Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are important respiratory viruses which cause significant morbidity and mortality, particularly in pediatric, elderly and immunocompromised patients. Sensitive molecular techniques show that patients with respiratory infections are frequently infected with more than one viral pathogen. In the case of RSV and HMPV co-infections, while some studies found no or limited impact of co-infection, other research strongly indicates that RSV-HMPV co-infection has a deleterious effect on the course of disease and severity of symptoms. However, few studies have examined the molecular details of co-infection by these two important pathogens. Thus, the impact of RSV/HMPV co-infections remains a critical area for continued study, but relatively little is known about the mechanisms by which co-infections may affect the steps of the viral lifecycle. Viral interference through triggering of innate immunity and also competition for cellular resources have been studied for their roles in co-infection, and very recent work suggests that hybrid viral particles can form between influenza and RSV. Our preliminary studies of HMPV/RSV co-infection demonstrate that inclusion bodies (IBs), which are key regions for viral transcription and replication, can contain both RSV and HMPV genomes in co-infected cells, indicating a shared replication compartment. Our overall hypothesis is that HMPV/RSV co-infection affects both virus replication and virus spread. To test this, we will pursue two Specific Aims, using a range of experimental approaches and the wealth of reagents we have created for the examination of pneumovirus infection. First, we will dissect the effect of HMPV/RSV co-infection on IB formation, dynamics and viral replication, including careful analysis of IBs over the course of infection and analysis of viral transcription and replication at the global and single cell level. Second, we will determine the effect of HMPV/RSV co-infection on potential formation of functional hybrid virus particles. In addition, we will examine whether a unique pathway for cell-to-cell spread which we have characterized for HMPV is also utilized by RSV during co-infection. These important experiments will elucidate new molecular consequences of viral co- infection, providing insight that may inform studies of additional respiratory viruses and lead to new ways to target respiratory virus infections.