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PubMed1978

Enzyme-linked immunosorbent assay for measurement of serological response to respiratory syncytial virus infection.

An enzyme-linked immunosorbent assay was applied to the detection of serum antibodies against respiratory syncytial virus. The end points of the various sera tested in the assay were approximately 100 times higher than in the complement-fixation test and 2 to 4 times higher than in the plaque reduction test. In addition, the immunosorbent assay appeared to be more efficient than the plaque reduction and complement-fixation techniques for detecting a serological response in young infants (1 to 6 months old) with serous respiratory syncytial virus lower respiratory disease. The simplicity, sensitivity, and rapidity of the enzyme-linked immunosorbent assay make it a useful tool for immunological studies with respiratory syncytial virus.

Antibodies, Viral↗
PubMed1981

Infectivity of respiratory syncytial virus by various routes of inoculation.

To understand the transmission of respiratory syncytial virus, we examined the frequency of infection in volunteers after inoculation by different routes with varying doses of virus. Thirty-two adult volunteers received serial dilutions of a safety-tested live strain of respiratory syncytial virus instilled into nose, eye, or mouth. The highest inoculum, 5.2 log10 50% tissue culture infective dose (TCID50), was administered to four groups of four subjects each, by nose to one group, by eye to one group, and by mouth to two groups. Subsequently, 1:100 and 1:1,000 dilutions of this inoculum were administered by nose and eye. At the highest inoculum, infection occurred in three of four subjects inoculated by nose and in three of four subjects inoculated by eye. Infection occurred in one of eight subjects inoculated by mouth, but this subject most likely was infected by secondary spread. With an inoculum of 3.2 log10 TCID50, the proportion of subjects infected by either route diminished to 25%. When the inoculum was further reduced to 2.2 log10 TCID50, no infections occurred by either route. Infections after the highest inoculum were characterized by earlier and greater shedding. These findings suggest that respiratory syncytial virus may infect by eye or nose and that both of these routes appear equally sensitive. In comparison, the mouth appears to be an insensitive route of inoculation. This is of potential import in infection control procedures and in the development of vaccines or other prophylactic measures.

Antibodies, Viral↗
PubMed1982

Development of antibody-dependent cell-mediated cytotoxicity in the respiratory tract after natural infection with respiratory syncytial virus.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured in nasopharyngeal secretions collected from 42 infants and young children at various intervals after primary or secondary infection with respiratory syncytial virus. ADCC was determined by specific immune release of 51Cr from respiratory syncytial virus-infected HEp-2 cell culture monolayers, with lymphocytes from adult volunteers as effector cells. Specific ADCC responses in nasopharyngeal secretions after primary infection were observed as early as 3 days after the onset of clinical symptoms, and peak activity was observed 14 to 29 days after the onset of illness. ADCC responses after reinfection were significantly greater in both the acute and convalescent phases (P less than 0.05) than were ADCC responses after primary infection. ADCC in secretions was mediated primarily by the immunoglobulin G isotype of respiratory syncytial virus antibody.

Acute Disease↗
PubMed1979

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.

Acute Disease↗
PubMed1983

Rapid immunoperoxidase assay for detection of respiratory syncytial virus in nasopharyngeal secretions.

Samples of nasopharyngeal secretions obtained from 70 infants and young children with acute respiratory disease were examined for the presence of respiratory syncytial virus by immunoperoxidase assay (IPA). The IPA was compared with the immunofluorescence assay and with cell culture isolation. Respiratory syncytial virus antigen-positive cells were detected by both IPA and immunofluorescence assay in 28 specimens; 25 samples were positive in cell culture. The agreement between virus isolation and IPA and IFA was 89%. The applicability of IPA to rapid viral diagnosis of respiratory syncytial virus infection is discussed.

Child↗
PubMed1983

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.

Antibodies, Monoclonal↗
PubMed1985

Monoclonal antibodies against respiratory syncytial virus and their use for rapid detection of virus in nasopharyngeal secretions.

We developed five monoclonal antibodies against respiratory syncytial virus. Three of these (23A3, 12A4, and 18B2) were used in an indirect fluorescent antibody test, and the results were compared with those of a similar indirect fluorescent test with commercial anti-respiratory syncytial virus serum. The results obtained with antibody 18B2 and commercial anti-respiratory syncytial virus serum were identical, whereas with antibodies 23A3 and 12A4 the incidence of positive identifications was around 50%.

Antibodies, Monoclonal↗
PubMed1989

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.

Antibodies, Monoclonal↗
PubMed1990

Evaluation of the Becton Dickinson Directigen test for respiratory syncytial virus in nasopharyngeal aspirates.

