A study of viral tissue tropism and cytokine expression in Bovine Ephemeral Fever

Abstract

While the clinical and pathological aspects of Bovine Ephemeral Fever (BEF) have been studied in cattle, major gaps still remain in our understanding of the pathogenesis of the disease. In particular, the cytokine networks that underlie fever and inflammation during acute BEF, and mechanisms that mediate the nascent adaptive immune response have not been empirically defined. In addition, the potential in vivo replication sites of BEFV, and the mechanistic events that underlie the paresis and chronic paralysis in some field BEF cases have also not been defined. The objectives of this research were: (1) to characterise the plasma kinetics of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and IL-10 during natural BEFV infections in cattle; (2) to evaluate the plasma kinetics of IL-2, IFN-γ, IL-6, and IL-10 during the period of innate-immune response transition; and (3) to determine the overall tissue tropism and potential replication sites, along with assessing the likelihood of neurotropism of BEFV in naturally infected cattle. For the cytokine expression studies, experiments were carried out on two different cohorts of animals raised at two farms at different time points. Briefly, plasma from three BEFV-infected and three uninfected cattle was tested by cytokine-specific cELISA, viraemia monitored by qRT-PCR, and virus neutralising antibody titres determined using a standard protocol. Similarly, plasma from another four virus-infected and uninfected negative control animals was tested for the four study cytokines (IL-2, IFN-γ, IL-6, and IL-10), and viraemia and virus neutralising antibody titres determined. Biological specimens from nine adult cattle that died or were euthanised at different time points following natural BEFV infections were tested for viral antigen and RNA by IHC and qRT-PCR, respectively. In addition, virus isolation in autogenously derived splenic and haemal node cultures, and electron microscopic examination of ultrathin ii sections from various tissues taken from the steer necropsied seven days after BEF diagnosis were performed. For the neurotropism studies, fresh brain, spinal cord, peripheral nerve, and other tissues were collected from four paralysed and six asymptomatic but virus-infected cattle and tested for viral RNA by qRT-PCR.Formalin-fixed tissues were routinely evaluated for histomorphological lesions and for viral antigen presence and distribution by IHC. Unlike the negative controls, plasma concentrations of IL-1β, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of these heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1β was the most strongly expressed cytokine, IL-6 and IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-...