ABSTRACT
BACKGROUND: Limited knowledge about the molecular mechanism of avian influenza H9N2 virus pathogenicity in birds as well as human hosts has limited the development of effective control against the disease. To overcome this issue detailed understanding of the infectious characteristics of the virus in host cells should be obtained. OBJECTIVES: In this study we examined the replication kinetics of H9N2 virus in a chicken hepatoma cell line to obtain insight into the pathogenesis of H9N2 viruses. MATERIALS AND METHODS: The kinetic replication of H9N2 influenza virus in chicken hepatoma and fibroblastic cells was studied in the presence and absence of supplemental trypsin. High viral titers observed in liver cells in a short time correlated with the degree of cytopathic effects. To determine whether the ultimate outcome of infection results in programmed cell death, the infected cells were observed by the cell viability assay, DNA fragmentation, caspase cascade activation, and quantified lactate dehydrogenase release. RESULTS: The degree of viability was significantly reduced in infected hepatoma cells. Observations of caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. The infected hepatoma cells released lactate dehydrogenase, which is consistent with cell death by necrosis. CONCLUSIONS: Taken together, these data reveal that cellular protease of chicken liver cells allows the replication of high yields of H9N2 virus in the absence of trypsin and also cell death in the infected cells is due to necrosis.
ABSTRACT
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.
