Identification of T cell and linear B cell epitopes on African horse sickness virus serotype 4 proteins VP1-1, VP2, VP4, VP7 and NS3.

The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics.

The impact of Escherichia coli contamination products present in recombinant African horse sickness virus serotype 4 proteins on the innate and humoral immune responses.

Transcriptome analysis was used to characterise the in vitro primary and secondary immune responses induced in horse peripheral blood mononuclear cells (PBMC) stimulated for 24 h with the individual recombinant proteins of a virulent AHSV serotype 4 (AHSV4) field isolate (rAHSV4 proteins) that were previously expressed in Escherichia coli (E. coli). The results showed that the E. coli contamination products greatly affected the innate and humoral immune response transcripts. Hence, the impact of E. coli contamination products present in the individual rAHSV4 proteins on the translational immune response was determined. The combined amplification effects of synergistic pattern recognition receptors (PRRs), TNF-α and IL-1ß signalling induced potent pro-inflammatory responses that were too overwhelming for the anti-inflammatory cytokines and regulators to control. In addition to inducing robust B cell and antibody-mediated responses, lipopolysaccharide (LPS) activation of the innate-like B cells and subsequent polyreactive (natural) antibody responses could potentially contribute to endotoxin tolerance.

Apoptosis versus survival of African horse sickness virus serotype 4-infected horse peripheral blood mononuclear cells.

Expanding on our previous work, this study used transcriptome analysis of RNA sequences to investigate the various factors that contributed to either inducing apoptosis that resulted in cell death or promoting the survival of African horse sickness virus serotype 4 (AHSV4)-infected horse peripheral blood mononuclear cells (PBMC) after 24 h. Apoptosis is a host defense mechanism that prevents virus replication, accumulation and spread of progeny viruses. AHSV4-infected PBMC were killed via the intrinsic and the perforin/granzyme pathways of apoptosis during the attenuated AHSV4 (attAHSV4) in vivo primary and secondary immune responses. Trained innate immunity played an important role in circumventing viral interference that resulted in the elimination of AHSV4-infected PBMC through the intrinsic and the extrinsic pathways of apoptosis during the virulent AHSV4 (virAHSV4) in vitro secondary immune response. Oxidative stress in conjunction with IRE1α pro-apoptotic signaling played a major role in the induction of the intrinsic pathway of apoptosis and cytotoxic lymphocytes induced the perforin/granzyme or extrinsic pathways of apoptosis. In contrast, AHSV4-infected PBMC survived during the virAHSV4 in vitro primary immune response, which allows unrestrained viral replication. The virAHSV4 interference with the innate immune response resulted in impaired NK cell responses and delayed immune responses, which together with the antioxidant defense system promoted AHSV4-infected PBMC survival.

Virulent African horse sickness virus serotype 4 interferes with the innate immune response in horse peripheral blood mononuclear cells in vitro.

African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a double stranded RNA (dsRNA) virus of the genus Orbivirus, family Reoviridae. For the development of new generation AHS vaccines or antiviral treatments, it is crucial to understand the host immune response against the virus and the immune evasion strategies the virus employs. To achieve this, the current study used transcriptome analysis of RNA sequences to characterize and compare the innate immune responses activated during the attenuated AHSV serotype 4 (attAHSV4) (in vivo) and the virulent AHSV4 (virAHSV4) (in vitro) primary and secondary immune responses in horse peripheral blood mononuclear cells (PBMC) after 24 h. The pro-inflammatory cytokine and chemokine responses were negatively regulated by anti-inflammatory cytokines, whereas the parallel type I and type III IFN responses were maintained downstream of nucleic acid sensing pattern recognition receptor (PRR) signalling pathways during the attAHSV4 primary and secondary immune responses. It appeared that after translation, virAHSV4 proteins were able to interfere with the C-terminal IRF association domain (IAD)-type 1 (IAD1) containing IRFs, which inhibited the expression of type I and type III IFNs downstream of PRR signalling during the virAHSV4 primary and secondary immune responses. Viral interference resulted in an impaired innate immune response that was not able to eliminate virAHSV4-infected PBMC and gave rise to prolonged expression of pro-inflammatory cytokines and chemokines during the virAHSV4 induced primary immune response. Indicating that virAHSV4 interference with the innate immune response may give rise to an excessive inflammatory response that causes immunopathology, which could be a major contributing factor to the pathogenesis of AHS in a naïve horse. Viral interference was overcome by the fast kinetics and increased effector responses of innate immune cells due to trained innate immunity and memory T cells and B cells during the virAHSV4 secondary immune response.

In vitro propagation and genome sequencing of three 'atypical' Ehrlichia ruminantium isolates.

Three isolates of Ehrlichia ruminantium (Kümm 2, Omatjenne and Riverside), the causative agent of heartwater in domestic ruminants, were isolated in Ixodes scapularis (IDE8) tick cell cultures using the leukocyte fraction of infected sheep blood. All stocks were successfully propagated in IDE8 cells, whereas initiation attempts using endothelial cell cultures were unsuccessful. Therefore, the new technique should be included in any attempt to isolate field strains of E. ruminantium to enhance the probability of getting E. ruminantium isolates which might not be initiated in endothelial cells. Draft genome sequences of all three isolates were generated and compared with published genomes. The data confirmed previous phylogenetic studies that these three isolates are genetically very close to each other, but distinct from previously characterised E. ruminantium isolates. Genome comparisons indicated that the gene content and genomic synteny were highly conserved, with the exception of the membrane protein families. These findings expand our understanding of the genetic diversity of E. ruminantium and confirm the distinct phenotypic and genetic characteristics shared by these three isolates.