Investigation for pathogenesis of influenza virus and SARS coronavirus

高致病性禽流感H5N1病毒(H5N1-Flu)史无前例的重新出现和严重急性呼吸综合征冠状病毒(SARS-CoV)的爆发对人类构成重大威胁。更好地了解这些高致病性病毒的基本知识和反向遗传学等新技术将有助于我们制定有效的预防和/或治疗策略来控制疾病负荷。我们建立了负链RNA病毒的反向遗传学，并分析了这些病毒复制和细胞致病的机制。从1岁的猪肺泡组织中分离出原代呼吸道上皮细胞，并研究了禽流感病毒在这些细胞中的生长动力学。H5N1病毒可显著诱导猪呼吸道上皮细胞死亡，尤其是细胞凋亡，而这三种病毒对鸡胚胎成纤维细胞的细胞毒性作用相似，提示H5N1禽流感病毒是…的主要致病因子。哺乳动物呼吸道上皮细胞由于诱导凋亡而导致的更严重的细胞死亡。细胞克隆#21是传染性SARS-CoV的长期制造者，尽管SPEKE(S)蛋白对病毒粒子的掺入率明显较低。序列分析表明，病毒结构蛋白在S蛋白和膜蛋白(M)中分别有4个和1个氨基酸替换。我们使用病毒样颗粒形成系统证明，S突变与S蛋白对病毒粒子的低掺入有关，尽管破坏糖基化的M突变与这一表型无关。进一步的突变实验发现，受体结合域中的两个突变Y442C和L472F可能是导致S掺入减少以及S与ACE2受体结合亲和力降低的关键因素。因此，这两个氨基酸的替换可能会导致S蛋白的构象变化，从而减少对病毒颗粒的掺入。较少

The unprecedented re-emergence of highly pathogenic avian influenza H5N1 viruses (H5N1-Flu) and the outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) pose a significant threat to humans. Better basic knowledge of these highly pathogenic viruses and novel technologies such as reverse genetics will help us to generate effective prophylactic and/or therapeutic strategies to control the disease load. We have developed the reverse genetics of negative strand RNA viruses and were analyzing the mechanisms behind the replication and cytopathicity of these viruses.Primary airway epithelial cells from alveolar tissues were prepared from 1-year-old pigs and the growth kinetics of avian H5 influenza viruses in these cells was investigated. H5N1 virus significantly induced cell death, especially apoptosis, in porcine airway epithelial cells although these three viruses induced similar cell toxicity in chicken embryonic fibroblasts, suggesting that avian H5N1 influenza virus leads … More severe cell death in mammalian airway epithelial cells due to the induction of apoptosis.A cell clone #21 is a long-term producer of the infectious SARS-CoV, although the incorporation rate of spike (S) protein into virions is significantly lower. Sequencing analysis of the viral structural proteins revealed four and one amino acid substitutions in the S and membrane (M) proteins, respectively. We demonstrated, using a viral-like particle formation system, that the S mutations were involved in lower incorporation of the S protein into virions, although the M mutation disrupting the glycosylation was not involved in this phenotype. Further mutational experiments identified two substitutions, Y442C and L472F, within the receptor binding domain that could be critical for the reduced S incorporation, as well as reduced binding affinity between the S and ACE2 receptor. Thus, these two amino acid substitutions might lead to a conformational change in the S protein, resulting in reduced incorporation into viral particles. Less