Journal of General Virology

Fig. 3. (A) Time-course analysis of the appearance of intron III-spliced mRNA species during the natural course of BDV infection. Vero NK cells were infected with BDV at an m.o.i. of about 1 f.f.u. per cell and RNA was harvested at the times indicated. RT–PCR analysis of BDV intron II- and intron III-spliced mRNAs was carried out as described in the legend to Fig. 1 (see also text). RT–PCR analysis of the synthesis of the BDV N mRNA was done by using BDV-specific primers 259F and 829R, as described previously (Sauder et al., 1996). As a control for RNA quality, cDNAs were also amplified with specific primers to generate a 609 bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment. RT–PCR analysis of BDV N and GAPDH was done by using 5 ng of cytoplasmic RNA from BDV-infected cells together with 5 µg of RNA from uninfected cells. Sizes of the corresponding PCR products are indicated. (B) Identification of intron II- and III-spliced mRNA species in different cell lines persistently infected with BDV. Cytoplasmic RNA was obtained from C6 and OL cells persistently infected with BDV-He80 (C6BV and OLBV, respectively) and from the corresponding uninfected control cells. RNA was analysed by RT–PCR as described in Fig. 1 to detect intron II- and III-spliced mRNA species. (C) Effect of osmotic shock on the pattern of BDV RNA splicing. Cells were either left untreated or exposed to 0.6 M sorbitol (Sorb) for 6 h. Total RNA was extracted and analysed by RT–PCR as described in Fig. 1.

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