To indirectly monitor A nucleotide insertions in GFP-polyA, Vero cells were inoculated with rhMPV/ΔM2-2/ GFPpolyA or one of the control viruses rhMPV/GFP, rhMPV/GFPpolyA or rhMPV/ΔM2-2/GFP, at MOI of 0.1, and viewed by fluorescence microscopy for 6 days. Fluorescence was readily observed throughout the monolayers Functional GFP expression in rhMPV/ΔM2-2/GFP6 poly A by A nucleotide insertion Figure 4 Functional GFP expression in rhMPV/ΔM2-2/GFP6 poly A by A nucleotide insertion. A) rhMPV and rhMPV/ΔM2-2 viruses containing the native GFP ORF or GFP-polyA sequences, harboring an engineered poly A tract that silenced the translation of GFP, formed comparable plaques in Vero cells. B) Western blots indicated F expression was comparable between viruses. C) A duplicate Western blot was probed with antibody directed to actin to serve as a loading control. D) GFP was detected by Western blot in viruses that contained native GFP ORFs. E) Fluorescence was robustly detected in viruses that contained native GFP ORFs, was readily detectable in some fluorescent foci in rhMPV/ΔM2-2/GFPpolyA, and was not detected in rhMPV/GFPpolyA. F) Nucleotide insertion of one A restored function of GFPpolyA ORF. Nucleotide insertion of 3 As would not restore functional GFPpolyA, but indicated heterogeneity at this polyA locus. of Vero cells infected with rhMPV/GFP or rhMPV/ΔM2-2/ GFP, but not in cells infected with rhMPV/GFPpolyA (Figure 4E ). Initially, no fluorescence was observed in cells infected with rhMPV/ΔM2-2/GFPpolyA. However, after two days, a few foci of fluorescent cells were observed in monolayers infected with rhMPV/ΔM2-2/GFPpolyA, suggesting that some cells were infected with GFP-expressing hMPV. One focus containing approximately a hundred infected fluorescent cells is shown ( Figure 4E ). The expression of GFP indicated that the reading frame of the GFP gene had been restored in some virions, and cell-to-cell spread within the focus of infection suggested that the restored GFP gene sequences were present in progeny virions. The low level of GFP expressed was only observable by fluorescence microscopy and not by Western blotting ( Figure 4D ).