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.