A reverse passive haemagglutination test for the detection of respiratory syncytial virus in nasal secretions from infants

A reverse passive haemagglutination (RPH) test has been developed for the detection of respiratory syncytial (RS) virus in nasal secretions, taken from infants with acute respiratory illness. In the final form of the procedure, RS virus was detected in 24 of 25 samples positive for RS virus by tissue culture and/or fluorescence antibody staining and in two samples negative for RS virus by these techniques. The simplicity of the technique and the rapidity with which it may be performed together with its apparently high degree of sensitivity should make RPH useful in the rapid diagnosis of RS virus.     

Cell-mediated immunity in respiratory syncytial virus disease

Systemic cell-mediated responses to respiratory syncytial (RS) virus were detected, using a whole blood transformation assay, in 10 of 28 infants and children with RS virus infections during the period 1–14 days postadmission. Cell-mediated responses were unrelated to the age of the patient or the severity of illness. No correlation was found between cellular responses and fourfold or greater rises in antibody titre to RS virus, as determined by a membrane immunofluorescence technique. Patients under 6 months of age had significantly lower levels of IgA and IgG antibody to RS virus compared to older patients, although cell-mediated responses were similar in both groups. The presence of cell-mediated reactivity to RS virus was also demonstrated in 5 of 95 samples of cord blood examined, and cellular responses failed to correlate with the levels of IgG antibody to RS virus.     

The coating of respiratory syncytial (RS) virus-infected cells in the respiratory tract by immunoglobulins.

During the course of 26 respiratory syncytial (RS) virus infections, infected cells in the respiratory tract become coated with immunoglobulins (IgA, IgG, and IgM), IgA being predominant. Methods are described for detecting both intracellular virus and coating immunoglobulin using a double staining technique and immunofluorescence. IgA coating antibody appears in small amounts very early in the illness. The relationship of coating antibody to pathogenesis, prevention, and recovery from RS virus infection is discussed.     

In vitro cell-dependent lysis of respiratory syncytial virus-infected cells mediated by antibody from local respiratory secretions.

Respiratory syncytial (RS) virus causes a local infection of the respiratory tract which is frequently severe in infants. We report the development in infected infants of antibodies in respiratory secretions capable of mediating in vitro destruction of RS virus-infected tissue culture cells in conjunction with non-immune lymphoid cells. The cytotoxic antibody activity was not detectable in nasal secretions from infants hospitalized with respiratory infections where RS virus was not identified. The rise in activity occurred concurrently with recovery from infection and the rise in specific IgG, IgM and IgA antibody levels measured by membrane immunofluorescence assay, but was dissociated from the development of plaque-neutralizing activity. In serum it appears that the cytotoxic antibody belongs to the IgG class as shown by its ability to cross the placenta and by neutralization with specific antiserum. These findings are discussed in relationship to secretory antibody responses in RS virus infection with respect to pathogenesis and recovery.     

Comparison of enzyme-linked immunosorbent assay and neutralization techniques for measurement of antibody to respiratory syncytial virus: implications for parenteral immunization with live virus vaccine.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect low levels of antibody to respiratory syncytial (RS) virus was compared with a tube dilution neutralization test (NEUT) on sera obtained from children who received a parenteral live RS virus vaccine. Among the children who developed antibody in response to live RS virus vaccine. ELISA was as sensitive as NEUT at detecting antibody increases. Some children who did not have detectable prevaccine ELISA antibody possessed NEUT antibody; these children were generally less than 12 months old, suggesting that they had low levels of maternal antibody. Low levels of NEUT or ELISA antibody were associated with the absence of antibody increases after injection of live RS virus vaccine. The quantity of antibody stimulated by this live RS virus vaccine was small compared with that which was stimulated by naturally acquired RS virus infection. We concluded that ELISA is a satisfactory test for determining antibody to RS virus in vaccine field trials, given the understanding that low levels of preexisting antibody are not detected in some instances.     

Human antibody-dependent cell-mediated cytotoxicity against target cells infected with respiratory syncytial virus.

