Abstract:
African horse sickness is a serious equid disease caused by the orbivirus African horse
sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding
seven structural and three non-structural proteins. Recently, an additional protein was
predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most
orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and
the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9
sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and
NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences
were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed
in cultured mammalian cells, with sizes close to the predicted 17–20 kDa. Fluorescence
microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution
that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen,
and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial
cells and mononuclear phagocytes in all of these tissues, localising to the both the
cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic
macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally,
nucleic acid protection assays using bacterially expressed recombinant AHSV NS4
showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be
required to determine the exact function of AHSV NS4 during viral replication.
