To compare the frequency of inserted A nucleotides in rhMPV/ΔM2-2 to that in rhMPV, RT-PCR products spanning nt4536 in F to nt5623 in SH were obtained from a passage 4 virus stock of rhMPV/ΔM2-2 or rhMPV. For this study, both positive sense and negative sense RNA were amplified using a one-step RT-PCR reaction. 1 kb RT-PCR fragments were inserted into pCR2.1 plasmids and 15 independent plasmids were sequenced. Surprisingly, 14 of the 15 (93%) cloned RT-PCR products of rhMPV/ΔM2-2 had an inserted A nucleotide at nt5060, nt5213 or nt5222 in the M2-1 gene ( Figure 3B ): there were 6 clones with insertion of A at nt5060, 2 with insertion at nt5213 and 6 with insertion at nt5222. Insertions of U, C or G Construction of cDNA for rhMPV/ΔM2-2, rhMPV/GFPpolyA and rhMPV/ΔM2-2/GFPpolyA Figure 1 Construction of cDNA for rhMPV/ΔM2-2, rhMPV/GFPpolyA and rhMPV/ΔM2-2/GFPpolyA. A) rhMPV/ΔM2-2 has a deletion in the M2-2 gene adjacent to a SwaI site. Nucleotides that were modified to introduce the SwaI site are underlined. Translational stop codons are bold and the intergenic (IG) sequence is bold italics. B) To construct rhMPV/GFPpolyA, an NheI site was introduced at the M2-2 stop codon of rhMPV and an NheI-N/P-GFP-polyA-NheI cassette was inserted. The modified nucleotides are underlined, the stop codon is bold and the IG sequence is bold italics. C) To construct rhMPV/ΔM2-2/GFPpolyA, an NheI site was introduced between the stop codon of M2-1 (bold) and the SwaI site (italics) in rhMPV/ΔM2-2 and an NheI-N/ P-GFP-polyA-NheI cassette was inserted. The modified nucleotides are underlined and the IG sequence is in bold italics. D) The reading frame of GFP is aligned with that of GFPpolyA to show the stop codon and frame shift resulting from the 11 nt insertion. were not observed. In sharp contrast, no A nucleotide insertions were detected in 15 cloned RT-PCR products derived from the identical region of rhMPV. Transitions, transversions, and deletions were also observed for rhMPV/ΔM2-2 in addition to insertions of A. For rhMPV/ ΔM2-2, 14 of 15 cloned RT-PCR sequences exhibited a total of 79 transition mutations, 2 transversions, and 1 deletion. Only 1 cloned sequence from rhMPV/ΔM2-2 had no nucleotide changes. In comparison, 7 of 15 cloned RT-PCR products of rhMPV showed a total of 17 transitions, 2 transversions, and 1 deletion. Eight cloned RT-PCR sequences from rhMPV had no nucleotide changes ( Figure 3B ). Thus, by passage 4, both rhMPV and rhMPV/ ΔM2-2 contained heterogeneous subpopulations and rhMPV/ΔM2-2 had a higher frequency of transition mutations and a propensity for insertion of A nucleotides in poly A tracts, compared to rhMPV.