Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory tract disease worldwide and is responsible for a huge burden of morbidity and significant mortality. A licensed vaccine or effective antiviral therapy is unavailable. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We developed a method for producing infectious recombinant RSV by the intracellular coexpression of cDNAs encoding a complete RSV replicative intermediate RNA (antigenome) and the N, P, L and M2-1 proteins, which together constitute a nucleocapsid that is fully competent for RNA synthesis. This provides an important tool for basic molecular and pathogenesis studies as well as a method for fine-tuning the level of attenuation of candidate vaccine viruses. We showed that RSV encodes ten mRNAs and eleven unique proteins (the M2 mRNA contains two overlapping ORFs encoding the M2-1 and M2-2 proteins). Five RSV genes, namely NS1, NS2, SH, M2-2 and G, could be """"""""knocked out"""""""" (deleted) singly and in some cases in combination without ablating the ability of the virus to grow in cell culture. However, in most cases growth was less efficient. Deletion of the NS1 and NS2 genes singly or in combination was found to be highly attenuating in mice and chimpanzees. These deletions are candidates for inclusion in a live-attenuated vaccine. The M2-2 deletion virus exhibited a shift favoring transcription over replication. This implicates this protein as a regulatory factor in RNA synthesis. This mutation has the novel property of reducing growth while increasing, rather than decreasing, gene expression, and thus might make a vaccine that is """"""""better than nature"""""""". The G knockout virus demonstrated cell-specific differences in growth efficiency, implying that it is using an alternative receptor whose distribution is cell-specific. Thus, these findings have important implications for virus assembly and receptor usage. Fully-viable chimeric viruses were constructed between human RSVs representing the two antigenic subgroups, making it possible to use a single attenuated backbone to express the major antigenic determinants of each of the subgroups. Fully-viable chimeras also were constructed between human RSV and bovine RSV, a virus that has a host range restriction that renders it highly attenuated in primates and thus represents a new method of attenuating an RSV vaccine. As another approach to making an RSV vaccine """"""""better than nature"""""""", recombinant RSV was engineered to express various cytokines and chemokines in order to enhance immunogenicity and, in some cases, attenuate the virus.
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