The Analysis Of Molecular Basis Of Pathogenicity Of Influenza A Virus By Using The Mouse As A Mammalian Model

The events of influenza A virus infection in humans occurred frequently and quickly in the recent years. These events include the human pandemic and the cross-transmission of avian influenza virus infection in humans, for example, the 2009 pandemic H1N1, the 1997 Hong Kong H5N1 highly pathogenic avian influenza virus infection in humans, and the 2013 H7N9 avian influenza virus infection in humans. The above events indicated that influenza A virus have posed a serious threat to the public health and safety. Therefore, it is very important to strengthen the exploring on the mammalian pathogenicity of influenza A virus. The studying of molecular basis of mammalian pathogenic of influenza A virus can provide molecular markers for monitoring the emergence of new highly pathogenic influenza viruses timely. Furthermore, it can improve our cognition of influenza A virus. The mouse model was widely used to study the pathogenicity of influenza A viruses in mammals. To study the molecular basis of pathogenicity of the 2009 pandemic H1N1 virus, the H1N2 avian influenza virus, the H3N2 avian influenza virus, the H5N5 avian influenza virus, and the H5N8 avian influenza virus in mammals, we performed the following studies:the serial passage of influenza viruses in mice, the whole genome sequencing, the identification of the key amino acid sites by reverse genetics technologies, the virulence evaluating in mouse, and the replication dynamics of influenza viruses in vitro and in vivo.Firstly, a 2009 pandemic H1N1 influenza virus was selected as the target in this study. In a previous study, we have obtained the highly pathogenic viruses through the serial passages of a 2009 pandemic H1N1 influenza virus in mice’s lungs. In this study, we performed the following works:recording the body weight change of mice, analyzing the survival of mice, analyzing the MLD50, analyzing the tissue tropism, and analyzing the replication dynamics of influenza viruses in vitro and in vivo. The results indicated that the morbidity and the mortality of a recombinant 2009 pandemic H1N1 influenza virus named rXD-PB1-296R increase significantly compared to the parental virus. The recombinant rXD-PB1-296R can be detected in the brains of mice. The mice’s virulence of rXD-PB1-296R enhanced 1000-fold higher compared to the parental virus. The above results suggested that PB1-T296R is the highly pathogenic molecular basis of the 2009 pandemic H1N1 virus in mice.On the basis of the above studies, we also selected the H1N2, H3N2, H5N5, andH5N8 avian influenza viruses as the targets to research the molecular basis of mammalian pathogenic of the avian influenza viruses. The mice were inoculated with the H1N2, H3N2, H5N5, and H5N8 avian influenza viruses, and these viruses were passaged in the mice’s lungs. We have obtained the H1N2, H3N2, H5N5, and H5N8 avian influenza mouse-adapted viruses, and the mice’s pathogenicity of these viruses are high. We analyzed the biological difference between the parental viruses and the mouse-adapted viruses. The replicate ability of H1N2, H3N2, H5N5, and H5N8 avian influenza mouse-adapted viruses are stronger compared to the parental viruses. The tissue tropism of H1N2, H3N2, H5N5, and H5N8 avian influenza mouse-adapted viruses are border compared to the parental viruses. The mice’s pathogenicity of H1N2, H3N2, H5N5, and H5N8 avian influenza mouse-adapted viruses are higher compared to the parental viruses. The whole genome sequencing and pathogenicity tests were carried on the different generations of mouse-adapted isolates. The mice’s virulence of the H1N2 avian influenza virus, which harbored PB2-D701N mutation, enhance at least 56-fold compared to the parental virus. The mice’s virulence of the H3N2 avian influenza virus, which harbored PB2-D701N mutation, enhance 56-fold compared to the parental virus. The mice’s virulence of the H5N5 avian influenza virus, which harbored PB2-E627K mutation, enhance 1000-fold compared to the parental virus. The mice’s virulence of the H5N8 avian influenza virus, which harbored PB2-E627K mutation, enhance 1000-fold compared to the parental virus. The above results suggested that the PB2-D701N is the molecular basis of high pathogenicity of the H1N2 or H3N2 avian influenza virus in mice, and the PB2-E627K is the molecular basis of high pathogenicity of the H5N5 or H5N8 avian influenza virus in mice.In summary, we found a series of virulent molecular markers for 2009 pandemic H1N1 influenza virus, H1N2 avian influenza virus, H3N2 avian influenza virus, H5N5 avian influenza virus, and H5N8 avian influenza virus in mice, including PB1-T296R, PB2-E627K, and PB2-D701N, etc. The results will deepen our understanding of the molecular basis of pathogenicity of influenza A virus in mammals. Our studies provide the theory basis for the monitoring of influenza A virus, and referential data for the studies of pathogenesis of influenza viruses.