A premarket trial of the Becton Dickinson Directigen respiratory syncytial virus membrane-based enzyme immunoassay compared the test with virus isolation for the detection of respiratory syncytial virus in 583 nasopharyngeal aspirates. After modification, the Directigen test showed a sensitivity of 83% and a specificity of 90%. It offers the potential for an efficient bedside test--without the need for any equipment--for the diagnosis of respiratory syncytial virus infection and requires only a 0.25-ml sample volume. However, for optimum reliability, freezing-thawing of samples and access to a confirmatory test were shown to be necessary.

Adolescent↗
PubMed1992

Immunoglobulin G antibody avidity in patients with respiratory syncytial virus infection.

The titer and avidity of respiratory syncytial virus-specific antibodies were measured in 196 serum specimens from 93 children with an acute, laboratory-confirmed respiratory syncytial virus infection. An enzyme immunoassay method based on the ability of urea to dissociate the bound antibodies with low avidity from the antigen was used. Three patterns of immune responses were observed. Children less than 6 months of age usually had low titers of antibodies with high avidity in their acute-phase serum samples. These antibodies were concluded to be of maternal origin, since their reaction pattern was similar to that of healthy adults. During the next few weeks, a slight increase in titers with a concurrent decrease in antibody avidity was observed. All children 6 to 24 months of age had low-avidity antibodies in their acute-phase serum samples, which matured to high avidity during the follow-up. On the contrary, about half of the children greater than 24 months of age had high-avidity antibodies already in the acute-phase serum samples. We conclude that the former children were experiencing primary infections with respiratory syncytial virus and the latter were experiencing reinfections. All adults with remote immunity had antibodies with high avidity.

Adult↗
PubMed1980

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.

Cell Line↗
PubMed1981

Respiratory syncytial virus proteins: identification by immunoprecipitation.

The proteins of respiratory syncytial virus have not been clearly identified due to the lability of the virus and difficulties in its purification. We have pulse-labeled respiratory syncytial virus with [35S]methionine and [35S]cysteine and analyzed cell lysates by polyacrylamide gel electrophoresis. Five 35S-labeled viral proteins ranging in molecular weight from 21,000 to 73,000 (VP73, VP44, VP35, VP28, and VP21) were easily discernable above background cellular proteins. Treatment of the infected cells with 0.15 M NaCl before labeling suppressed host cell protein synthesis and allowed clearer visualization of the five viral proteins by polyacrylamide gel electrophoresis. Three glycoproteins (VGP 92, VGP 50, and VGP 17) were also identified after labeling with [3H]glucosamine. Five of these polypeptides (VP51, VP44, VP35, VP28, and VGP92) were shown to be antigenically active because they could be immunoprecipitated with anti-respiratory syncytial virus antibody produced in New Zealand white rabbits, cotton rats, and humans before analysis by polyacrylamide gel electrophoresis.

Antigen-Antibody Complex↗
PubMed1982

Analysis of proteins synthesized in respiratory syncytial virus-infected cells.

The spectrum of respiratory syncytial virus-encoded proteins was examined in infected cell extracts by standard polyacrylamide gel electrophoresis and by two-dimensional gel analysis. Polyacrylamide gel electrophoresis analysis of a variety of respiratory syncytial virus-infected, actinomycin D-treated cell lines revealed the presence of as many as nine virus-encoded proteins. Seven of these nine proteins were immunoprecipitated by anti-respiratory syncytial serum. Only one major band of [3H]glucosamine was detected in infected cell extracts (Vp86), whereas the reported major virion glycoprotein (Vp48-53) was difficult to detect in infected cells when carbohydrate labels were employed. Two-dimensional gel analysis easily identified seven viral proteins, and one other was tentatively identified. The reported major virion glycoprotein again was not consistently detected. The results of this study confirm the existence of a virus-coded glycoprotein (Vp86) in infected cell extracts. The existence of this glycoprotein in the purified virion has been in dispute, but the apparent low methionine content of this protein may be the reason for this controversy.

Animals↗
PubMed1984

Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes.