A chromium release assay was established to study human antibody-dependent cell-mediated cytotoxicity (ADCC) of HEp 2 cells infected with respiratory syncytial (RS) virus. Human peripheral blood lymphocytes in the presence of specific antibody to RS virus caused in vitro lysis of RS virus infected target cells. ADCC was detected in sera of infants with RS virus infections and in specimens of colostrum. The ability of serum or colostrum to mediate the cytotoxic reaction appeared to be related to the level of specific IgG, or IgA antibody to RS virus, as detected by membrane fluorescence. Separation of effector cells by their glass adherence properties showed that the ability to produce cytotoxicity resided in non-adherent effector cells.     

Immunofluorescence for routine diagnosis of respiratory syncytial virus infection

A comparison of immunofluorescent tests for the diagnosis of respiratory syncytial (RS) virus infections was carried out on 42 hospitalized cases of respiratory infection in childhood. Respiratory syncytial virus was detected in 22 (52%) cases, the most sensitive method of detection being by indirect immunofluorescence of Bristol HeLa tissue cultures inoculated with nasopharyngeal aspirates. The highest detection rate was in bronchiolitis cases (92%). Detection of antibody rises in paired sera, eight days apart, confirmed RS virus infection in 13 of 16 cases, the most sensitive test being detection of a specific rise in IgG antibody by indirect immunofluorescence. A serodiagnosis was made in all 10 non-bronchiolitis cases. Recommendations are made for the application ofimmunofluorescence to routine diagnosis of RS virus infection.     

Epitope specificities of human serum antibodies reactive with respiratory syncytial virus fusion protein

Summary Respiratory syncytial (RS) virus continues to cause serious human respiratory disease and no prophylactic vaccine is yet available. Serum antibodies to RS virus fusion protein (F) that have the appropriate specificities and activities could confer protection against severe RS virus infections. To explore human serum antibody responses to RS virus F we first characterised four epitopes on F and then measured the concentrations of human serum antibodies to these sites for 389 sera. Individuals varied in serum antibody concentration to the epitopes. The distribution patterns of the concentrations of antibodies reactive to each epitope were different. Antigenic variation of F at these epitopes in Southampton RS virus isolates was examined by immunofluorescence. The F proteins from different isolates varied within and between RS virus subtypes which co-circulated in the outbreak of winter 1985–1986. Variations in F detected by immunofluorescence were consistent with differences between the strains' susceptibilities to monoclonal antibody antiviral action.     

Isolation,characterization, and pathogenicity studies of a bovine respiratory syncytial virus

Summary The isolation and characterization of a bovine respiratory syncytial (RS) virus is described. Serological studies indicate that bovine RS virus is widespread in Iowa cattle and that it is involved in some outbreaks of respiratory disease. Experimental infection in calves indicates that the virus can cause illness in calves, particularly those with serum neutralizing antibody.     

Systemic cell-mediated and antibody responses in infants with respiratory syncytial virus infections

In order to investigate the possible role of immunity in lower respiratory tract disease of infants produced by respiratory syncytial (RS) virus, 18 hospitalized infants were tested for cell-mediated immune (CMI) responses in a whole blood culture assay utilizing a gamma emitting tracer, 5(¹²⁵ I) Iodo-2′-deoxyuridine [¹²⁵ IUdR] to quantitate cellular proliferative responses to virus antigen. Class-specific antiviral antibody titres were determined in an indirect membrane immunofluorescence test. One infant showed a CMI response in the acute phase of illness whereas 72% responded one month later. Of the 18 infants, 14 were tested for antibody responses and 71% showed significant rises of antiviral IgG. IgM was detectable in only one acute phase specimen. A tendency for higher CMI responses following severe infection with RS virus was noted but little difference in antibody responses was respect to severity was seen. These findings are discussed in relationship to the pathogenesis of RS virus.     

