Abstract
In vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. However, these experiments must be complemented with in vitro and ex vivo experiments. Here, we describe a protocol for the preparation and ex vivo infection of lung slices from different mammalian host species with various respiratory paramyxoviruses expressing fluorescent reporter proteins, and suggest follow-up experiments including immunohistochemistry, flow cytometry and confocal microscopy.
Highlights
Ex vivo models provide an important bridge between in vitro and in vivo experiments
The combination of these viable lung slices with recombinant viruses expressing fluorescent reporter proteins [7,8,9] allows for accurate, sensitive and reproducible assessment of respiratory virus infection and dissemination over time. Use of these recombinant viruses allows for real time monitoring of infection processes, using multiple methods for measurement of fluorescence
We have validated this technique by infecting lung slices of multiple host species with various paramyxoviruses expressing fluorescent reporter proteins (measles virus (MV), canine distemper virus (CDV), human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV)) [10]
Summary
Ex vivo models provide an important bridge between in vitro and in vivo experiments. The use of agarose-inflated lung slices for respiratory virus pathogenesis studies has been described previously [1,2,3,4,5,6]. We describe a protocol in which agarose-inflated lung slices can be kept viable in culture for at least seven days post-necropsy of an experimental animal The combination of these viable lung slices with recombinant viruses expressing fluorescent reporter proteins [7,8,9] allows for accurate, sensitive and reproducible assessment of respiratory virus infection and dissemination over time. Lung slices are suitable for analysis by immunohistochemistry, thereby visualizing virus cell tropism and spatial localization of infected cells within the tissue We have validated this technique by infecting lung slices of multiple host species (cotton rats, ferrets, dogs and macaques) with various paramyxoviruses expressing fluorescent reporter proteins (measles virus (MV), canine distemper virus (CDV), human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV)) [10]. This technique, is directly transferable to different host species and different viruses [11]
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