Vero cells were inoculated at MOI of 0.1 with rhMPV/GFP, rhMPV/GFPpolyA, rhMPV/ΔM2-2/GFP or rhMPV/ΔM2-2/ GFPpolyA as well as the control viruses rhMPV and rhMPV/ΔM2-2. Viruses were harvested on day 4 for Western blot analysis. The Western blot was probed for expression of hMPV F and GFP. Actin was also probed as a loading control ( Figure 4C ). The levels of hMPV F as detected by Western blot were considered equivalent among the GFP-viruses ( Figure 4B ). As expected GFP protein was detected by Western blot only in rhMPV/GFP and rhMPV/ΔM2-2/GFP, and not in rhMPV/GFPpolyA and rhMPV/ΔM2-2/GFPpolyA ( Figure 4D ). These data indi-Chromatogram and frequency of A insertions and point mutations in rhMPV/ΔM2-2 compared to rhMPV Figure 3 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. Gene: cate that insertion of the GFP cassette at this genome position was well tolerated by hMPV in vitro and insertion of the CGA 6 TTA sequences in the N terminus of the GFP ORF effectively silenced GFP expression.