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PubMed1975

Effect of acetone fixation on infectivity and antigenicity of respiratory syncytial virus and adenovirus in the fluorescent antibody test.

An investigation was made on the effect of acetone fixation on infectivity and immunofluorescent antigenicity of respiratory syncytial virus and adenovirus type 5, lipid- and nonlipid-containing viruses, respectively. Viruses were allowed to replicate in HEp-2 cells, and the cells were then fixed in acetone at 5 C for periods ranging from 30 s to 7 days. Treatment for 10 min was sufficient to inactivate respiratory syncytial virus, whereas infectious adenovirus type 5 could be isolated from cells immersed in acetone for 7 days. There was a gradual reduction, to 50% of that observed at 30 s, in the intensity of fluorescent antibody staining of both viruses with increasing fixation time, but no significant decreases in fluorescent antibody end-point titers of antisera to either virus were observed.

Acetone↗
PubMed1981

Sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus.

A sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus was developed in Vero and MA 104 cell cultures. The tests were performed in semimicrotoiter trays containing 24 wells, 16 mm in diameter. Parainfluenza virus type 1 formed plaques in Vero and MA 104 cells only when trypsin was added to the overlay medium. Plaquing of parainfluenza virus type 1 was more sensitive and technically reproducible in MA 104 cells than in Vero cells. Parainfluenza virus types 2 and 3 and respiratory syncytial virus readily formed plaques in Vero cells. Plaques with all viruses were necrotic in character, except for plaques produced by parainfluenza virus type 3, which appeared red due to an increased uptake of neutral red by infected cells. Different conditions for plaquing of the four viruses had to be used to obtain plaques of suitable size. Antibody titers of commercially prepared guinea pig typing sera were 5- to 50-fold higher by the plaque neutralization test than by complement fixation. The addition of guinea pig immunoglobulin G antiglobulin to the serum-virus mixtures enhanced the conventional neutralization test 5- to 10-fold. The sensitivity and specificity of the plaque neutralization test was also determined with sera of marmosets experimentally infected with parainfluenza virus types 1 and 3. The generally low postinfection titers could be enhanced, on the average, 40-fold by using human immunoglobulin G antiglobulin in the neutralization test. A low degree of cross-reactivity was shown between parainfluenza virus types 1 and 3 both in the conventional neutralization test and in the anti-immunoglobulin enhanced neutralization test.

Animals↗
PubMed1982

Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: application to clinical samples.

An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus antigens was applied to the rapid diagnosis of acute infections in children and was compared with viral culture and immunofluorescence tests. The ELISA test employed commercially available reagents and was run on a day-to-day basis as specimens were received in the laboratory. Sensitivity and specificity by ELISA were 82 and 95%, respectively, compared with culture. In the same specimens, the sensitivity and specificity by immunofluorescence were 86 and 96%, respectively. Nasopharyngeal aspirates were proven to be a better source of viral antigen than were nasopharyngeal swabs. ELISA-positive samples remained positive even when left unrefrigerated for a week or mailed to the laboratory in plastic containers. Respiratory syncytial virus ELISA, like culture, became negative as the disease progressed and showed no superiority over culture for diagnosis late in the illness.

Antigens, Viral↗
PubMed1984

Use of the biotin-avidin system to study the specificity of antibodies against respiratory syncytial virus.

To characterize the specificity of monoclonal antibodies against respiratory syncytial virus, we developed a technique which combined the biotin-avidin system, immunoprecipitation, and transblot electrophoresis. The viral proteins were first biotinylated and then immunoprecipitated with a monoclonal antibody or respiratory syncytial virus immune serum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrophoretically transferred to nitrocellulose paper. The protein bands were located on the paper with avidin-peroxidase plus a precipitable substrate. This procedure gave reproducible results qualitatively similar to those obtained by radioimmunoprecipitation but without the cost, risk of radiation exposure, or disposal problems of radioisotopes. This procedure should have widespread applications.

Antibodies, Monoclonal↗
PubMed1985

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.

Antigens, Viral↗
PubMed1985

Comparison of ortho respiratory syncytial virus enzyme-linked immunosorbent assay and HEp-2 cell culture.

Two hundred seventy nasopharyngeal aspirates were tested in duplicate with the Ortho Diagnostics, Inc. (Raritan, N.J.), respiratory syncytial virus antigen enzyme-linked immunosorbent assay. The test was sensitive (80 to 82%) and specific (96%) when compared with cell culture. The enzyme-linked immunosorbent assay detected seven antigen-positive specimens not among the 71 specimens that were positive for respiratory syncytial virus in cell culture. A blocking test confirmed those specimens as true positives (specificity, 100%).

Antigens, Viral↗
PubMed1986

Enhanced isolation of respiratory syncytial virus in cell culture.