Local antibody production and respiratory syncytial virus infection in children with leukaemia

Children undergoing therapy for acute lymphoblastic leukaemia (ALL) are at increased risk of severe viral respiratory infection, and some find it difficult to terminate virus secretion. This increased severity may result from a defect in the mucosal immune response. To test this hypothesis, nasal immunoglobulin secretion and specific antiviral antibody responses to infection with respiratory syncytial (RS) virus in children with ALL have been compared with those in a normal age-matched comparison group. Children with leukaemia secreted normal levels of IgA and slightly raised IgM levels. IgG levels were depressed. Following RS virus infection, the majority of children with leukaemia secreted normal amounts of IgA and IgG nasal antibody and successfully cleared the virus. However, three of the 13 children studied made poor or undetectable nasal antibody responses, which correlated with their inability to clear the virus.     

A study of an inhibitor in bovine serum active against respiratory syncytial virus

Summary An inhibitor for respiratory syncytial virus was demonstrated by a neutralization technique. Twenty (83%) of 24 adult bovine sera had a titre of 1/8 or greater. Some physical and chemical properties of the inhibitor were compared with those of parainfluenza 3 HI antibody and -inhibitor for influenza A virus, and RS inhibitor resembled the antibody rather than the -inhibitor. RS inhibitor was insensitive to trypsin, was not inactivated by 2-mercaptoethanol, and was found in the G serum fraction after Sephadex G200 gel filtration.The incidence of RS inhibitor in bovine sera increased with age. Rises in inhibitor titre were detected in paired sera from 3 of 33 cattle with respiratory illness of unknown aetiology.     

Production of antiserum to respiratory syncytial virus polypeptides: application in enzyme-linked immunosorbent assay.

By use of crossed immunoelectrophoresis techniques, respiratory syncytial (RS) virus-specific precipitates were produced between RS virus cellular antigen [solubilized in tris(hydroxymethyl)aminomethane-glycine buffer, pH 9] and antiserum raised in rabbits against semipurified RS virus. When these precipitates were employed as antigens for further immunizations in rabbits, antibodies (anti-RSV-precip.I) were produced which reacted with only one RS virus antigen when tested by the crossed immunoelectrophoresis technique. Precipitates obtained between RS virus cellular antigen (labeled with L-[35S]methionine) and anti-RSV-precip.I were examined by polyacrylamide gel electrophoresis, which showed that anti-RSV-precip.I precipitated RS virus polypeptides of molecular weights 28,000 to 84,000. Anti-RSV-precip.I was employed as capture antibodies in the enzyme-linked immunosorbent assay, in which RS virus cellular antigen was used as the second layer. Determination of human RS virus immunoglobulin G antibodies by this enzyme-linked immunosorbent assay technique showed a high degree of sensitivity, specificity, and reproducibility.     

Detection of respiratory syncytial virus serum antibodies by an ELISA system

An ELISA test for respiratory syncytial (RS) virus assay was adapted and standardized; it gave 10-15 times higher antibody titres than complement fixation (CF) but was not a more sensitive test for detecting recent RS virus infection in persons above 1 year of age. In testing normal-population sers, ELISA revealed twice more positive sera than the CF test. Owing to its high sensitivity and apparent ability to detect long-persisting antibodies, ELISA is the test of choice for sero-epidemiological surveys on RS virus infections.     

Location and quantitation of antigen on the surface of virusinfected cells by specific bacterial adherence and scanning electron microscopy

A modification of the SABA (surface analysis by bacterial adherence) technique (Huang and Okorie, 1978, 1979) is described which allows precise location and quantitation of antigen on the surface of virus-infected cells by scanning electron microscopy. Experiments with several viruses and host cells have established that the procedure is sensitive and specific. Appearance of antigen on the surface of cells infected by vesicular stomatitis virus (VSV) could be detected by 2.5 h after infection and 1 h before the release of virions. Bacterial adherence was observed with homologous antiserum, and not with heterologous antisera, in experiments with Chandipura virus (CHV), herpes simplex virus (HSV), murine pneumonia virus (PVM), respiratory syncytial (RS) virus and vesicular stomatitis virus.Development of the technique was facilitated by the observation that potoroo kidney cells (PTK-2) were susceptible to infection by RS virus and PVM. Uninfected PTK-2 cells were almost devoid of surface feature, which allowed early discrimination of the filaments uniquely associated with pneumovirus infection (Parry et al., 1979). Linear adherence of staphylococcal cells along these filaments after exposure to homologous antibody demonstrated the localisation of pneumovirus antigen in these filaments.     

