During the preparation of viral stocks, we noted several mutations in rhMPV/ΔM2-2. To further assess the genetic stability of rhMPV/ΔM2-2, one-step RT-PCR was performed on a virus stock that was serially passaged 4 times in Vero cells. Sequence analysis of an RT-PCR product spanning the M2 and SH genes (nt4536 to nt6205) revealed nucleotide polymorphisms in several poly A tracts (sense direction) in the M2-1 and SH genes. Figure  3A shows a representative chromatogram of the sequence of an RT-PCR product generated from a rhMPV/ΔM2-2 virus stock. The wild-type sequence AGAGAAACTGA 6 TT is shown overlapping another sequence containing an inserted A in the poly A 6 tract. Three independently derived virus stocks of rhMPV/ΔM2-2 had major subpopulations with inserted A nucleotides (nts) at nt5060, nt5166 or nt5222 in M2-1, each of which would cause a premature translation termination in the M2-1 ORF. (See figure 3D for numbering of A insertions). Subpopulations with inserted A's were also detected at nt5551 or nt5572 in SH that would result in premature translation termination in the SH ORF.