Abstract
Paramyxoviruses, including measles virus (MV), human metapneumovirus (HMPV), human respiratory syncytial virus (HRSV) and canine distemper virus (CDV), are transmitted via the respiratory route. Despite their close phylogenetic relationship, the pathogenesis of these viruses is very different. To study viral tropism and replication ex vivo, a protocol for the inflation of lungs with low-melting-point agarose mixed with culture medium was established. Lung slices were prepared and remained viable in culture at 37°C for at least 7 days. Lung slices obtained from different animal species were infected with recombinant paramyxoviruses expressing enhanced green fluorescent protein (EGFP). Progression of infection was monitored in real time by detection of EGFP fluorescence. Flow cytometric analysis of cells emigrating into the culture medium proved to be a valuable tool for qualitative and quantitative assessment of infection over time. MV replicated optimally in lung slices of macaques, HMPV and HRSV in lung slices of cotton rats, and CDV in lung slices obtained from dogs and ferrets. In conclusion, these ex vivo lung slice cultures are discriminatory models for the study of respiratory virus infections and can be used to inform the design of future in vivo experiments, thereby reducing numbers of animals required.
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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