Thailand-Japan Zoonotic Diseases Collaboration Center, Kasetklang, Chatuchak, Bangkok 10900, Thailand.
J Gen Virol. 2010 Apr;91(Pt 4):938-48. doi: 10.1099/vir.0.016691-0. Epub 2009 Dec 9.
Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptor-binding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) alpha2,6galactose (Gal) or a Neu5Acalpha2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Acalpha2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Acalpha2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galbeta1,3/4GlcNAcalpha2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The alpha2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Acalpha2,3Gal more abundantly than Neu5Acalpha2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) alpha2,6Gal and Neu5Acalpha2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.
流感病毒在鸡胚中分离时其受体结合特性的改变尚未得到很好的研究。在本研究中，我们研究了仅在鸡胚或 Madin-Darby 犬肾（MDCK）细胞中分离的经典 H1 流感病毒的受体结合特性。序列分析显示，在血凝素（HA）蛋白中，鸡胚分离株发生了 D190V/N 或 D225G 的取代，而 MDCK 分离株保留了与临床样本中原始病毒相同的 HA 基因。具有 D190V/N 或 D225G 取代的鸡胚分离株对鼠和绵羊红细胞的血凝活性增加，但对兔红细胞的活性降低。此外，具有 D225G 取代的鸡胚分离株对鸡红细胞的血凝活性增加。使用具有 5-N-乙酰神经氨酸（Neu5Ac）α2,6 半乳糖（Gal）或 Neu5Acα2,3Gal 键的唾液酸化糖聚合物进行的直接结合测定表明，本研究中使用的鸡胚分离株对 Neu5Acα2,3Gal 受体的结合活性高于 MDCK 分离株。鸡胚分离株对 Neu5Acα2,3Gal 受体的结合活性增加也通过 Galβ1,3/4GlcNAcα2,3-唾液酸转移酶重新唾液酸化的鸡红细胞的血凝测定得到证实。流式细胞术和红细胞 N-糖链分析进一步证实了这些观察结果。α2,3 连接的唾液酸主要表达在鼠和绵羊红细胞的表面。鸡红细胞表达 Neu5Acα2,3Gal 多于 Neu5Acα2,6Gal，而兔红细胞表达 5-N-羟乙酰神经氨酸（Neu5Gc）α2,6Gal 和 Neu5Acα2,6Gal。我们的研究结果清楚地表明，经典 H1 流感病毒在鸡胚中分离和传代时，HA 蛋白中的氨基酸取代会导致其受体结合活性发生改变。
