Chromatogram and frequency of A insertions and point mutations in rhMPV/ΔM2-2 compared to rhMPV. A) A chromatogram of the RT-PCR product derived from P4 of rhMPV/ΔM2-2, spanning nt4536 in F to nt6205 in NCR of SH, contained this sequence showing two subpopulations. One population is the correctly cloned sequence; the second population has one inserted A nt (sense direction) at nt5222 in the M2-1 gene. B) To assess the relative frequency of mutations, RT-PCR fragments spanning nt4536 in F to nt5623 in SH were obtained from rhMPV/ΔM2-2 or rhMPV using one-step RT-PCR, and were cloned into pCR2.1 plasmids. Among 15 independent plasmids the number of inserted As, single nt deletions, and point mutations (transition or transversion) for each virus were tabulated. 14 of the 15 (93%) rhMPV/ΔM2-2RT-PCR products had an inserted A (sense direction) nucleotide. No fragments containing A nucleotide insertions were detected in any of the 15 RT-PCR fragments spanning the identical region in P4 of rhMPV. C) To study frequency of mutations in genomic RNA, RT-PCR fragments spanning nt4536 to nt5623 were obtained from rhMPV/ΔM2-2 using two-step RT-PCR, and were cloned into pCR2.1 plasmids. Nucleotide insertions were predominantly T (genomic antisense direction), with one A, and were distributed among 8 locations in the fragments. D) To describe the position where insertion of an A was observed, the nt number of the last A in the poly A tract is used, though it is not known which A residue in the poly A tract is the inserted residue. The example shown is A inserted at nt5166.