Investigation for pathogenesis of influenza virus and SARS coronavirus

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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