Rice stripe virus (RSV), the type species of the tenuivirus group, contains four RNA segments as its genome. Sequence analyses of the three smaller segments indicated that all of them have ambisense coding strategies. To examine the ambisense nature of the genomic RNAs, we synthesized the RNAs carrying the putative open reading frames (ORFs) by transcribing cDNA clones for RNA segments 2, 3 and 4 in both directions using T7 RNA polymerase and translated each RNA using two systems: reticulocyte lysates and wheatgerm extracts. We detected the proteins encoded by the ORFs present in the 5′-proximal regions of both viral RNAs (vRNAs) and their complementary RNAs (cRNAs). Translation of total vRNA generated proteins encoded by the ORFs present in the 5′ regions of vRNAs. The overall results are consistent with the prediction that RSV RNAs, at least up to segment 2, are ambisense in their coding strategy.


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