Virus titer determination
Virus titers were determined in the lung homogenates and nasal washes.
The lungs were removed into RPMI and homogenized by gentleMACS (Miltenyi
Biotec). Nasal washes were collected by flushing with 150 µl PBS. The
nasal washes and lung homogenates were stored at −80 °C, and the RNA was
isolated by MagNA Pure 24 Total NA Isolation Kit on the MagNA Pure 24
instrument (Roche). The quality of the RNA was determined using
NanoDrop™ 2000/2000c (Thermo Fisher Scientific). Virus titers were
determined by the virotype Influenza A RT-PCR Kit (Indical Bioscience,
Leipzig, Germany), using 70 ng of RNA. RT-PCR was performed on a
LightCycler® 480 (Roche) using AbsQuant 2nd Derivative Max for obtaining
the Ct value. PCR conditions were 10 min at 45 ℃, 10 min at 95 ℃,
followed by 40 cycles of 15 s at 95 ℃ and 1 min at 60 ℃. The relative
mRNA amount was obtained by the ΔΔCt method12, using
β-actin as housekeeping gene.