Abstract:
African horse sickness virus (AHSV) is a member of the genus Orbivirus in the family
Reoviridae and is the causative agent of African horse sickness (AHS), an acute disease in horses
with a high mortality rate. AHSV consists of two concentric capsids that enclose the viral
double-stranded RNA genome. The outer capsid is composed of two major structural proteins of
the virion, VP2 and VP5. A focus of this investigation was on the functional characterization of
the VP5 protein, which is known only to play a supportive role to VP2 in enhancing the
protective immune response in horses and is cytotoxic when expressed in Spodoptera frugiperda
insect cells. Silencing of VP5 gene expression in AHSV-infected mammalian cells by short
hairpin RNA (shRNA) and small interfering RNA (siRNA) proved inefficient as means to
determine the in vivo functional role of the VP5 protein. Subsequently, characterization of a
series of baculovirus-expressed N- and C-terminal truncated VP5 proteins in S. frugiperda cells,
as well as relevant peptides based on the predicted structural features of the VP5 protein, indicated that the N-terminal 43 amino acids of the VP5 protein correlated with increased
membrane permeabilization. These results suggest that this property of VP5 may be of
importance during the initial stages of virus entry into susceptible host cells by facilitating the
release of core particles from early endosomes.
Infection of mammalian cell cultures with AHSV is known to result in dramatic cytopathic
effects (CPE), but no CPE is observed in infected insect cell cultures despite productive virus
replication. The basis for this phenomenon has not yet been investigated, but is suggestive of
apoptosis being induced following virus infection of the mammalian cells. A second focus of this
investigation was therefore to determine whether AHSV can induce apoptosis in infected
mammalian cells and by which mechanism. To investigate, Culicoides sonorensis (KC) insect
cells and BHK-21 mammalian cells were infected with AHSV-9 and analyzed for morphological
and biochemical hallmarks of apoptosis. In contrast to KC cells, infection of BHK-21 cells with
AHSV-9 resulted in ultrastructural changes and nuclear DNA fragmentation, both of which are
associated with the induction of apoptosis. Results also indicated that AHSV-9 infection of
BHK-21 cells resulted in activation of caspase-3, a key agent in apoptosis, and in mitochondrial
membrane depolarization. Cumulatively, the data indicate that the intrinsic pathway is activated
in AHSV-induced apoptosis.
