Gene expression
To test whether the inferred genes are expressed under conditions known to induce type
I interferons, we designed primers to Pteropus IFNB and to a control housekeeping gene (PPIA) and a gene (OAS2) induced by paracrine IFNB signaling. The latter two genes were also inferred by the methods described.
Fresh Pteropus PBMCs from two individual animals were cultured under four different conditions: with lipopolysaccharide (LPS), with the synthetic RNA poly(I:C), with Vesicular Stomatitis Virus (VSV), and with culture medium alone.
With LPS or poly(I:C) treatment, IFNB mRNA expression was increased 20-50 fold by 2 hours and decreased slowly to near the starting value by 24 hours. OAS expression lagged behind that of IFNB, reaching its peak value by 8 hours, declining from the peak level only partially by 24 hours. In contrast, cells treated with VSV had peak levels of IFNB appear by 8 hours and remain almost unchanged to 24 hours; OAS expression rose more slowly, and had not reached a maximum level by 24 hours (Figure 5).
Figure 5 Gene expression time course by qRT-PCR in fresh P.vampyrus PBMCs stimulated in culture by the indicated compound. Expression levels are relative to the housekeeping gene PPIA. The error bars represent standard errors of the mean over biological duplicates.