Frozen respiratory samples stored in UTM are thawed, combined 1:1 with DMEM (Dulbecco’s Minimal Essential Medium; Lonza) supplemented with 5% FBS and 100 μg/ml Normocin (InvivoGen), and subsequently filtered through a 0.45 μm filter. The filtrate is used to infect HEp-2 cells (60% confluent) in T25 flasks for 1 h at 33 °C and 5% CO2. The supernatant is replaced with fresh DMEM supplemented with 5% FBS and 100 μg/ml Normocin and placed back into the 33 °C humidified, 5% CO2 incubator. The viral culture is harvested upon reaching approximately 70% cytopathic effect (CPE) by centrifugation at 247×g for 10 min and combining the supernatant with 50% sucrose in dPBS (sterile filtered). The viruses are stored in 1 ml aliquots at − 80 °C.
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