To further investigate the role of hMPV M2-2, we compared the transcription and genome replication of rhMPV/ΔM2-2 with rhMPV in Vero cells. First, we compared the amounts of rhMPV/ΔM2-2 viral transcripts with that of rhMPV by Northern blotting. Northern blot analysis was performed using hMPV-specific anti-sense DIGlabeled riboprobes to detect M2, SH, N, F, or G mRNA. At 24-hr intervals, RNA was extracted from Vero cells inoculated with rhMPV or rhMPV/ΔM2-2 at an MOI of 0.1, and Northern blot analysis was performed in 6 replicates. The M2 and SH riboprobes each detected two RNA species from rhMPV-infected cells (Lanes 1, 3, 5 and 7 of Figure  5A and 5B). The size of the minor species is consistent with the monocistronic transcript while the size of the major species coincided with the predicted size of the M2/ SH read-through product. No monocistronic M2 transcripts were observed at 24 or 48 hours post rhMPV/ΔM2-2 infection in Vero cells. The predicted M2/SH readthrough product showed a reduction in size in rhMPV/ ΔM2-2 infected cells consistent with the deletion of M2-2 (compare lanes 1 and 2 of Figure 5A ). The levels of bicistronic compared to monocistronic SH transcripts were higher in both rhMPV and rhMVP/ΔM2-2 infected cells, but the difference was more pronounced in rhMPV/ΔM2-2 infected cells ( Figure 5B ). This increased level of readthrough was unexpected since we had sought to preserve the native M2/SH noncoding sequences. One explanation could be that transcription termination at the M2 gene end sequences required nucleotides in the coding region of M2-2 that had been inadvertently removed and/or the M2 termination signal was altered by the introduction of the Swa I site. Next we compared the amounts of M2 transcripts in cells infected with rhMPV or rhMPV/ΔM2-2 at days 1 to 4 postinfection (p.i.). At day 1 p.i., the levels of transcripts were equivalent between both viruses (lanes 1 and 2 in Figure  5A ). By day 2 p.i., the relative levels had changed markedly. The amount of transcripts in cells infected with rhMPV/ΔM2-2 was several-fold higher compared to cells infected with rhMPV (lanes 3 and 4 in Figure 5A ). The upregulation was maintained up to day 4, when peak titers were observed (lanes 7 and 8 in Figure 5 ). More SH, N, F, and G transcripts were also observed in cells infected with rhMPV/ΔM2-2 compared to rhMPV ( Figure 5B ,C,D and 5E). Therefore, M2-2 deletion resulted in up-regulation of viral transcripts of genes upstream (N, F) and downstream (SH, G) of the M2 gene. However, the increased levels of viral transcripts produced by the rhMPV/ΔM2-2 mutant were not accompanied by an increase in virus titer. On days 2 and 3, rhMPV/ΔM2-2 had higher levels of transcripts but equivalent or lower titers compared to rhMPV ( Figure 5H) . Neither was there a concomitant increase in protein expression, at least for the F gene ( Figure 4B , lanes 1 and 2). Thus, the higher levels of viral transcripts produced by the M2-2 deletion mutant did not yield a greater number of infectious rhMPV/ΔM2-2 virions compared to rhMPV. We noted that the levels of rhMPV transcripts peaked at day 3 (lanes 1, 3, 5 and 7 in Figure 5 ), while the levels of rhMPV/ΔM2-2 transcripts remained the same on days 3 and 4 (lanes 6 and 8 of Figure 5 ).