Serum clot activator tube

Female 7-8 weeks old BALB/c mice (Janvier, France) were randomly allocated to individually ventilated cages of 9 animals each. Food (Carfil, Belgium) and drinking water were available ad libitum. Prior to RSV challenge, the mice were anesthetized with 5% isoflurane (Halocarbon ® , New Jersey, USA) and subsequently intranasal inoculated with 2x10 5 PFU of pelleted WT or glycomutant RSV diluted in 100 µL HBSS. Mice were sacrificed by CO2 at 4, 6 and 8 days p.i. and the lungs were excised. The left lung was homogenized in HBSS containing 20% sucrose and clarified by centrifugation (4°C, 15 min, 1000 x g) for further titration by plaque assay in Vero cells as described (Schepens et al., 2014) . The right lung was fixed by formaldehyde (PF) for the preparation of paraffin slides (see below). To study antibody responses after infection, 6 animals/recombinant virus were included and blood was collected 3 and 5 weeks p.i. by retro-orbital bleeding after anesthetization with 5% isoflurane. The blood was left to clot in a serum clot activator tube (Sarstedt, Nümbrecht, Germany) at room temperature for 30 min and supernatant was collected after centrifugation (5 min, 10,000 x g). The animal studies were approved by the Animal Ethical Committee of the University of Antwerp (UA-ECD 2015-63).