM and 156 (41.5%) for HPV type 2 IgM. HPV 1 IgM antibody was significantly more likely to be associated with plantar warts than warts elsewhere (P less than 0.0001). HPV 2 IgM was present in 45 (34.1%) patients with plantar warts and 99 (45.6%) patients with warts at other sites (P = 0.1). Evidence of multiple infection by HPV types 1 and 2 was demonstrated by the finding of HPV 1 and 2 IgM antibodies in the sera of 16 (4.3%). HPV 4 was found in only 1 out of 30 biopsies and HPV 4 IgM was undetectable in 50 randomly chosen sera.

Low and high molecular weight cytomegalovirus-specific immunoglobulin M antibody in renal transplant patients with cytomegalovirus infections.

Sera from 27 renal transplant patients with primary and recurrent CMV infections and which were known to contain CMV-specific IgM antibodies were investigated by indirect immunofluorescence for the presence of virus-specific high molecular weight IgM (19S IgM) and low molecular weight IgM (7S IgM). After sucrose gradient fractionation of the sera, 19S IgM was found in all 27 patients, whereas 7S IgM was present in 11 out of 19 (56%) patients with primary CMV infection and in 1 out of 8 (12%) patients with recurrent CMV infection. The presence of 7S IgM was unrelated to the titre of the virus-specific IgM in whole serum. The presence of IgM rheumatoid factor was monitored by a sensitive fluorescence assay using measles virus antigen/antibody complexes. The absorption of the serum fractions with heat-aggregated gamma globulin failed to remove the specific IgM staining indicating that it was not due to IgM rheumatoid factor. On the other hand adsorption with protein A/sepharose removed the specific IgM staining from the 7S IgM fractions but not from the 19S IgM fractions. This suggests that specific 19S and 7S IgM antibodies may belong to different subclasses of IgM.

Cytomegalovirus-specific antibody responses in renal transplant patients with primary and recurrent CMV infections.

Cytomegalovirus (CMV) specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody responses were measured before and after renal transplantation in 20 patients with primary CMV infection and in 16 patients with recurrent CMV infection. In primary CMV infection IgG antibody titres to late antigen (IgG-LA) measured by indirect fluorescence (IFA) were approximately seven times higher than those obtained by the complement fixation test (CFT). In contrast, in recurrent CMV infection this difference was found to be about twofold. Virus-specific IgM antibody to late antigen (IgM-LA) was detected in 100 percent of patients with primary CMV infection and in only 50 percent of patients with recurrent CMV infection. The IgM-LA titres were highest in primary CMV infection and reached peak levels at approximately 10 weeks post transplantation, whereas in recurrent CMV infection the IgM-LA titres were lower and reached peak levels at three months post transplantation. Moreover, IgM-LA was found to persist in patients from both groups at nine months post transplantation. IgM antibody to early antigen (IgM-EA) was not detected in any patient in this study. However, significant fourfold titre rises in IgG antibody to EA (IgG-EA) were detected in 100 percent of patients with recurrent CMV infection and in 50 percent of patients with primary CMV infection. These results clearly show the difference in antibody responses to the various antigens of CMV in patients with primary and recurrent CMV infection.

Morphology and development of Rift Valley fever virus in Vero cell cultures.

Rift Valley fever virus (RVFV) grown in vero cell cultures has a completed replication cycle within 13 hours. The first signs are the appearance of intranuclear fibrillar rods, followed by aggregations of precursor viral material in host cell cytoplasm and viral nucleocapsids budding into vacuoles associated with the Golgi apparati. Mature particles, liberated by the disintegration of vero cells, contained ribosomelike structures within the nucleocapsid, which was surrounded by a typical unit membrane through which were inserted some 350-375 surface spikes whose inner ends were incorporated into the nucleocapsid structure. In the negatively stained material, the overall diameter of the virion was 90-110 nm; the spikes were 10-18 nm in length and 5 nm in diameter.

Viral antibody titers. Comparison in patients with multiple sclerosis and rheumatoid arthritis.

Higher titers of antibodies to measles virus envelope antigens, hemolysin and hemagglutinin, Epstein-Barr virus (EBV) capsid antigen and nuclear antigen, and rubella virus hemagglutinin were demonstrated in serum samples of patients with multiple sclerosis and rheumatoid arthritis than in age- and sex-matched control subjects. A significant correlation was observed between antibodies to measles and rubella viruses both in patients with multiple sclerosis and rheumatoid arthritis, but such a correlation was not observed between antibodies to EBV and measles or rubella viruses. Whether elevated levels of antibodies to EBV are due to reactivation of the virus, or elevated levels of antibodies to all the enveloped viruses result from cross-reactions between viruses and host tissue, or perhaps reflect defects in immunoregulation, needs further investigation.

Antibodies specific for measles virus envelope antigens and autoantibodies in patients with chronic active hepatitis.

