Virus strains and HA vaccine
The type A influenza viruses used in this study were the mouse-adapted A/Puerto Rico/8/1934 (H1N1) [PR8] viral strains obtained from ATCC. Type A influenza viruses were propagated in MDCK cells in serum-free media in the presence of TPCK-trypsin and the median tissue culture infective dose (TCID50) and median lethal dose (LD50) of the viruses were calculated. The HA vaccines (viral vaccines) were prepared from influenza viruses, including A/Puerto Rico/8/1934 (A/PR8:H1N1) strains, according to the method of Jian et al . Briefly, we performed a plaque assay with MDCK and a hemagglutination assay with serum to calculate the titer of virus stock. The titer of virus stock showed that 1 hemagglutination unit (HAU) was equal to 103 PFU. The PR8 virus stock were exposed to ultraviolet light (UV) for 45 minutes.(Jian, Chang, Lin, Yang, & Chuang, 2013) The loss of infectivity of the UV‐inactivated PR8 virus was confirmed by the plaque assay.