Influenza vaccines represent the most effective preventive strategy to control influenza virus infections; however, adaptive mutations frequently occur in the hemagglutinin (HA) glycoprotein during the preparation of candidate vaccine virus and production of vaccine in embryonated eggs. In our previous study, we constructed candidate vaccine virus (HA-R) to match the highly pathogenic avian influenza H7N9 viruses A/Guangdong/17SF003/2016 as part of a pandemic preparedness program. However, mixed amino acids (R, G, and I) were presented at position 220 (H3 numbering) in HA during passage in embryonated eggs. The residue at position 220 is located close to the receptor-binding site and the biological characteristics of this site remain to be elucidated. Therefore, in this study, using reverse genetics, we constructed two viruses carrying the single substitution in position 220 of HA (HA-G and HA-I) and evaluated the biological effects of substitution (R with G/I) on receptor binding, neuraminidase (NA) activity, growth characteristics, genetic stability, and antigenicity. The results revealed both mutant viruses exhibited lower HA binding affinities to two receptor types (sialic acid in alpha2,3- and alpha2,6-linkage to galactose, P < 0.001) and significant better growth characteristics compared to HA-R in two cells. Moreover, under similar NA enzymatic activity, the two mutant viruses eluted more easily from agglutinated erythrocytes than HA-R. Collectively, these results implied the balance of HA and NA in mutant viruses was a stronger determinant of viral growth than the individual amino acid in the HA position 220 in HA-R without strong binding between HA and sialylated receptors. Importantly, both the substitutions conferred altered antigenicity to the mutant viruses. In conclusion, amino acid substitutions at position 220 can substantially influence viral biological properties.

流感疫苗是控制流感病毒感染最有效的预防策略；然而，在制备候选疫苗病毒和在含胚鸡蛋中生产疫苗过程中，血凝素（HA）糖蛋白中经常发生适应性突变。在我们之前的研究中，我们构建了候选疫苗病毒 (HA-R) 以匹配高致病性禽流感 H7N9 病毒 A/Guangdong/17SF003/2016，作为大流行防范计划的一部分。然而，混合氨基酸（R、G 和 I）出现在 HA 中的第 220 位（H3 编号）在含胚卵中传代期间。220位的残基靠近受体结合位点，该位点的生物学特性仍有待阐明。因此，在本研究中，利用反向遗传学，我们构建了两种携带 HA 220 位单一取代的病毒（HA-G 和 HA-I），并评估了取代（R 与 G/I）对受体结合、神经氨酸酶 (NA) 活性、生长特性、遗传稳定性和抗原性。结果显示，两种突变病毒对两种受体类型（与半乳糖的 α2,3- 和 α2,6- 连接中的唾液酸、P  < 0.001) 和在两个细胞中与 HA-R 相比显着更好的生长特性。此外，在相似的 NA 酶活性下，这两种突变病毒比 HA-R 更容易从凝集的红细胞中洗脱。总的来说，这些结果表明突变病毒中 HA 和 NA 的平衡是比 HA-R 中 HA 位置 220 中的单个氨基酸更强的病毒生长决定因素，而 HA 和唾液酸化受体之间没有强结合。重要的是，这两种替换都赋予突变病毒改变的抗原性。总之，第 220 位的氨基酸取代可以显着影响病毒的生物学特性。