Abstract: 【Objective】 The antigenicity of influenza virus is mainly determined by the hemagglutinin (HA), a surface glycoprotein of the virus. Our previous study indicated that amino acid changes at positions 190, 230 and 269 (H3 numbering) of HA protein resulted in antigenic escape by using the monoclonal antibody (mAb) against the HA protein of Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV). These three amino acid substitutions widely existed in the HA protein of the recently isolated EA H1N1 SIVs. This study aimed to explore which amino acids play a key role in the antigenicity of the virus and further provide scientific basis for the control of influenza.  【Method】 In this study, A/swine /Liaoning /SY72 / 2018 (H1N1) (SY72) was selected as the model virus, and its reverse genetic system (RGS) was established. Then, using SY72 virus as backbone, three reassortant viruses were rescued by introducing the respective single amino acid mutation at position 190, 230, and 269 into HA protein. Antisera were raised by inoculating the rSY72-inactivated vaccine into specific-pathogen-free (SPF) chickens and non-immunized pigs.  The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the rSY72virus. Then their effects on viral replication capacities and receptor binding properties were further evaluated.  【Result】Sequence analysis showed that the HA of SY72 virus carried 190D, 230M, and 269R, respectively. The viruses, including rSY72, rSY72HA/D190N, rSY72HA/M230I, and rSY72HA/R269M were rescued by RGS. The results of neutralization test showed that all the three mutant viruses could react with two mAbs, to some extent, compared to the rSY72 virus.  The HI results indicated that the HI antibody titers of the rSY72HA/D190N reacted with the rSY72–immunized chicken and pig sera were 4- and 8-fold lower than those of the rSY72 virus with the respective sera. However, the rSY72HA/M230I and rSY72HA/R269M virus reacted well with these two sera. The results indicated that residue 190 in the HA had the important effects on the viral antigenicity. Results of the viral plaque assay and growth curve experiments demonstrated that plaque sizes generated by the rSY72HA/D190N virus were smaller than those by the rSY72 virus in MDCK cells. and the replication ability of the rSY72HA/R269M virus was significantly decreased in MDCK cells, compared with that of the rSY72 virus. Furthermore, the three amino acid mutations had no impact on the viral receptor-binding preference.【Conclusion】 The amino acid at position 190 of HA protein plays an important role in determining the antigenicity of EA H1N1 SIV. The mutation of amino acid at position 269 reduces the viral replication ability in MDCK cells. Above these results suggested that more attentions should be paid for monitoring these residue changes in the influenza surveillance, so as to improve early warning of influenza in animals. 

Key words: swine influenza virus, HA protein, antigenicity