Research Center in Infectious Diseases, CHUQ-CHUL and Laval University, Québec City, Canada.
J Infect Dis. 2011 Oct 1;204(7):1008-16. doi: 10.1093/infdis/jir483.
The D222G (H1 numbering) hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pH1N1) infections. We investigated the impact of this mutation in vitro and in animal models using recombinant pH1N1 viruses.
The recombinant D222G HA mutant was generated from a wild-type (WT) clinical strain by using reverse genetics and site-directed mutagenesis. Replicative capacities were determined in MDCK and MDCK-α2,6 cells. Antigenicity was characterized by HA inhibition and microneutralization assays. HA titers were determined using human, chicken, and resialylated turkey red blood cells (RBCs). Virulence and contact-transmissibility were analyzed in mice and ferrets.
The recombinant D222G virus grew to significantly higher titers and generated larger viral plaques compared with the WT in MDCK but not in MDCK-α2,6 cells. The mutant also showed a significant reduction in HA titers using α2,6-expressing RBCs. The 2 recombinants were antigenically similar. The D222G mutant virus induced higher lung viral titers and alveolar inflammation in mice whereas the 2 recombinants had similar impacts in ferrets.
The D222G HA mutation alters receptor binding specificity, resulting in higher lung titers in mice. This could contribute to the higher case fatality rates reported in humans.
在 2009 年甲型 H1N1 大流行（pH1N1）感染的严重病例中，更高频率地检测到受体结合位点内的 D222G（H1 编号）血凝素（HA）突变。我们使用重组 pH1N1 病毒在体外和动物模型中研究了该突变的影响。
通过反向遗传学和定点诱变，从野生型（WT）临床株中生成了 D222G HA 突变体。在 MDCK 和 MDCK-α2,6 细胞中测定复制能力。通过 HA 抑制和微量中和测定来表征抗原性。使用人、鸡和去唾液酸化火鸡红细胞（RBC）测定 HA 滴度。在小鼠和雪貂中分析了毒力和接触传染性。
与 WT 相比，重组 D222G 病毒在 MDCK 中生长到明显更高的滴度并产生更大的病毒斑，但在 MDCK-α2,6 细胞中没有。该突变体使用表达α2,6 的 RBC 也显示出 HA 滴度的显著降低。两种重组体在抗原性上相似。D222G 突变病毒在小鼠中引起更高的肺部病毒滴度和肺泡炎症，而两种重组体在雪貂中具有相似的影响。
D222G HA 突变改变了受体结合特异性，导致小鼠肺部滴度更高。这可能导致人类报告的更高病死率。
