To study the epidemiology of respiratory syncytial virus (RSV) infections during the year, the incidences of primary infections and reinfections were monitored by titrating antibodies to bovine RSV (BRSV) in cattle above 2 months of age in 6 dairy herds in the Netherlands. From August 1990 until September 1991, 884 cattle were sampled at one-month intervals. A total of 155 cattle, most under two years of age, had a primary antibody response. Antibody rises were found in 259 cattle of all ages. The highest incidences of BRSV infections were found in one period either in autumn or winter. In other seasons, primary infections were rare, whereas reinfections were not uncommon. In 5 out of the 6 herds, two seronegative sentinel calves were introduced at the end of the winter and none developed specific antibodies before the next winter. The observations strongly suggest that, in spite of regular reinfections, BRSV circulates during spring or summer at a very low level or not at all. Persistent BRSV infection in a number of cows might be a means for the virus to survive during summer, but a steady rate of reinfection of seropositive cows throughout the year at a low level might also maintain a reservoir of infectious virus. This study adds to the knowledge of frequency and timings of primary infections and reinfections of BRSV and it might contribute to the study of these issues of human RSV.

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.

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Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein E rns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the E rns protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed. To identify type-specific pestivirus antibodies, a liquid-phase peptide ELISA, with a labeled, specific classical swine fever virus (CSFV) peptide and an unlabeled bovine viral diarrhea virus peptide to block cross-reactivity, was developed. Specificity and sensitivity of the liquid-phase peptide ELISA for CSFV were 98 and 100%, respectively. Because the peptide is a fragment of the E rns protein, it can be used to differentiate between infected and vaccinated animals when a vaccine based on the E2 protein, which is another pestivirus envelope protein, is used.

To reproduce experimentally clinical bovine respiratory syncytial virus (BRSV) infections in cattle, we isolated BRSV from a calf in the field that suffered from acute respiratory disease. Cell culture passage of the virus was avoided to prevent any modification of the biological properties of the virus. The isolated BRSV was passaged in specific-pathogen-free (SPF) calves. Lung lavage fluids of these calves, which contained at least 10(3) TCID50/ml BRSV and which were found to be free of other known respiratory pathogens, were collected and pooled for experimental infection. To reproduce a clinical BRSV infection, two groups of six SPF calves were inoculated intranasally with 2 ml of 10(3.9) TCID50/ml BRSV of the obtained virus stock. Another five calves, which were persistently infected with bovine virus diarrhoea virus (BVDV), were given the same inoculum. One group of six calves served as mock-infected controls. Clinical signs were closely monitored from 1 week before until 16 days after inoculation. Reproducible clinical signs consisting of significantly (p < 0.05) increased respiratory rates and elevated body temperatures were recorded but not in all BRSV-inoculated calves. Although clinical signs were induced by experimental infection with non-cell-culture-passaged BRSV, the respiratory signs were not as serious as in the most severe cases in the field.