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.

Animals↗
PubMed1986

Serum immunoglobulin G antibody subclass responses to respiratory syncytial virus F and G glycoproteins after primary infection.

Because the immunoglobulin G (IgG) response to carbohydrate antigens is typically from the IgG2 subclass and the IgG response to protein antigens is typically from the IgG1 and sometimes the IgG3 subclass, two respiratory syncytial virus glycoproteins, F and G, which differ substantially in the amount of glycosylation, were used as antigens in an enzyme-linked immunosorbent assay to determine IgG subclass responses in 20 infants and young children with naturally acquired respiratory syncytial virus infection. Both glycoproteins elicited primarily IgG1 and IgG3 responses, indicating that the protein moieties of the glycoproteins may be immunodominant in this age group.

Antigens, Viral↗
PubMed1987

Peroxidase-antiperoxidase assay for rapid detection of respiratory syncytial virus in nasal epithelial specimens from infants and children.

A peroxidase-antiperoxidase (PAP) assay for the rapid detection of respiratory syncytial virus was compared with the indirect immunofluorescence method and with viral culture. Nasal epithelial specimens from 147 infants and children with acute respiratory infections were obtained and evaluated for the presence of respiratory syncytial virus antigens. Sensitivity, specificity, and accuracy by PAP were 91.7, 84.8, and 87.1%, respectively, and 87.0, 88.5, and 88.0%, respectively, by immunofluorescence compared with viral culture. The PAP assay was found to be as accurate as the indirect immunofluorescence method and more convenient to perform, since the color reaction and cell morphology were more easily observable by light microscopy. A new specimen collection method is reported; gentle scraping of the superficial nasal mucosa by the Rhino-probe method provided sufficient numbers of epithelial cells to perform multiple assays.

Adolescent↗
PubMed1987

Comparison of three rapid diagnostic techniques for detection of respiratory syncytial virus from nasal wash specimens.

We report results of three rapid tests for respiratory syncytial virus antigen detection. An immunofluorescence assay using commercial antibody and two commercial enzyme immunoassays (Ortho Diagnostics, Inc., Raritan, N.J., and Abbott Laboratories, North Chicago, Ill.) were applied to 199 nasal wash specimens. The Abbott enzyme immunoassay was the most sensitive technique, with a sensitivity of 93.8%. The specificities of the three techniques were comparable and greater than 95%. The availability of reliable rapid diagnostic techniques will allow for better care of infants with severe respiratory syncytial virus infection.

Antigens, Viral↗
PubMed1987

Freeze-drying of respiratory syncytial viruses for transportation and storage.

The stabilities of eight strains of respiratory syncytial virus were compared after the strains were freeze-dried in the presence and absence of the stabilizer SPGA, which contains 218 mM sucrose, 7.1 mM dipotassium hydrogen phosphate, 3.76 mM potassium dihydrogen phosphate, 4.9 mM sodium glutamate, and 1% (wt/vol) bovine albumin. Strains freeze-dried in the presence of SPGA showed only small-to-negligible losses at 4 degrees C and losses of approximately 2.0 log10 infectious units at 25 degrees C when held for 45 weeks. Losses at 37 degrees C for one strain were approximately 10-fold greater when the strain was freeze-dried in the absence of SPGA. These results indicate that respiratory syncytial virus strains freeze-dried in the presence of a suitable stabilizer can be transported as unrefrigerated samples without undue losses in infectivity.

Excipients↗
PubMed1990

Comparison of directigen RSV with viral isolation and direct immunofluorescence for the identification of respiratory syncytial virus.

An enzyme immunoassay membrane test (Directigen RSV) for the detection of respiratory syncytial virus in clinical specimens was compared prospectively with isolation in cell culture and direct immunofluorescence (IF). A total of 315 nasopharyngeal wash specimens from pediatric patients were examined. Directigen RSV was 86.1% sensitive and 91.3% specific for specimens positive by isolation in cell culture and/or IF, with 88.6% agreement. The false-positive rate was 16%; 2 of 20 specimens giving false-positive reactions by Directigen RSV were true-positives by blocking assay. Twenty-seven specimens (8.5%) whose results were initially uninterpretable by Directigen RSV due to filtration difficulties were diluted and upon retesting produced acceptable results. Sixty-three viral isolates and/or IF identifications of virus antigens representing seven virus groups other than respiratory syncytial virus were also found; cross-reactions between Directigen RSV and other viruses were not observed. Directigen RSV will be useful as an immediate procedure and in facilities lacking a comprehensive virology laboratory.

Bronchiolitis↗
PubMed1990

Rapid detection of respiratory syncytial virus in nasopharyngeal specimens obtained with the rhinoprobe scraper.

