Maternal antibody and respiratory syncytial virus infection in infancy

One hundred newborn infants were studied prospectively for 1 year for evidence of infection with respiratory syncytial virus (RSV). The indirect membrane fluorescence technique was used to determine specific antibody in sera. Infection was shown in 29 cases. In 31 infants exposed to an RSV epidemic season, there was no evidence of infection. Maternal antenatal sera were also tested, and a wide range of IgG antibody to RSV was found. Mean titre of maternal IgG antibody to RSV was significantly higher (P < 0.001) in those mothers whose babies remained uninfected than in those whose babies had proved RSV infection before 6 months of age. Babies born to mothers with high levels of IgG antibody to respiratory syncytial virus were protected against infection with this virus during the first months of life when the risk of severe disease was greatest.     

The detection of respiratory syncytial virus in nasopharyngeal aspirates: assessment, formulation, and evaluation of monoclonal antibodies as a diagnostic reagent

Comparisons were made between standard methods of cell culture, indirect immunofluorescence (IF) using hyperimmune respiratory syncytial virus (RSV) antiserum, and indirect IF using mouse monoclonal antibodies directed against various epitopes of RSV for the detection of RSV in nasopharyngeal aspirates. The monoclonal antibodies were used singly and in pools of different specificities which in turn were tested in both direct and indirect IF. In a preliminary study, aspirates from 227 infants were examined for RSV by standard methods. The results were compared with the detection of RSV in these aspirates using nine separate monoclonal antibodies and a pool consisting of five monoclonal antibodies. Respiratory syncytial virus was detected in 64 (28%) by cell culture, in 68 (30%) by indirect IF using bovine polyclonal antibody (BPA), and in 75 (33%) by indirect IF using the monoclonal antibody pool. The nine individual monoclonal antibodies when tested separately were less sensitive, detecting between 8 and 77% of all aspirates found to be positive by culture. After statistical analysis of the results obtained in the preliminary study, a refined monoclonal antibody pool was prepared and in a further study was tested by both direct and indirect IF in parallel with our two standard methods. Slides prepared from 303 nasopharyngeal aspirates collected between 1981 and 1984 and either tested the same day or stored at -20 degrees C were used to evaluate these reagents. Overall agreement between the four tests was found in 274 (90%) specimens. Cell culture detected RSV in 68 (22%) specimens, indirect IF with BPA in 67 (22%), indirect IF with monoclonal antibody in 72 (24%), and direct IF with monoclonal antibody in 79 (26%). The pool of monoclonal antibodies used in direct or indirect IF was thus more sensitive than our standard methods for the detection of RSV in nasopharyngeal aspirates, and direct IF tests could be completed in 40 minutes.     

Role of complement in neutralization of respiratory syncytial virus

Serum neutralizing antibody titers to respiratory syncytial virus (RSV) are higher when assayed with guinea pig complement. A number of different mechanisms have been suggested for enhancement of neutralization by complement. The most straightforward is that complement-antibody complexes present a greater steric hindrance to viral entry than with antibody alone. To define the implications of measuring serum neutralizing antibody with and without complement, sera from adults, young children, infants, and cord bloods were run in plaque neutralization assays with representative viruses of the RSV A and B subgroups. Although titers of neutralizing antibody were higher in the presence of complement, the addition of complement did not increase the ability to detect antibody rises after natural infection. Some of the complement effect may be attributable to an inhibition of RSV replication by complement alone. While these observations do not address the role for complement in the pathogenesis of RSV infection, they suggest that neutralization assays performed without complement may be most reflective of physiologic conditions in the respiratory tract.     

Bovine respiratory syncytial virus antibodies in non-bovine species

Summary To study the role of non-bovine species in the epidemiology of bovine respiratory syncytial virus (RSV) infections, sera obtained from 9 non-bovine animal species and from humans were examined for bovine RSV specific antibodies. Sera were mainly from animals and humans which had been in contact with cattle. Forty sera of each species were tested in an RSV specific whole virus ELISA as well as in a peptide based ELISA, that was developed to measure antibodies specific for bovine RSV. Antibodies directed against RSV were detected in over 50% of sera obtained from sheep, goat, cattle and human beings, and anti-RSV activity was also found in some roe and dogs and one horse. Antibodies to bovine RSV were found in sera of all tested cattle, 11 (27.5%) goats and in some other individual animals: 3 horses, 2 roe, 1 cat and 1 dog. These results indicate that of the investigated species, besides cattle only goats might play a role in the epidemiology of bovine RSV.     