Distinctly increased levels of antibodies to measles virus envelope antigens haemolysin and haemagglutinin were found in the sera of patients with chronic active hepatitis compared with a normal control group, using immunofluorescence and functional tests. Similarly, a higher incidence of smooth muscle antibody of both IgG and IgM classes was observed in the patients and an important correlation was found between haemolysin antibodies specific for measles virus and smooth muscle antibody of IgG and IgM classes. In contrast, there was no such correlation between the virus specific haemolysin antibodies and antinuclear antibodies. The increased levels of antibodies to measles virus envelope antigens and of autoantibodies may reflect defects in immunoregulation rather than persistent infection with measles virus.

Recognition of host-membrane antigens in the envelope of measles virions.

Trypsin- and acetone-treated virions from either of two strains of measles virus grown in Vero cells stimulated the production in guinea pigs of (i) virus-specific antibodies to polypeptide (P) of molecular weight 70,000 (70K) and to the portion of the HA spike embedded in the viral envelope, but not to M protein, and (ii) antibodies to two host cell membrane antigens which were identified as glycoproteins with molecular weights of 108K and 104K. These host cell antigens were present in increased amounts in infected cells and were intimately associated with the virus. Untreated measles virions grown in Vero cells also stimulated the production of antibody to the 108K glycoprotein. The host polypeptides were less antigenic in virus derived from HEp2 cells, which apparently contained less of these antigens.

Immunoelectron microscopic studies on haemagglutinin and haemolysin of measles virus in infected HEp2 cells.

The antigenic determinants of the haemagglutinin and haemolysin antigens of measles virus were located at the surface of HEp2 cells infected with measles virus and on measles virions released from these cells, using immunoelectron microscopy. Antisera specific for haemagglutinin or haemolysin antigen and peroxidase-conjugated antiglobulin were used. Treatment of the infected cells with trypsin removed the virus spikes and prevented binding by the anti-haemagglutinin serum, while the reaction with anti-haemolysin serum was unaltered. This suggests that the antigenic determinants for measles haemagglutinin reside on the spike, while the antigenic determinants for haemolysin reside on, or are close to, the virus membrane.

Antigen and polypeptide synthesis by temperature-sensitive mutants of respiratory syncytial virus.

A revised nomenclature for the polypeptides of respiratory syncytial (RS) virus has been devised on the basis of comparison of the Long, A2 and RSN-2 strains by slab-gel electrophoresis. Seven polypeptides, now designated VP200, VGP48, VPN41, VPP32, VPM27, VP25 and VP10, were observed in preparations of all three strains of RS virus, irrespective of the host cell of origin. In addition, a slowly migrating glycopolypeptide GP1 was prominent in partially purified RS virus of the Long and A2 strains obtained from Hep-2 cells, and to a lesser extent from BS-C-1 cells. In the case of the RSN-2 strain, this polypeptide was only resolved clearly in virus obtained from Hep-2 cells. GP1 was an atypical glycopolypeptide in that 35S-methionine incorporation was poor relative to 3H-glucosamine incorporation. The ts mutants of RS virus exhibited four distinct phenotypes with respect to intracellular polypeptide synthesis and antigen production of 39 degrees C. Mutants ts 17 (complementation group B') and ts 19 (group E) were almost completely restricted, suggesting defective early functions. Mutants ts A1 (group A), ts A7 (group C) and ts 1 (group D) synthesized antigen and polypeptides normally, but the amount of antigen at the cell surface was reduced, suggesting maturation defects. In addition, the VPP32 of ts 1 (group D) exhibited an aberrant mobility, confirming its viral specificity. The remaining mutants, representing groups B, F and G exhibited generally impaired synthesis at 39 degrees C. Absence of surface filaments in ts mutant-infected cells at 39 degrees C confirmed their virus-specific nature.

Examination of the immunological relationship between measles virus and canine distemper virus using monospecific measles antisera.

Indirect immunofluorescence titrations were performed with measles virus, the Rockborn strain of canine distemper virus (CDV), and a large plaque variant of the Onderstepoort strain of CDV (Ond-LP) using monospecific antisera prepared against either the haemagglutinin (anti-HA), the haemolysin (anti-HL), or the ribonucleoprotein (anti-RNP) of measles virus. Tests with anti-HA showed that the Rockborn strain of CDV was more closely related to measles virus than Ond-LP. The ribonucleoprotein antigens of the CDV strains were closely related to each other but were both related to and distinct from measles virus RNP. The use of measles anti-HL serum demonstrated that CDV possesses an antigenically related acetone-sensitive component equivalent to the haemolysin of measles virus. Absorption of human convalescent serum with excess quantities of acetone-fixed CDV antigens had no effect on measles-specific anti-HA, HL, or RNP activity in the serum. Absorption with measles antigens on the other hand, totally removed all measles and CDV-specific HA and RNP activity. CDV was not neutralised by any of the monospecific antisera when tested either as individual antisera or as mixtures. Our results demonstrate the occurrence of antigenic variation between different strains of CDV, they also reveal unique antigenic determinants in both measles virus and CDV.

Virus-specific and anticellular antibodies in molluscum contagiosum.

