Bovine model of respiratory syncytial virus infection.

Bovine respiratory syncytial virus (BRSV), which is an important cause of respiratory disease in young calves, is genetically and antigenically closely related to human (H)RSV. The epidemiology and pathogenesis of infection with these viruses are similar. The viruses are host-specific and infection produces a spectrum of disease ranging from subclinical to severe bronchiolitis and pneumonia, with the peak incidence of severe disease in individuals less than 6 months of age. BRSV infection in calves reproduces many of the clinical signs associated with HRSV in infants, including fever, rhinorrhoea, coughing, harsh breath sounds and rapid breathing. Although BRSV vaccines have been commercially available for decades, there is a need for greater efficacy. The development of effective BRSV and HRSV vaccines face similar challenges, such as the need to vaccinate at an early age in the presence of maternal antibodies, the failure of natural infection to prevent reinfection, and a history of vaccine-augmented disease. Neutralising monoclonal antibodies (mAbs) to the fusion (F) protein of HRSV, which can protect infants from severe HRSV disease, recognise the F protein of BRSV, and vice versa. Furthermore, bovine and human CD8(+) T-cells, which are known to be important in recovery from RSV infection, recognise similar proteins that are conserved between HRSV and BRSV. Therefore, not only can the bovine model of RSV be used to evaluate vaccine concepts, it can also be used as part of the preclinical assessment of certain HRSV candidate vaccines.

Virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus.

Tachykinins, an evolutionary conserved family of peptide hormones in both invertebrates and vertebrates, are produced by neuronal cells as inactive preprotachykinins that are post-translationally processed into different neuropeptides such as substance P, neurokinin A, and neurokinin B. We show here that furin-mediated cleavage of the bovine respiratory syncytial virus fusion protein results in the release of a peptide that is converted into a biologically active tachykinin (virokinin) by additional post-translational modifications. An antibody directed to substance P cross-reacted with the C terminus of mature virokinin that contains a classical tachykinin motif. The cellular enzymes involved in the C-terminal maturation of virokinin were found to be present in many established cell lines. Virokinin is secreted by virus-infected cells and was found to act on the tachykinin receptor 1 (TACR1), leading to rapid desensitization of this G protein-coupled receptor as shown by TACR1-green fluorescent protein conjugate translocation from the cell surface to endosomes and by co-internalization of the receptor with beta-arrestin 1-green fluorescent protein conjugates. In vitro experiments with isolated circular muscle from guinea pig stomach indicated that virokinin is capable of inducing smooth muscle contraction by acting on the tachykinin receptor 3. Tachykinins and their cognate receptors are present in the mammalian respiratory tract, where they have potent effects on local inflammatory and immune processes. The viral tachykinin-like peptide represents a novel form of molecular mimicry, which may benefit the virus by affecting the host immune response.

Identification of CD4+ T cell epitopes on the fusion (F) and attachment (G) proteins of bovine respiratory syncytial virus (BRSV).

To gain insight into the antigenic structure of the F and G proteins of BRSV, we have mapped CD4+ T cell epitopes on these proteins using synthetic peptides and lymphocytes from vaccinated, naturally infected or experimentally infected calves, in proliferation assays. Bovine CD4+ T cells recognised epitopes that were distributed predominantly within the F1 subunit of the F protein, some of which were adjacent to previously identified B cell epitopes. Bovine CD4+ T cell epitopes within the G protein were mainly located within the cytoplasmic tail. Several immunodominant bovine T cell epitopes within the F protein, that were recognised by calves with different haplotypes, are also recognised by human T cells. Thus, cattle and humans appear to recognise similar T cell epitopes on the F protein. Studies using antibodies to bovine MHC class II and BoLA DR-transfected CHO cells as antigen-presenting cells indicated that immunodominant regions of the F and G proteins contained both DR- and DQ-restricted epitopes. The finding that there was little recognition of the extracellular domain of the G protein by T cells has important implications for vaccine design based on the soluble form of this protein.

Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P and P-L interactions using a minigenome system.

The interaction of bovine respiratory syncytial virus (BRSV) phosphoprotein (P) with nucleocapsid (N) and large polymerase (L) proteins was investigated using an intracellular BRSV-CAT minigenome replication system. Coimmunoprecipitation assays using P-specific antiserum revealed that the P protein can form complexes with N and L proteins. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with N and L proteins. The results indicate that two independent N-binding sites exist on the P protein: an internal region of 161-180 amino acids and a C-terminal region of 221-241 amino acids. The L-binding site was mapped to a region of P protein encompassing amino acids 121-160. The data suggest that N and L protein binding domains on the P protein do not overlap.

Bovine respiratory syncytial virus (BRSV): a review.

Bovine respiratory syncytial virus (BRSV) infection is the major cause of respiratory disease in calves during the first year of life. The study of the virus has been difficult because of its lability and very poor growth in cell culture. However, during the last decade, the introduction of new immunological and biotechnological techniques has facilitated a more extensive study of BRSV as illustrated by the increasing number of papers published. Despite this growing focus, many aspects of the pathogenesis, epidemiology, immunology etc. remain obscure. The course and outcome of the infection is very complex and unpredictable which makes the diagnosis and subsequent therapy very difficult. BRSV is closely related to human respiratory syncytial virus (HRSV) which is an important cause of respiratory disease in young children. In contrast to BRSV, the recent knowledge of HRSV is regularly extensively reviewed in several books and journals. The present paper contains an updated review on BRSV covering most aspects of the structure, molecular biology, pathogenesis, pathology, clinical features, epidemiology, diagnosis and immunology based on approximately 140 references from international research journals.

Expression in Escherichia coli and purification of soluble forms of the F protein of bovine respiratory syncytial virus.

Six fragments of the F gene from bovine respiratory syncytial virus (BRSV) were engineered into the pMAL-c2 Escherichia coli expression vector and expressed as C-terminal maltose-binding protein (MBP) fusion products. The resulting polypeptides were partially soluble and single-step purified by affinity chromatography. These fusion proteins were recognized in Western blots by several MAbs directed against human respiratory syncytial virus F protein. In addition, rabbit polyclonal antisera raised against two purified MBP-derived proteins reacted with the BRSV-F protein.

Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P interaction using a two-hybrid system.

Specific interactions between the nucleocapsid protein (N) and the phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) have been investigated using a yeast-based two-hybrid system. Plasmids encoding the yeast GAL4 DNA binding domain fused with the N gene and GAL4 activation domain fused with the P gene were cotransfected into competent yeast cells. The ability of the N and P proteins to interact in vivo was measured by activation of the lacZ reporter gene by the GAL4 transactivation region. Results indicated that the N and P proteins interact very strongly in vivo. When interactions between N and various deletion mutants of the P protein were examined, an internal region (aa 132-168) and the highly acidic C-terminal region (aa 236-241) of the P protein were found to be essential for N-P interaction. In addition, the highly basic N-terminal region (amino acids 1-40) was found to be involved in N-P interaction to a lesser extent.

Structural difference in the fusion protein among strains of bovine respiratory syncytial virus.

The polypeptides of different strains of bovine respiratory syncytial virus (RSV) were compared. Altered electrophoretic migrations were observed in the G, F, P, M and 22 kDa polypeptides. The molecular weight of the F2 fragment in human RSV (Long strain) and bovine RSV (A51908 and Md-X strains) was approximately 20 kDa whereas it was approximately 15.5 kDa in caprine RSV and bovine RSV (FS-1 and VC-464 strains). The size difference of the F2 subunit was due to difference in the extent of glycosylation.