Respiratory syncytial virus infection in mice

The A2 strain of human respiratory syncytial virus replicated in the nose and lung of BALB/c mice, with virus growing to higher titers in older animals than in younger animals. Virus was recovered from the nose between days 2 and 7 with peak titers on days 3 and 4, and from the lungs between days 2 and 9, with peak titers on days 4 through 6. Serum antibody developed 2 weeks after infection. Viral antigen was demonstrated in the alveolar cells of the lung by immunofluorescence. Histopathological changes included infiltration by mononuclear cells of the peribronchiolar and perivascular tissue, some interstitial thickening, and formation of multinucleated giant cells. Virus could not be recovered from the respiratory tract of mice inoculated with bovine strains of respiratory syncytial virus. Growth of the A2 strain of human respiratory syncytial virus in different cell lines affected its infectivity for mice. Infection of BALB/c mice with respiratory syncytial virus provides a highly reproducible model for the study of the pathogenesis of and mechanisms of immunity to this virus.     

Morphological variation of a syncytial virus from lymphosarcomatous and apparently normal cattle

Summary Double-membrane enveloped, endoplasmic reticulum-associated (ERA) viral particles occurred in the cytoplasm of bovine embryonic splenic cell cultures previously infected with bovine syncytial virus (BSV) that had been isolated from lymphosarcomatous and apparently normal cattle. Previously, BSV was seen only in budding forms at the plasma membrane.It is proposed that the double membrane enveloped, ERA viral-like particle is an aberrant form of BSV because: (1) the ERA viral particle had a nucleocapsid morphologically similar to BSV; (2) it was associated with BSV immunofluorescence; (3) it occurred in the same cells and cell cultures as BSV; and (4) it was not demonstrated in cell cultures in the absence of BSV.     

Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: development and description

An indirect enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV) antigens was developed, using commercially available antisera. Horse anti-RSV and calf antiserum to bovine RSV were used as capture and detector antibodies, respectively. The assay could detect as few as 50 PFU of unpurified RSV per ml in infected cell culture supernatant fluids and as little as 10 ng of affinity-purified RSV antigen per ml. No cross-reactions were observed with heterologous virus types. Freeze-thaw treatment had no effect on RSV enzyme-linked immunosorbent assay titers, but viral transport medium inhibited RSV enzyme-linked immunosorbent assay titers from 10- to 100-fold. The assay can be easily performed in 24 h and is a sensitive and specific method for the detection of RSV antigens.     

Commercial monoclonal antibodies for rapid detection of respiratory syncytial virus by direct immunofluorescence

A commercially available direct immunofluorescence (IF) test using a reagent consisting of a pool of two monoclonal antibodies against selected surface and internal proteins of respiratory syncytial virus (RSV) was evaluated in comparison with the indirect IF technique using a commercial bovine anti-RSV hyperimmune serum for the rapid detection of RSV in nasopharyngeal aspirates from 228 hospitalized children. Overall agreement between the two IF methods was 95%. The direct IF test was quicker to perform and easier to interpret than the indirect IF test.     

Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5' Taq nuclease assays with fluorescent 3' minor groove binder-DNA probes

Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqMan)MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5' Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.     

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.     

Identifying infections with respiratory syncytial virus by using specific immunoglobulin G (IgG) and IgA enzyme-linked immunosorbent assays with oral-fluid samples

Currently, respiratory syncytial virus (RSV) infection is identified in epidemiological studies by virus antigen or nucleic acid detection in combination with serology. Oral-fluid specimens may provide a noninvasive alternative to blood, and oral fluid is more suitable for sampling outside of the clinic setting. We evaluated an indirect enzyme-linked immunosorbent assay for the detection of RSV-specific immunoglobulin G (IgG) and IgA by using oral-fluid samples collected from individuals with RSV infections confirmed by an immunofluorescent antibody test. For five children sampled repeatedly from birth, antibody profiles in oral fluid quite consistently tracked those in paired sera, and RSV infections were detected by rising titers of antibodies of at least one Ig class. Specific IgG responses were generally more reliable than IgA responses, except in early infancy, where the reverse was sometimes true. For a further five young children from whom oral fluid was collected weekly following RSV infection, boosted antibody responses, frequently of a transient nature, lasting a few weeks, were observed; specific IgG responses were of longer duration and more pronounced than specific IgA responses. Our data show significant promise for the use of oral fluid alone in RSV infection surveillance. The observed rapid dynamics of the antibody responses are informative in defining study sampling intervals.     

