Measurement of viral titers in the mouse lung tissues
Mouse lung tissue samples were collected four days after the viral challenge, homogenized in PBS containing antibiotics, and centrifuged at 12,000×g. The supernatants were 10-fold serially diluted, inoculated into 95% semi-confluent MDCK cells, and incubated at 37 °C for 1 h. The culture media were then discarded, a low-temperature melting agarose solution was added over the cells, and the cells were incubated at 37 °C for three days. The cells were then fixed with 4% paraformaldehyde, stained with crystal violet, and the viral plaques formed were counted.