Introduction and objectives Respiratory Syncytial Virus (RSV) is a major cause of lower respiratory tract infection during infancy. Neutrophils are the predominant cell type within the RSV-infected airway, 80% of inflammatory cells from BAL of intubated infants being neutrophils. Despite extensive research unpicking innate viral responses to RSV little is known about the specific role the neutrophil plays. The aim of this work was to investigate the response of neutrophils to RSV using an in vitro model utilising neutrophils from adult volunteers. To investigate whether relative immaturity of infant neutrophils leads to impaired responses we are now comparing adult neutrophils with neonatal neutrophils, isolated from cord blood.

Methods Highly purified neutrophils from whole blood of healthy adult donors were incubated with RSV. Samples were taken at 2, 4, and 20 h for QT-PCR of RSV N gene, western blot analysis and cytospin slides for confocal imaging of RSV F protein. Experiments were then replicated using neutrophils purified from cord blood, collected from the placenta following elective caesarean section, of healthy term neonates. Supernatants were stored for measurement of the cytokine response of the neutrophil to RSV.

Results Uptake of RSV by adult neutrophils was shown by both western blot and quantitative RT-PCR. Maximal uptake was at 4 h with a reduction by 20 h. Confocal microscopy was undertaken, using a primary monoclonal antibody to RSV fusion protein. This showed that RSV was internalised inside the cytoplasm in a distribution suggestive of endosomal uptake (see figure). Preliminary data suggests that neonatal neutrophils are also capable of this viral uptake but work is ongoing to determine differences between the two models.

Conclusions We have shown that neutrophils may be involved in viral clearance as part of the immune response to viral invasion. They appear to take up virus with kinetics suggestive of endocytosis. Work is ongoing to establish the mechanism of entry using a panel of inhibitors. Initial results would suggest that neonatal neutrophils may respond similarly but work continues to establish whether this is the case and if there is any functional impairment that may explain infants’ propensity to severe disease.

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Abstract S87 Figure 1

Indirect fluorescent confocal microscopy was carried out on cytospins of neutrophils purified from adult blood and incubated with RSV for a 2 h in the presence of GM-CSF. Z stacks were taken using a Leica confocal and Z-projection images produced using Imaris software. RSV (white arrow) is identified in all depths of the cytoplasm in this orthogonal view throughout the cell in discrete pockets suggestive of endosomes