Immune responses of lambs experimentally infected with bovine respiratory syncytial virus and Pasteurella haemolytica.

The immune responses of lambs experimentally infected with bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica were compared with lambs infected with bovine RSV or Pasteurella haemolytica alone. Superinfection with P. haemolytica aggravated the reduction in the number of CD5+, CD4+ and LCA p220+ (B) lymphocytes caused by bovine RSV, but it had no effect on the neutralizing antibodies against P. haemolytica cytotoxin. Serum samples obtained from lambs experimentally infected with bovine RSV and P. haemolytica had significantly lower amounts of neutralizing antibodies to bovine RSV than those obtained from lambs infected with bovine RSV alone.     

Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by a commercial enzyme immunoassay.

A commercial enzyme immunoassay (EIA) for the rapid detection of respiratory syncytial virus (RSV) in respiratory secretions was evaluated by comparison with both virus isolation in HEp-2 cells and indirect immunofluorescence (IFA) staining of exfoliated respiratory cells. Initial examination of 80 nasopharyngeal aspirates collected from infants with acute respiratory illness showed that the RSV EIA was positive for 21 of 24 specimens positive by virus isolation or IFA (87.5% sensitivity) and negative for 53 of 56 specimens negative by virus isolation and IFA (95% specificity). The EIA appears to be an acceptable and more rapid test than virus isolation for the detection of RSV, especially for laboratories in which prompt inoculation of specimens is not always possible. IFA staining with commercial bovine anti-RSV serum was found to be the most sensitive and rapid test for the detection of RSV. However, three of four specimens positive by IFA and negative by virus isolation were not cultured under optimal conditions. In addition, the IFA test requires a highly trained technologist to interpret the staining results.     

Antigenic analysis of the F protein of the bovine respiratory syncytial virus: identification of two distinct antigenic sites involved in fusion inhibition

Summary From two independent fusions, fifteen MAbs directed to the F protein of the bovine respiratory syncytial virus (BRSV) were characterized by radioimmunoprecipitation assays. Competition binding assays among these MAbs identified two distinct antigenic sites (A and B) and one overlapping site (AB). All of the MAbs specific to epitopes belonging to site A neutralized the infectivity of the virus in vitro and recognized human and bovine RSV strains. Only two out of the five MAbs directed to epitopes of site B were neutralizing and three reacted with all of the RSV strains tested, suggesting that the epitopes constituting this domain present heterogeneous characteristics. In each of sites A and B, one of the neutralizing MAbs also inhibited cell fusion. The biological relevance of these domains was established by competing representative MAbs and sera from BRSV-infected calves.     

Prevalence of neutralizing antibody to respiratory syncytial virus in sera from mothers and newborns residing in the Gambia and in The United States.

The prevalence of maternal respiratory syncytial virus (RSV)-neutralizing antibodies has been documented in developed countries, but there is little information from developing countries. We assessed the prevalence of RSV-neutralizing antibody in sera from Gambian women and their newborns and compared them with their American counterparts during a similar period. The geometric mean titers of maternal antibodies to RSV subgroup A in the two populations were similar, while titers of antibodies to RSV subgroup B in Gambian mothers were significantly higher (8.7 +/- 1.4 versus 7.9 +/- 1.3 [mean +/- standard deviation], P < 0.001). The titers of neutralizing antibody in newborns in both populations correlated with the neutralizing-antibody titers of their mothers. Thus, the status of neutralizing antibody to both major RSV subgroups was comparable among infants and mothers in a developing country, The Gambia, and those in a developed country, the United States.     

Rapid diagnosis of respiratory syncytial virus infection by antigen immunofluorescence detection with monoclonal antibodies and immunoglobulin M immunofluorescence test.

During a respiratory syncytial virus (RSV) infection outbreak in a pediatric hospital, diagnosis was made by immunofluorescence on smears by using an anti-RSV monoclonal antibody (IFm). Immunoglobulins M and G were titrated by indirect immunofluorescence on HEp-2 cells infected with an RSV strain. The IFm was sensitive (89%) and specific (75%) when compared with the cell culture method. We showed that the specimens which were found positive by IFm and negative by cell culture were truly positive. Under these conditions, the IFm test appears more sensitive and more specific than cell culture, particularly when no care is taken to maintain the specimens in the cold during transport. In this study the immunoglobulin M immunofluorescence test had a low sensitivity (34%), especially on serum samples taken on days 0 to 4 after the onset of illness.     

