Establishment Of A Mouse-adapted Seasonal Influenza A H3N2 Virus Strain And Preliminary Inquiry Its Molecular Mechanism

Influenza virus is a single negative strand RNA virus, which belongs to the Orthomyxoviridae family. According to the nucleoprotein and matrix proteins, it can be sorted into A, B and C types. In addition, influenza A virus can also be categorized into different subtypes according to the hemagglutinin and neuraminidase of virion surface. Since its mass epidemic in 1918, influenza has brought great loss of life and property to human society, and put heavy burden on public health in the 20 century. Even in the 21st century, though with the progress of science and technology, we cannot curb the ravages of influenza virus. With the highly pathogenic avian influenza outbreaks, and the 2009 novel H1N1 flu pandemic in Mexico, influenza virus has become a major threat to worldwide public health security.Experimental animal are an important tool for studying influenza virus, which provides important assistance in the study of pathogenesis, immune response, drug and vaccine evaluation of influenza virus. Now the main animal models for influenza virus are:ferrets, swines, mice, and non-human primates. Each animal model has its own advantages and shortcomings. BALB/c mice are small, easy feeding, and after infection with influenza virus, can develop similar clinical symptoms and pathological changes in the lungs with human infected the flu, thus BALB/c mice become a popular choice in researching influenza virus. However, natural human influenza virus cannot infect BALB /c mice, so we need to continuously passage influenza viruses in BALB/c mice to obtain mouse adapted influenza virus strains.In this study, we continuously passage human seasonal influenza A virus (A/Hong Kong/1968 (H3N2)) in BALB/c mice for 7 generations. Through clinical observation, viral load and virus titer measuring, pulmonary pathological change observation and cytokine detection in BALB/c mice, we found that serially passage of influenza A virus in BALB/c mice lung boosted its virulence. Viral load increased by nearly four folds of magnitude, and the virus titer increased 2.51gCCID50 in Mice lungs. Virus growth curve test also proved that adaptation process in mice strengthened the virulence of virus.Meanwhile, the pathological changes in lungs of mice became rather more serious with the increasing of virus passages. To the mice that infected with 7th generation virus, massive hemorrhages and severe structure damage in lungs were observed, and with the virus adaption progress went on, more lesions of lung in infected mice can be seen. In addition, after sequencing of the fourth paragraph of the influenza virus (encoding HA protein) and section VI, paragraph (NA protein coding),we found the seventh generation of these two segments have show sense of point mutations, which may related to the virulence increase and adaptation in mice.