Human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are cytoplasmic enveloped RNA viruses of the pneumovirus family. Their genomes are single strands of negative-sense RNA of 15.2 kb (RSV) or 13.3 kb (HMPV) that encode 10 mRNAs and 11 unique proteins (RSV) or 8 mRNAs and 9 unique proteins (HMPV). Each virus encodes a nucleoprotein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, major glycoprotein G, fusion glycoprotein F, polymerase factors M2-1 and M2-2, and the polymerase protein L. In addition, RSV encodes 2 nonstructural proteins NS1 and NS2. Murine pneumonia virus (MPV, previously known as pneumonia virus of mice, PVM) is a close relative of RSV whose natural host is the mouse and which provides a convenient permissive animal model. We recently evaluated the strategy of codon-pair deoptimization (CPD) as a means to attenuate RSV. In this strategy, one or more ORFs are recoded to introduce under-represented codon-pairs, with no changes in amino acid coding, codon usage, or nucleotide composition. This can cause suboptimal translation, among other effects, resulting in attenuation. Recoding potentially can involve hundreds or thousands of changes and thus should be refractory to de-attenuation. We made 4 CPD derivatives of RSV: Min A (CPD NS1, NS2, N, P, M, and SH), Min B (CPD G and F), Min L (CPD L), and Min FLC (CPD all ORFs except M2-1 and M2-2). In vitro, the CPD RSVs replicated more slowly and to lower titers than wt RSV even at the normally permissive temperature of 32C, with the following order of growth efficiency: WT>Min L>Min A>Min FLC>Min B. At higher incubation temperatures, all 4 of the CPD viruses were temperature-sensitive (ts), with the order of increasing sensitivity Min A
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