Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

Bovine respiratory syncytial virus (BRSV) is an enveloped, nonsegmented, negative-stranded RNA virus and is a major cause of respiratory disease in young calves (44). BRSV is closely related to human RSV (HRSV), which is a major cause of respiratory disease in young children (10), and the epidemiology and pathogenesis of infection with these viruses are similar (44). These features make BRSV infection in calves a good model for the study of HRSV. HRSV and BRSV belong to the Pneumovirus genus within the Paramyxoviridae family. One of the major differences between this genus and all the other Paramyxoviridae is the presence of two nonstructural (NS) genes called NS1 and NS2. These genes code for two proteins, which are abundantly transcribed in virus-infected cells. Comparison of the sequence of the NS proteins of BRSV with that of HRSV subgroup A and B reveals amino acid identities of 69 and 68% for the NS1 protein and 84 and 83% for the NS2 protein, respectively (8, 39).The role(s) of the NS proteins is not fully defined. They are not essential for virus replication in vitro, although the growth of recombinant HRSV and BRSV lacking these proteins is attenuated in cell culture (8,25,42,51). There is evidence that the HRSV NS1 protein coprecipitates with the M protein (19), and in experiments using HRSV minigenomes, the NS1 protein appears to be a strong inhibitor of viral RNA transcription and replication (1). The NS2 protein also appears to be a transcriptional inhibitor but at a lower level than is the NS1 protein (1). The NS2 protein colocalizes with the P and N proteins in infected cells (60) but does not coprecipitate with any viral protein (19). In addition, the NS1 and NS2 proteins of BRSV and HRSV mediate resistance to the antiviral action of alpha/beta inferferons (IFN-␣/␤) (3, 42).Anti-IFN activity has been described for accessory proteins for a number of other negative-stranded RNA viruses. Of those characterized to date, some block the IFN response by hindering the late-stage activation of antiviral genes. For example, the C protein of Sendai virus inhibits STAT1 activation by hampering phosphorylation and by increasing instability (21, 64) and the V protein of simian virus 5 inhibits the activation of IFN-responsive genes by targeting STAT1 for proteasome-mediated degradation (13). Other viral accessory proteins, such as influenza A virus NS1 protein and Bunyamwera virus NSs protein, inhibit the production of IFN-␣/␤ (58, 61).

The role of T-cell subsets in respiratory syncytial virus (RSV) infection was investigated by using monoclonal antibodies (MAbs) to selectively deplete gnotobiotic calves of CD4+, CD8+, or WC1+ gamma delta T-cell receptor+ lymphocytes. Injection of these MAbs produced specific reductions of the target cell populations in the circulation and tissues. Ten days after RSV infection, immunoglobulin M (IgM), IgG1, and IgA antibodies were detected in sera and lung washings from control calves. Depletion of CD8+ T cells had no effect on either the serum or local antibody responses to RSV, whereas depletion of CD4+ T cells suppressed the antibody responses in two of three calves. The IgM and IgA responses were significantly increased in the lung washings of calves from which WC1+ T cells were depleted. Depletion of CD4+ or WC1+ T cells caused no significant delay in virus clearance, although an increase in the extent of pneumonic consolidation was observed in anti-CD4-treated calves. Nasopharyngeal excretion of RSV was prolonged in calves depleted of CD8+ T cells, and virus was isolated in high titers from lung washings of these animals 10 days after infection, whereas virus had been cleared from lung washings of all other animals. The delayed virus clearance was associated with an increase in the severity of pneumonic consolidation in three of four of the calves from which CD8+ T cells were depleted. This study shows that CD8+ T cells play a dominant role in the recovery of calves from RSV infection.

The immunogenicity and protective efficacy of recombinant vaccinia viruses (rVV) encoding the F, G, N or M2 (22K) proteins of bovine respiratory syncytial virus (BRSV) were evaluated in calves, the natural host for BRSV. Calves were vaccinated either by scarification or intratracheally with rVV and challenged 6 to 7 weeks later with BRSV. Although replication of rVV expressing the F protein in the respiratory tract was limited after intratracheal vaccination, the levels of serum and pulmonary antibody were similar to those induced following scarification. The serum antibody response induced by the F protein was biased in favour of IgG1 antibody, whereas the G and the N proteins induced similar levels of IgG1 : IgG2, and antibody was undetectable in calves primed with the M2 protein. The F protein induced neutralizing antibodies, but