The characteristics of the humoral response to respiratory syncytial viral infection in adult patients with different forms of bronchitis]

The levels of specific IgG and IgM were determined in 72 patients with verified respiratory syncytial (RS) virus infection: 11 patients with acute delayed bronchitis (ADB), 26 with recurring bronchitis (RB) 35 with chronic obstructive bronchitis (ChOB), by indirect enzyme immunoassay; RS virus was detected by direct immunofluorescent technique. The activity of the inflammatory process in the bronchi was shown to depend upon RS infection activity in cases of ChOB and RB. RS infection was observed to be significantly dependent on obstruction and bronchospasms. The specific humoral response was reduced and delayed in ChOB and RB as compared to acute bronchitis. The possibility of long-term persistence of RS virus antigen in chronic and recurring forms of bronchitis was demonstrated.     

Cellular reactivity to respiratory syncytial virus in human colostrum and breast milk

Colostrum and breast-milk samples were taken from 23 mothers between 2 days and 7 weeks postpartum and were examined for the presence of cellular reactivity to respiratory syncytial (RS) virus using a lymphocyte transformation assay. Positive responses were detected in nine of the 23 (39%) samples taken at 2-5 days postpartum, but this reactivity was undetectable at 3 weeks. Positive responses developed in a further three mothers during the 3-7-week period of lactation, suggesting a response to virus infection. Colostral whey was found to suppress the cellular response to RS virus and inhibition was related to the level of specific IgA antibody to RS virus present in the whey. The role of colostral cellular reactivity in protection of breast-fed infants from RS virus bronchiolitis is discussed.     

Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection

Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated.     

Respiratory syncytial virus detection by immunofluorescence in nasal secretions with monoclonal antibodies against selected surface and internal proteins

Specimens containing respiratory tract epithelial cells from infants and children with acute respiratory disease were evaluated by using an indirect immunofluorescence technique with two specific respiratory syncytial virus monoclonal antibodies. One (RS/HN 13-1) was directed against a cell surface viral antigen, and the other (RS/HN 25-2) was directed against viral antigen present in large cytoplasmic inclusions. The same results on presence or absence of respiratory syncytial virus were obtained by cell culture and immunofluorescence in 93% of 252 patients tested adequately by both methods. The sensitivity of indirect immunofluorescence was approximately equal to that of cell culture. A total of 84 specimens were positive for RSV by immunofluorescence; 82 of them were positive with both monoclones, and the remaining 2 were positive only with the monoclone directed against the internal protein. The fluorescence pattern of the latter monoclone was unique and easily recognized. Indirect immunofluorescence testing with monoclonal antibodies to respiratory syncytial virus proved to be a very useful diagnostic technique, and results could be obtained within 4 h of specimen collection.     

应用MAC-ELISA方法检测呼吸道合胞病毒IgM抗体

建立酶联免疫吸附试验(MAC-ELISA)检测呼吸道合胞(RS)病毒特异性IgM抗体方法,并与中和试验(NT)进行对比。 取35例患毛细支气管炎婴幼儿的急性期及恢复期双份血清共70份标本进行研究。MAC-ELISA测得急性期血清中RS病毒特异性IgM抗体的阳性率为27/35(77.14％),而恢复期血清中和抗体呈≥4倍升高者为23/35(65.71％)。凡NT阳性病例,MAC-ELISA也呈阳性;凡MAC-ELISA阴性病例,NT也呈阴性。另4例NT阴性而MAC-ELISA呈阳性。两者阳性符合率为88.57％。 本研究也观察了IgM抗体与年龄的关系。在6个月龄以前,MAC-ELISA的阳性率远远高于NT。 实验说明采用MAC-ELISA检测RS病毒特异性IgM是可靠的快速诊断方法。