HOST RANGE VARIATION OF EMERGING AND REEMERGING INFLUENZA VIRUSES IN ASIA

在中国南方，目前正在发生禽流感病毒在人群中的持续传播。在我们为期两年的研究中（1999-2000），我们发现了亚洲流感病毒宿主介导变异的几个重要机制。1)H3人流感病毒的血凝素（HA）尽管起源于禽类，但不支持病毒在鸭肠中的复制。在人A/Udorn/307/72 （H3N2）的HA中，226位的Leu-to-Gln突变和228位的Ser-to-Gly突变允许重组病毒在鸭子中复制。2)用neu5gc2 - 3gal特异性抗血清进行免疫荧光检测，主要在鸭结肠隐窝上皮细胞上检测到该片段。这种具有生物学结果的识别表明，对Neu5Gc2-3Gal片段的识别在禽流感病毒的亲肠性中起重要作用。3)猪气管上皮细胞具有结合鸟类和人类A型流感病毒的受体涎糖链（Neu5Ac2-6Gal）。这些结果表明，猪可能在自然界中充当人禽流感病毒和禽流感病毒的混合容器。A病毒血凝素受体结合特异性的宿主范围选择发生在病毒在表达不同受体唾液糖链的不同宿主细胞中的维持过程中。4)甲型流感病毒神经氨酸酶（受体破坏酶）的底物特异性表明，所有病毒对Neu5Ac2-3Gal具有相似的特异性，而对Neu5Gc2-6Gal的活性介于边缘（以禽流感和早期N2人类病毒为代表）和高（尽管仅为Neu5Ac2-3Gal活性的三分之一）之间（以猪和最近的N2人类病毒为代表）。通过位点特异性诱变，我们在最早的人类病毒中发现了Neu5Ac2-6Gal特异性可检测到的增加，275位（Leu到Val）的变化增强了该底物的特异性。5) HA识别Neu5Ac2-6Gal但不识别Neu5Ac2-3Gal或Neu5Gc2-3Gal的流感病毒在马体内复制失败。生化和免疫组织化学分析以及凝集素结合试验表明，马气管上皮细胞中Neu5Gc2-3Gal片段的丰度表明，对该片段的识别对马的病毒复制至关重要。上述新结果表明本课题的研究目标取得了令人满意的进展，为阐明亚洲流感病毒血凝素和神经氨酸酶新亚型的宿主范围变异和新亚型的出现机制，为控制流感病毒变异建立基本思路提供了基础实验结果。少

In south China, the continuous transmission of avian influenza viruses into human population is currently occurring. In our two year's investigation (1999-2000), we found several important mechanisms on the host mediated variation of influenza viruses in Asia.1) The Hemagglutinin (HA) of H3 human influenza viruses does not support viral replication in duck intestine despite its avian origin. A Leu-to-Gln mutation at position 226 and a Ser-to-Gly mutation at 228 in the HA of human A/Udorn/307/72 (H3N2) permit a reassortant virus to replicate in ducks.2) Immunofluorescense assays with Neu5Gc2-3Gal-specific antiserum detected this moiety primarily on the crypt epitherial cells of duck colon. Such recognition with biological results suggests that recognition of the Neu5Gc2-3Gal moiety plays an important role in the enterotropism of avian influenza viruses.3) Epitherial cells in pig trachea has receptor sialosugar chains that binds both of bird and human influenza A viruses (Neu5Ac2-6Gal fo … More r humans aud Neu5Ac2-3Gal for birds). These results indicate that pig may play as a mixing vessel of human and bird influenza viruses in nature. The host range selection of the receptor binding specificity of the A virus hemagglutinin occurrs during the maintenance of the virus in different host cells which express different receptor sialo-sugar chains.4) Substrate specificity of influenza A virus neuraminidase (receptor destroying enzyme) showed that all viruses had similar specificities for Neu5Ac2-3Gal, while the activities for Neu5Gc2-6Gal ranged marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for Neu5Ac2-3Gal), as represented by swine and more recent N2 human viruses. Using site specific mutagenesis, we identified in the earliest human virus with a detectable increase in Neu5Ac2-6Gal specificity a change at position 275 (Leu to Val) that enhanced the specificity for this substrate.5) An Influenza virus with an HA recognizing Neu5Ac2-6Gal but not Neu5Ac2-3Gal or Neu5Gc2-3Gal failed to replicate in horses. Biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the Neu5Gc2-3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses.The above new results show that the aim of this project is satisfactory progressed, and the fundamental experimantal results to elucidate the mechanism of host range variation and emerging of the new subtype of the hemagglutinin and neuraminidase of influenza viruses in Asia and to establish the fundamental idea for the control of the influenza virus variation. Less