Understanding the growth dynamics of influenza viruses is an essential step in virus replication and cell-adaptation. The aim of this study was to elucidate the growth kinetic of a low pathogenic avian influenza H9N2 subtype in chicken embryo fibroblast (CEF) and chicken tracheal epithelial (CTE) cells during consecutive passages. An egg-adapted H9N2 virus was seeded into both cell culture systems. The amount of infectious virus released into the cell culture supernatants at interval times post-infection were titered and plaque assayed. The results as well as cell viability results indicate that the infectivity of the influenza virus was different among these primary cells. The egg-adapted H9N2 virus featured higher infectivity in CTE than in CEF cells. After serial passages and plaque purifications of the virus, a CTE cell-adapted strain was generated which carried amino acid substitutions within the HA stem region. The strain showed faster replication kinetics in cell culture resulting in an increase in virus titer. Overall, the present study provides the impact of cell type, multiplicity of infection, cellular protease roles in virus infectivity and finally molecular characterization during H9N2 virus adaptation procedure.
To understand human response to avian H9N2 influenza, we investigated the effects of the viral infection on A549, HepG2, and HeLa cells at low and high MOIs. To identify virus-host interplay, expression of Mx and NP genes was measured in the cells supernatants. Cell viability and apoptosis were evaluated by MTT assay, DNA fragmentation, and florescent staining. The virus titration and NP gene transcript levels indicate lower susceptibility of HeLa cell to H9N2 replication than other cells. Although H9N2 did produce a faster CPE in HepG2, high dose of the virus induced apoptosis within early stage of A549 infection. The DNA laddering was enhanced in the cell correlated with increase in virus transcripts. The undetectable to different regulation levels of Mx gene were observed in response to H9N2 infection suggesting that an insufficient antiviral defense in the noncompetent-IFN HepG2 cell promotes efficient viral replication. These results showed that the permissivity of HepG2 for H9N2 is comparable with A549; however, liver cells are not target tissue respond to the infection. These data revealed that the H9N2 virus induced apoptosis signaling via mitochondrial pathway in human alveolar epithelial cells, indicating that the induction may be associated with a dose-dependent manner.
Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1 (HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte (CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin (HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.
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