Pathogenesis of the novel avian-origin influenza A (H7N9) virus Influenza H7N9 virus in human lower respiratory tract

Abstract

Background: As of May 2013, 131 laboratory-confirmed human infections with a novel influenza H7N9 virus had been reported from China. The source of human infection appears to be poultry. There is so far no evidence of sustained human-to-human transmission. Genetic analysis revealed that all eight gene segments of H7N9 were of avian origin; six internal gene segments from avian influenza H7N9 viruses, while hemagglutinin and neuraminidase genes were derived from influenza viruses circulating in ducks and wild ducks, respectively. The emergence of the H7N9 influenza virus catches global attention about whether the new virus could spark another pandemic. The majority of the infected patients were hospitalized and suffered from ARDS, with a fatality rate of about 37%. Our study aimed to determine the mechanism contributing to the pathogenesis of the H7N9 virus. A panel of proinflammatory cytokines and chemokines will be examined upon influenza H7N9 virus infection in alveolar epithelial cells in order to examine if these mediators were induced differentially when compared with the highly pathogenic avian influenza (HPAI) H5N1 and the 2009 pandemic H1N1 virus. Moreover, because cleaved caspase 3 is commonly employed as a marker for the indication of apoptosis, we further examined the extensiveness of cleaved caspase 3 in influenza virus infection in human lung ex vivo cultures. Materials and Methods: Fresh biopsies of human lung tissue were obtained from patients undergoing surgical resection of lung tissues. Lung tissue fragments were cultured with F12K medium incubated at 37°C. For viral infection experiments, influenza viruses A/Shanghai/1/2013 (SH1, H7N9), A/Shanghai/2/2013 (SH2, H7N9), A/Hong Kong/483/97 (H5N1), and A/California/07 (Ca07, H1N1pdm) at a viral titer of 106 TCID50/mL were used for ex vivo lung culture infection. Infected lung tissues were collected in 10% formalin at 24, 48, and 72 hpi for immunohistochemical staining. Costaining of cleaved caspase 3 and influenza virus nucleoprotein was carried out for the detection of apoptosis. Furthermore, primary culture of human alveolar epithelial cells was isolated from human lungs by mincing the lung, followed by filtration and centrifugation. Human alveolar epithelial cells were infected with the novel influenza H7N9, the HPAI H5N1, and the pandemic H1N1 virus. Virus replication was monitored by measuring infectious viral particles using TCID50. mRNA and protein expression of proinflammatory cytokines and chemokines were quantified by real time qPCR and ELISA. Results: We found extensive apoptosis in influenza H7N9 (both SH1 and SH2) and H5N1, but not H1N1pdm infected ex vivo lung tissues, suggesting that both avian influenza viruses can induce apoptosis and cause severe cell death in human lung tissue. Furthermore, unlike HPAI H5N1 which induces dysregulated proinflammatory cytokine responses, the novel influenza H7N9 virus elicited poor proinflammatory cytokine responses, inducing type I and III interferon in ex vivo human lung explant cultures. The novel influenza H7N9 virus is an intrinsically more potent inducer of proinflammatory cytokine than the H1N1pdm virus but less than the H5N1 virus. Conclusions: The proinflammatory cytokine and chemokine responses may contribute modestly to the severity of human H7N9 disease, but it is likely that direct viral cytopathology is probably playing a more important role in pathogenesis of human H7N9 diseases. The recognition of the role of cleaved caspase 3 in severe human infection of avian influenza virus can provide insights on the development of novel therapeutic approaches for the preparedness of the future outbreak of pandemics.