We compared the Rhinoprobe scraping technique for collection of superficial nasal mucosa epithelial cells and rapid detection of respiratory syncytial virus by immunofluorescence with paired, swab-collected specimens for virus culture from 1,257 infants and children with acute respiratory infections. Compared with viral culture as the reference test, the sensitivity, specificity, and accuracy of the immunofluorescence test were 83.6, 93.6, and 91.3%, respectively. We found the Rhinoprobe method safe, easy to use, and helpful in obtaining large quantities of epithelial cells for detection of respiratory syncytial virus and other respiratory viruses.

Adolescent↗
PubMed1991

Antigenic characterization of respiratory syncytial virus group A and B isolates in Rio de Janeiro, Brazil.

The antigenic characteristics of 87 strains of respiratory syncytial virus isolated in Rio de Janeiro, Brazil, from 1982 to 1988 were determined with a panel of monoclonal antibodies (MAbs) in an enzyme immunoassay. Four of these MAbs immunoprecipitated the fusion protein, and five immunoprecipitated the large glycoprotein. On the basis of the patterns of reaction of these MAbs to respiratory syncytial virus isolates in an enzyme immunoassay, we were able to separate isolates into the two major groups, A and B, and also to identify four variants within group A and three within group B. Strains from groups A and B were isolated each year, and the prevalence of the two groups varied over the seven study years.

Antibodies, Monoclonal↗
PubMed1991

Prevalence of respiratory syncytial virus subgroups A and B in France from 1982 to 1990.

A fluorescence antibody test with monoclonal antibodies was used to determine the subgroup (A or B) of respiratory syncytial virus from infants hospitalized in Caen, France, over eight consecutive epidemics from 1982 to 1990. From 1982 to 1985, 27 (30%) out of 90 frozen nasal slides were classified as subgroup A strains and 63 (70%) were classified as subgroup B. B strains predominated over A in 1983-1984 and 1984-1985. From 1985 to 1990, 284 respiratory syncytial virus field strains were reisolated from frozen materials; 115 (40.5%) were typed as subgroup A and 169 (59.5%) were typed as subgroup B. In 1985-1986, 1986-1987, and 1988-1989, both subgroups were present in almost equal numbers; subgroup A (88.3%) predominated in 1987-1988, and subgroup B (84.5%) predominated in 1989-1990. In conclusion, both subgroups occur together each year, and one subgroup rarely predominates, e.g., subgroup A in 1987-1988 and subgroup B in 1983-1984 and 1989-1990. Therefore, there is a gradual change of the predominant subgroup into another over a period of about 5 years; the relative frequency of subgroup A strains increased from 1983 to 1988, whereas the percentage of subgroup B decreased during the same period.

Antibodies, Monoclonal↗
PubMed1991

Synthetic oligonucleotide probes differentiate respiratory syncytial virus subgroups in a nucleic acid hybridization assay.

A new approach to respiratory syncytial virus subgroup differentiation has been developed on the basis of the hybridization of subgroup-specific synthetic oligonucleotides with viral mRNA. Two oligonucleotide probes were designed from the nucleotide sequences of an A and a B subgroup respiratory syncytial virus glycoprotein G gene. All 28 virus isolates tested were correctly classified by subgroup with these probes.

Base Sequence↗
PubMed1992

Identification of subgroups of bovine respiratory syncytial virus.

The occurrence of antigenic variation among nine isolates of bovine respiratory syncytial virus (BRSV) was determined by examining their reaction patterns to human respiratory syncytial virus (HRSV) subgroup A and B monoclonal antibodies (MAbs) by enzyme immunoassay and radioimmunoprecipitation with fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using MAbs and polyclonal antisera to HRSV and BRSV. Shared epitopes were demonstrated on four of five structural proteins between BRSV and both subgroups A and B of HRSV. The nine isolates of BRSV showed different patterns of reactivity in enzyme immunoassays with panels of MAbs to HRSV subgroups A and B. Major variations in the molecular weights of the P (phosphoprotein) and F (fusion protein) proteins were demonstrated among the BRSV isolates tested. These results suggest that BRSV belongs to a different antigenic grouping than HRSV and that BRSV is composed of two distinct subgroups.

Animals↗
PubMed1993

Utility of a respiratory virus panel containing a monoclonal antibody pool for screening of respiratory specimens in nonpeak respiratory syncytial virus season.

An indirect immunofluorescence respiratory virus panel containing monoclonal antibodies directed against respiratory syncytial virus, parainfluenza virus types 1, 2, and 3, adenovirus, and influenza viruses A and B was used to screen specimens in the nonpeak respiratory syncytial virus seasons in 1989 and 1990. The results indicate that the respiratory virus panel is fairly sensitive (79%) and very specific (99%) for the detection of respiratory viruses directly in clinical specimens during these time periods.

Adenoviruses, Human↗