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 remains to be developed. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of the infection in experimental animals, and the difficulty of achieving an appropriate balance between immunogenicity (which depends on reasonable levels of virus replication) and attenuation (which depends on reduced levels of virus replication). We recently 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. RSV encodes ten mRNAs encoding eleven proteins (the M2 mRNA contains two overlapping ORFs encoding two separate proteins, M2-1 and M2-2). We investigated whether individual RSV genes could be ?knocked out? (deleted) without ablating the ability of the virus to grow in cell culture. To date, four RSV genes have been individually knocked out without loss of infectivity, namely NS1, NS2, SH, and G. Deletion of the NS2 gene is highly attenuating in vitro and in vivo and represents a very useful mutation for vaccine purposes. The NS1 and SH knockout virus are moderately attenuating, and also are candidates for inclusion in a live-attenuated vaccine. The G knockout virus grows well in certain cells but not others, implying that it is using an alternative receptor whose distribution is cell-specific. The ability to recover a G knockout virus shows that G is not essential for the formation or transmission of infectious virus, findings which have important implications for virus assembly and receptor usage. Finally, we previously showed that RSV can accept and express an added foreign gene. Here, we have expressed certain cytokines in recombinant RSV as a possible method to improve the immune response to a live-attenuated vaccine. - Virus, vaccine, live-attenuated viral vaccine, pediatrics, infectious disease, respiratory tract disease, recombinant DNA

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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