In humans and mice, γδ T cells represent <5% of the total circulating lymphocytes. In contrast, the γδ T cell compartment in ruminants accounts for 15–60% of the total circulating mononuclear lymphocytes. Despite the existence of CD4+CD25high Foxp3+ T cells in the bovine system, these are neither anergic nor suppressive. We present evidence showing that bovine γδ T cells are the major regulatory T cell subset in peripheral blood. These γδ T cells spontaneously secrete IL-10 and proliferate in response to IL-10, TGF-β, and contact with APCs. IL-10–expressing γδ T cells inhibit Ag-specific and nonspecific proliferation of CD4+ and CD8+ T cells in vitro. APC subsets expressing IL-10 and TFG-β regulate proliferation of γδ T cells producing IL-10. We propose that γδ T cells are a major regulatory T cell population in the bovine system.

Viruses routinely employ strategies to prevent the activation of innate immune signalling in infected cells. RSV is no exception, encoding two accessory proteins (NS1 and NS2) which are well established to block Interferon signalling. However, RSV-encoded mechanisms for inhibiting NF-κB signalling are less well characterised. In this study we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins, and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV infected cells we demonstrated that the P65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, puncta which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured P65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following TNF-α stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalise RSV N protein and P65 within bRSV IBs; granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterisation of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signalling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate – a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells. Importance Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalise their life cycle to provide favourable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterised. In this paper we show that bovine RSV (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signalling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that carries enormous economic consequences. CD8 + cytotoxic T lymphocytes play an important role in protection and disease outcome in viral infections but, to date, the role of the CD8 + T-cell immune response to FMDV remains unclear. This study aimed to investigate major histocompatibility complex (MHC) class I-restricted CD8 + T-cell responses to FMDV in vaccinated and in infected cattle. An in vitro assay was used to detect antigen-specific gamma interferon release by CD8 + T cells in FMDV-infected cattle of known MHC class I genotypes.A significant MHC class I-restricted CD8 + T-cell response was detected to both FMDV strain O1 BFS and a recombinant fowlpox virus expressing the structural proteins of FMDV. Antigen-specific MHC class I-restricted CD8 + T-cell responses were also detected in cattle vaccinated with inactivated FMDV. These responses were shown to be directed, at least in part, to epitopes within the structural proteins (P12A region) of the virus. By using mouse cells expressing single cattle MHC class I alleles, it was possible to identify the restriction elements in each case. Identification of these epitopes will facilitate the quantitative and qualitative analysis of FMDV-specific memory CD8 + T cells in cattle and help to ensure that potential vaccines induce a qualitatively appropriate CD8 + T-cell response.

Targeting dendritic cells (DC) is؉ T cell responses were significantly enhanced when using rhuAdV5-FMDV-A 22 in adjuvant. Therefore, the interaction between viral vectors and afferent lymph DC ex vivo can predict the outcome of in vivo immunization and provide a means of rapidly assessing the effects of vector modification.

Bovine respiratory syncytial virus, a major cause of respiratory disease in calves, is closely related to human RSV, a leading cause of respiratory disease in infants. Recently, promising human RSV-vaccine candidates have been engineered that stabilize the metastable fusion (F) glycoprotein in its prefusion state; however, the absence of a relevant animal model for human RSV has complicated assessment of these vaccine candidates. Here, we use a combination of structure-based design, antigenic characterization, and X-ray crystallography to translate human RSV F stabilization into the bovine context. A “DS2” version of bovine respiratory syncytial virus F with subunits covalently fused, fusion peptide removed, and pre-fusion conformation stabilized by cavity-filling mutations and intra- and inter-protomer disulfides was recognized by pre-fusion-specific antibodies, AM14, D25, and MPE8, and elicited bovine respiratory syncytial virus-neutralizing titers in calves >100-fold higher than those elicited by post-fusion F. When challenged with a heterologous bovine respiratory syncytial virus, virus was not detected in nasal secretions nor in respiratory tract samples of DS2-immunized calves; by contrast bovine respiratory syncytial virus was detected in all post-fusion- and placebo-immunized calves. Our results demonstrate proof-of-concept that DS2-stabilized RSV F immunogens can induce highly protective immunity from RSV in a native host with implications for the efficacy of prefusion-stabilized F vaccines in humans and for the prevention of bovine respiratory syncytial virus in calves.

Bovine respiratory syncytial virus (BRSV) causes inflammation and obstruction of the small airways, leading to severe respiratory disease in young calves. The virus is closely related to human (H)RSV, a major cause of bronchiolitis and pneumonia in young children. The ability to manipulate the genome of RSV has provided opportunities for the development of stable, live attenuated RSV vaccines. The role of the SH protein in the pathogenesis of BRSV was evaluated in vitro and in vivo using a recombinant (r)BRSV in which the SH gene had been deleted. Infection of bovine epithelial cells and monocytes with rBRSVΔSH, in vitro, resulted in an increase in apoptosis, and higher levels of TNF-α and IL-1β compared with cells infected with parental, wild-type (WT) rBRSV. Although replication of rBRSVΔSH and WT rBRSV, in vitro, were similar, the replication of rBRSVΔSH was moderately reduced in the lower, but not the upper, respiratory tract of experimentally infected calves. Despite the greater ability of rBRSVΔSH to induce pro-inflammatory cytokines, in vitro, the pulmonary inflammatory response in rBRSVΔSH-infected calves was significantly reduced compared with that in calves inoculated with WT rBRSV, 6 days previously. Virus lacking SH appeared to be as immunogenic and effective in inducing resistance to virulent virus challenge, 6 months later, as the parental rBRSV. These findings suggest that rBRSVΔSH may be an ideal live attenuated virus vaccine candidate, combining safety with a high level of immunogenicity.

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The cattle major histocompatibility complex (MHC) region contains a variable number of classical class I genes encoding polymorphic, ubiquitously expressed molecules with a role in antigen presentation. Class I cDNA sequences have previously been reported that are thought to derive from putative nonclassical class I genes. We have located four nonclassical class I genes within the cattle genome; three are close to the MIC genes, and one is close to the classical class I genes. The genomic position relative to anchor genes is very similar to the arrangement reported in the pig MHC region. We have designed gene-specific oligonucleotide primers with which to investigate the presence of these genes in distinct and well-defined MHC haplotypes and to assess transcription in different cell types. Analysis and comparison of all sequences allows an assessment of allelic variation in each case. Partial characterisation gives an indication of the possible role and likely importance of each of these genes.