Amino Acid of 225 in the HA Protein Affects the Pathogenicities of H1N1 Subtype Swine Influenza Viruses

The pathogenicities of influenza viruses are determined by multiple viral genes. The results of our previous study indicated that hemagglutinin (HA) gene substitutions of the two genetically similar H1N1 swine influenza viruses altered their pathogenicities in mice. This study aimed to further identify the key amino acids affecting viral pathogenicity.

After analyzing the amino acid differences of HA protein between the two H1N1 viruses, the reassortant viruses bearing the single amino acid mutations were constructed using the site-directed mutagenesis primers, and their EID₅₀ values were determined. To determine the growth of the parental, reassortant and mutant viruses in vitro, MDCK cells and A549 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001 and 0.1, respectively. The BALB/c mice was further intranasally (i.n.) inoculated with 10⁶ EID₅₀ of each virus, and three mice were euthanized at 3 days post-infection (dpi). The organs, including brain, nasal turbinate, lung, kidney and spleen, were collected from the mice and titrated in eggs to evaluate the viral replication abilities in vivo. The MLD₅₀ values of the indicated viruses were determined by inoculating i.n. groups of five mice with 10¹-10⁶ EID₅₀ of viruses. The body weight was measured daily for 14 dpi, and the mice that lost more than 25% of their original weight were euthanized for humane reasons.

The HA proteins of the ZD71 and SY130 viruses differed at four amino acids at positions 4, 138, 144, and 225 (H3 numbering). Four reassortants were rescued, followed by whole-genome sequencing to ensure the absence of unwanted mutations. The viral replication abilities of the reassortant viruses (rZD71-HA/G225E and rSY130-HA/E225G) were significantly affected in MDCK, as well as in A549 cells, when G225E and E225G substitutions were introduced into the rZD71 and rSY130 virus, respectively. In contrast, the mutations of the other three amino acids had little effect on viral replication in vitro. Further mouse infection experiments also demonstrated that amino acid substitutions at site 225 of HA protein significantly affected the viral pathogenicities in mice. In particular, the substitution G225E increased the pathogenicity of rZD71-HA/G225E virus, with the MLD₅₀ value of rZD71-HA/G225E virus decreasing from 4.32 log₁₀EID₅₀ to 3.0 log₁₀EID₅₀, compared with that of rZD71 virus. And the virus replicated well not only in the nasal turbinate and lung, but also in the spleen and kidney.

A single amino acid at position 225 in the HA protein significantly affects the viral replication capacity and virulence of these two H1N1 swine influenza viruses. It is suggested that close monitoring for this residue should be paid in the future virological surveillance, so as to provide a scientific basis for better prevention and control of animal influenza, and even human influenza pandemic.