(A) Neuraminidase activity of patient-derived HPIV3 HNs. HEK293T cells were transfected with plasmids containing corresponding sequences of clinically isolated HNs. Neuraminidase activity was quantified by measuring amount of cleavage of 4-MUNANA. Results depict representative experiments from 3 biological replicates. Data indicate the mean ± SD. A 1-way ANOVA was performed to determine significance; P values are specified as follows: *P ≤ 0.05; ****P ≤ 0.0001. FC, fold change. (B) Release kinetics of patient-derived HPIV3 HNs binding sialic acid–containing RBCs at 37°C. HEK293T cells transiently expressing HNs were incubated with RBCs at 4°C for 30 minutes, washed, and transferred to 37°C. Supernatant was collected at 0, 5, 10, 15, 45, and 60 minutes and percentage of RBCs released at each time point was determined by quantification of relative absorbance at 410 nm. Results depict representative experiments from 3 biological replicates.