BALB/c鼠和裸鼠呼吸道合胞病毒感染比较研究

目的 比较普通BALB/c鼠和裸鼠呼吸道合胞病毒(RSV)感染免疫及炎症反应特点.方法 BALB/c鼠和裸鼠感染RSV后不同时间空斑形成试验检测肺组织病毒滴度,计数支气管肺泡灌洗液(BALF)白细胞总数和分类,HE染色分析肺组织病理学改变,免疫组化检测肺组织F4/80+细胞和CD49b+细胞.ELISA检测BALF中TNF-α、IFN-r、IL-12和IL-10浓度.结果 BALB/c鼠和裸鼠感染RSV后肺组织病毒滴度在第3天达峰值,感染裸鼠带毒时间更长,在感染后各天病毒滴度明显高于BALB/c鼠(P＜0.05),肺组织病理改变也更重.感染BALB/c鼠和裸鼠BALF白细胞总数明显升高,分类以淋巴细胞为主.感染裸鼠与感染BALB/c鼠比较,肺组织检测到更多的F4/80+巨噬细胞和CD49b+NK细胞(P＜0.05),BALF中TNF-α、IL-12和IL-10水平更高(P＜0.05).结论 RSV感染裸鼠与BALB/c鼠比较,病毒复制水平更高,时间更持久,炎症反应更重.单核巨噬细胞和NK细胞是RSV感染重要的免疫细胞和炎症细胞,炎症反应强度并不一定与T细胞免疫应答平行.     

Rapid detection of respiratory syncytial virus with a monoclonal antibody.

We developed an indirect fluorescent-antibody test employing a mouse monoclonal antibody directed against the nucleoprotein of RSV for rapid detection of respiratory syncytial virus (RSV). This test demonstrated distinctive fluorescent inclusions in HEp-2 cells infected with 24 RSV isolates collected during 6 previous years but not in cells infected with 13 other respiratory viruses. Examination of nasal cells of 100 infants with acute respiratory illness showed that the indirect fluorescent-antibody test employing the monoclonal antibody was 79% sensitive and 100% specific, as compared with the combination of both culturing and a similar indirect fluorescent-antibody test with commercial anti-RSV serum. This monoclonal antibody is an easily produced, well-characterized, sensitive, and specific reagent for the rapid detection of RSV antigen.     

Simplified enzyme-linked immunosorbent assay for specific antibodies to respiratory syncytial virus.

A simplified and reliable enzyme-linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against respiratory syncytial virus (RSV). RSV-infected cells were fixed and dried on 96-well microtiter plates and kept at 4 degrees C. The titers of reference sera were determined by endpoint dilution. A linear relation was found between the titers and the logarithm of absorbance values of sera diluted to 1:1,000 (r = 0.93, P less than 0.001). Measurement of RSV antibodies was done by using a single serum dilution (1:1,000) in conjunction with a standard curve. A strong correlation was found between complement fixation and ELISA results (r = 0.89, P less than 0.001). In addition, the ELISA method exhibited higher titers and a greater sensitivity than did complement fixation, although the applicability of the assay is limited with positive serum samples of low titer.     

Primary cytotoxic T-cell responses to bovine respiratory syncytial virus in calves.

Bovine respiratory syncytial virus (RSV) is a major cause of respiratory disease in young calves. Recent studies in calves, in which different T-cell subsets were depleted, have shown that CD8+ T cells play a central role in recovery from RSV infection. The present study demonstrates that RSV-specific, major histocompatibility complex-restricted cytotoxic T cells appear in the peripheral blood of gnotobiotic calves 7-10 days after infection with bovine RSV and were also detected in the lungs 10 days after infection. The cytotoxic T lymphocytes (CTL) recognized antigenically distinct strains of bovine RSV. There was no correlation between either the level of CTL activity in the lung or the development of CTL in the peripheral blood and the extent of pneumonic consolidation. The demonstration of CD8+ CTL in the lungs at a time when bovine RSV has been cleared confirms the importance of these cells in recovery from infection.