In principle, altered BMV-directed Rluc expression observed in the 42 candidate genes might result from general defects in RNA4 translation or viral RNA synthesis, or from selective defects in replication, expression or function of the Rluc reporter gene. To identify genes that specifically affected BMV RNA synthesis and/ or accumulation, secondary validation testing of the 42 candidate genes was performed using Northern blotting to analyze RNA3 replication and RNA4 production, as described in the next two sections.