Persistence of H1, H3, and H5 pseudotypes in water.

<p><b>A.</b> P24 capsid ELISA was performed on three independent production of HA bearing pseudotypes in triplicate. PBS and pseudotypes produced in the absence of HA and NA proteins were used as negative controls (PBS and HA-/NA- respectively). <b>B.</b> Quantitative real-time RT-PCR targeting the luciferase genome was performed on three independent production of HA bearing pseudotypes (expressed as the mean Log₁₀ RNA copies.mL⁻¹ ± SD of triplicate repeats). H₂0 and pseudotypes produced in the absence of HA and NA proteins were used as negative controls (H₂0 and HA-/NA- respectively). <b>C.</b> Pseudotypes were diluted in water (1∶2) in the presence (35 g.L⁻¹) or absence (0 g.L⁻¹) of NaCl and were exposed to 4 and 35°C. At each time interval, single-cycle infectivity assays were performed. Loss of infectivity was estimated by calculating the difference between the RLUs after 48 hours and the initial RLU titer. <b>D.</b> Pseudotypes from two independent production stocks were diluted in water (1∶2) in the presence (35 g.L⁻¹) or absence (0 g.L⁻¹) of NaCl and were exposed to 35°C. At each time interval, single-cycle infectivity assays were performed. Loss of infectivity was estimated by calculating the difference between the RLUs after 48 hours and the initial RLU titer. Experiments were carried in triplicates and error bars indicate standard errors of the mean; ***p<0.0001 and NS (not significant) (unpaired Student’s t-test). <b>E.</b> Transmission electronic microcopy of negatively stained H1 pseudotypes, which have been exposed to 35°C and 35 g.L⁻¹ of NaCl for 72 hours.