H9N2亚型流感病毒血凝素受体结合区定点突变株拯救及其受体亲和性鉴定

Rescue of site-directed mutants of the HA receptor-binding domain of H9N2 subtype influenza virus and the characterization of viral receptor affinity

利用重叠PCR技术对H9N2亚型流感病毒Y280亚系毒株A/Chicken/Jilin/DH102/2012(H9N2)的血凝素受体结合区220-loop中相对保守的226位和227位进行了氨基酸置换,并通过反向遗传技术拯救了定点突变病毒。固相酶联试验结果显示:226位为L时,病毒偏嗜结合人型受体;226位为Q时,病毒偏嗜结合禽型受体;226位为M时,病毒同时具有与2种类型受体结合的能力,但偏嗜结合人型受体;227位为Q或M时,病毒均偏嗜结合人型受体。因此,相比于227位,Q226L突变足以改变病毒的受体偏嗜性。由于近年来90%以上的H9N2病毒分离株HA226位氨基酸都是L,推测226L是H9N2病毒感染哺乳动物的重要位点。

The binding doma tant determin were substitu first step of viral infection is the binding between the hemagglutinin （HA） receptorin of influenza A virus and the sialic acid receptors on the cell, which is also an imporant factor of viral host spectrum. In this study,the relatively conservative amino acids ted at positions 226 and 227 within the 220-1oop at tbe HA receptor-binding domain of A/Chicken/Jilin/DH102/2012 （HgN2） in the Y280 like sublineage by the overlapping PCR and the mutants were rescued by the reverse genetics technique. The resuhs of solid-phase enzyme re- ceptor-binding assay showed that L-226-containing virus prefers binding to human-like receptors; Q-226-containing virus binds to avian-like receptors in priority;M-226-containing virus can bind to both two types of receptors,despite of preferentially binding to humandike receptors. For those vi- ruses possessing Q or M at position 227, they prefer human-like receptors. Therefore, the Q226L mutation is enough to alter the receptor preference of virus compared with the substitution at posi tion 227. It is speculated that 226L is an important determinant site of H9N2 virus infection in mammals,given that the amino acid at position 226 of HA is L,which was displayed in more than 90% of H9N2 virus isolates in recent years.