Sera from patients with molluscum contagiosum showed a higher incidence of anticellular and fibrillar anticellular antibodies of IgM class as compared to sera from control subjects. Most patients with anticellular IgM antibodies also had molluscum contagiosum virus-specific antibodies. In a comparative study, there was a higher incidence of anticellular IgM antibodies to normal human epidermis and fibrillar anticellular IgM antibodies in molluscum contagiosum than in warts of psoriasis. The incidence in psoriasis was lower than warts and was similar to that found in control subjects.

Pneumoviruses: the cell surface of lytically and persistently infected cells.

Human embryonic lung (MRC-5), feline embryo (FEA), mink lung (Mv1Lu) and monkey kidney (BSC-1) cells infected by respiratory syncytial virus showed characteristic morphological changes when viewed by scanning electron microscopy. The surfaces of respiratory syncytial virus-infected cells developed a profusion of slender filaments after 48 h incubation at 31 degrees C. Similar changes in surface morphology were observed in BSC-1 cells infected by murine pneumonia virus. Filament production therefore appears to be a common property of pneumo-viruses. Filaments were not observed in cells infected with either syncytial and non-syncytial herpes simplex virus, the cytocidal vesicular stomatitis and Batai (Bunyaviridae) viruses, or the focus-inducing rabbit fibroma virus. Filament production was not observed in cells infected with ts mutants of respiratory syncytial (RS) virus during incubation at the restrictive temperature, or in a persistently infected culture of BSC-1 cells at 37 degrees C. The persistently infected cells (the RS ts 1/BSC-1 line) had some of the characteristics of cells transformed by oncogenic viruses, namely ability to overlap adjacent cells and agglutination by a low concentration of concanavalin A. The pseudo-transformed phenotype was temperature-dependent, however, and suppressed by raising the temperature of incubation to 39 degrees C. The presence of virus antigen at the cell surface was similarly temperature-dependent in these cells, diminished at high temperature (39 degrees C) and enhanced at low temperature (31 degrees C), suggesting that the changes in the host cell were the result of insertion of virus protein into the cell membrane. Evidently, persistent infection by a cytoplasmic virus can produce alterations in the host cell usually associated with transformation by nuclear viruses.

The sensitivity of measles virus haemolysin to acetone and the preparation of mono-specific human anti-haemolysin by absorption.

The haemolysin of measles virus, either in the virion or in infected cells, is functionally and antigenically sensitive to acetone. Absorption of human sera with acetone-treated, measles virus-infected cells removes antibodies to all measles virus structural antigens except haemolysin. The antibody titres of absorbed sera give good correlation in HLI, neutralization and fluorescent antibody staining on unfixed infected cells.

Measles virus-specific antibodies and immunoglobulin M antiglobulin in sera from multiple sclerosis and rheumatoid arthritis patients.

When rheumatoid factor in rheumatoid arthritis and multiple sclerosis sera was titrated by the fluorescent antibody method on measles virus-infected cells, there was a marked and variable drop in titer on acetone-fixed cells as compared with unfixed cells. This was accounted for by the failure of measles virus hemolysin-inhibiting (HLI) antibody of the immunoglobulin G class to bind to acetone-fixed infected cells. It was shown by staining unfixed and acetone-fixed measles virus-infected cells that rheumatoid factor in most rheumatoid arthritis sera combined with measles virus-specific hemagglutinin-inhibiting and HLI antibodies, whereas rheumatoid factor in multiple sclerosis sera combined only with HLI antibody. Rheumatoid factor of similar specificity was also observed in normal sera and occasionally in rheumatoid arthritis sera. Both rheumatoid arthritis and multiple sclerosis sera showed almost identical increases in average titer above normal of measles virus-specific fluorescent staining immunoglobulin G and HLI antibodies.

Affinity for measles virus anti-haemolysin of a residual immunoglobulin M in sera of some patients with multiple sclerosis.

HEp2 cells persistently infected with measles virus were treated with trypsin to remove haemagglutinin (HA) and examined unfixed or fixed in acetone by fluorescent antibody methods, comparing those sera specific for structural antigens of the virus. Staining patterns combined with the blocking of specific immunofluorescence indicated that IgG specific for measles virus haemolysin could be recognized in multiple sclerosis (MS) sera and that in some sera from which rheumatoid factor had been removed, a residual IgM (MS-IgM) was absorbed to measles virus-infected cells and showed the same specificity in blocking tests as measles virus anti-haemolysin. MS-IgM could be removed from sera by absorption with latex particles coated with human IgG and would seem to be anti-globulin with preferential affinity for anti-haemolysin.

Initiation and maintenance of persistent infection by respiratory syncytial virus.

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (39 degrees C) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (ts1) from group D and a noncomplbmenting mutant produced persistent infections. The persistently infected culture initiated by mutant ts1 (RS ts1/BS-C-1) has been maintained in serial culture for greater than 100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS ts1/BS-C-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 37 degrees C. The RS ts1/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS ts1/BS-C-1 culture differed from that of unifected cells.