Nine mRNAs, their cDNA clones, and a genome transcriptional map have been reported previously for respiratory syncytial virus (P. L. Collins and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 80:3208-3212, 1983). We report here the identification of a 10th viral mRNA, designated mRNA 2b (molecular weight [MW] ca. 0.39 X 10(6)), that was detected by RNA (Northern) blot hybridization with cDNA clones. Analysis of a polycistronic readthrough transcript was used to deduce the position in the viral transcriptional map of the gene encoding the newly identified mRNA. The polypeptide coding assignments of 9 of the 10 respiratory syncytial virus mRNAs were determined. Individual viral mRNAs were purified by hybridization selection with nine unique, nonoverlapping cDNA clones and analyzed by translation in vitro. Each of the nine mRNAs encoded a single polypeptide chain. The coding assignments were as follows: RNA 1a (MW ca. 0.24 X 10(6)), a 9,500-dalton (9.5K) protein; RNA 1b (MW 0.26 X 10(6)), an 11K protein; RNA 1c (MW 0.26 X 10(6)), a 14K protein; RNA 2a (MW 0.38 X 10(6)), the 34K phosphorylated (P) protein; RNA 2b (MW 0.39 X 10(6)), a 36K protein; RNA 3a (MW 0.40 X 10(6)), the 26K matrix (M) protein; RNA 3b (MW 0.40 X 10(6)), a 24K protein; RNA 4 (MW 0.47 X 10(6)), the 42K major nucleocapsid (N) protein; and RNA 5 (MW 0.74 X 10(6)), a 59K protein. The cDNA clones used for the hybridization selections were respiratory syncytial virus specific and did not hybridize with uninfected-cell mRNA; therefore the proteins synthesized with the selected mRNAs were virus specific. The 9.5K, 11K, 14K, 24K, M, P, 36K, N, and 59K proteins were encoded by different mRNAs; therefore these nine proteins are all unique. The 9.5K, 11K, 14K, 24K, M, P, and N proteins synthesized in vitro with hybrid-selected mRNAs each had counterparts with the same electrophoretic mobilities in extracts of virus-infected cells. The in vitro polypeptides and their authentic counterparts were shown to be closely related by limited digest peptide mapping. The 36K and 59K polypeptides lacked counterparts with the same electrophoretic mobilities in infected cells and therefore are candidates for the unprocessed precursors of the viral F and G glycoproteins. The 10th viral mRNA, the 2,500K RNA 7, was not tested directly but is the only known mRNA of the appropriate size to encode the 200K large (L) protein of the viral nucleocapsid. These assignments account for all 10 of the reported viral mRNAs and bring to 10 the number of known unique viral proteins.

Animals↗
PubMed1984

Sequence analysis of the respiratory syncytial virus phosphoprotein gene.

A recombinant cDNA plasmid (pRSA3) containing an almost full-length copy of the mRNA encoding respiratory syncytial virus phosphoprotein was identified in a cDNA library prepared with mRNA from respiratory syncytial virus-infected cells. The cDNA insert was sequenced, and a protein of 27,150 daltons was deduced from the DNA sequence. The protein is relatively acidic, containing two clusters of acidic amino acids, one in the middle of the molecule and the other at the C-terminus. It is devoid of both cysteine and tryptophan. There was no other potential reading frame within the phosphoprotein gene of respiratory syncytial virus. This situation is unlike that with Sendai virus, a paramyxovirus, which has a nonstructural C protein encoded by a second overlapping reading frame near the 5' end of the mRNA for phosphoprotein.

Amino Acid Sequence↗
PubMed1986

Neonatal respiratory syncytial virus infection: role of transplacentally and breast milk-acquired antibodies.

The effect of transplacentally and breast milk-acquired antibodies on respiratory syncytial virus infection was studied in neonatal and 2-month-old cotton rats. Adult female rats infected intranasally with live virus regularly produced virus-specific antibodies in the serum, colostrum, and breast milk. By using foster feeding techniques, we showed that both transplacentally and breast milk-acquired antibodies were effective in reducing the replication of respiratory syncytial virus in the lungs of neonatal animals when they were challenged with live virus via the nasal route at 3 days of age. However, the protection provided by these antibodies was rather brief. There was no difference in the replication of respiratory syncytial virus in the lungs of 2-month-old animals that were delivered and nursed by seropositive (immunized) or seronegative (control) cotton rats.

Animals↗
PubMed1990

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.

Animals↗
PubMed1968

Application of immunofluorescent antibody technique in rapid diagnosis of respiratory syncytial virus infection.

Seventy-eight unselected children under the age of 2 years suffering from acute respiratory infections were investigated by the fluorescent antibody technique and a comparison was made with conventional isolation and serological methods. Sixty-nine per cent. of children with bronchiolitis were diagnosed as suffering from respiratory syncytial virus infection on the day of admission by examination of nasopharyngeal secretions. There were 44 children with respiratory syncytial virus infection diagnosed by conventional methods over a month, but by using fluorescent antibody techniques on tissue culture 53% were diagnosed by the second day, 71% by the fourth day, and 82% by the seventh day. The method of choice for a rapid diagnosis of respiratory syncytial virus infection is by examining nasopharyngeal secretions, when 90% of those with this infection can be diagnosed on the day of admission to hospital.

Animals↗