Risk assessment on human transmission potential of avian influenza H7N9 virus using ex vivo cultures of the human respiratory tract

Abstract

Background: A novel avian-origin influenza H7N9 virus has emerged in Eastern China in February 2013, has re-emerged resulting in second and third waves of zoonotic transmission and has become enzootic in mainland China. Zoonotic H7N9 disease is a pandemic with approximately 30% fatality rate. The HA protein of H7N9 isolates from humans and poultry from Mainland China possesses T160A and Q226L (H3 numbering) and this would be expected to enhance the receptor binding specificity towards α-2,6 sialic acid receptors, enabling the virus to transmit from birds to humans. In the PB2 gene of H7N9 viruses, lysine was found at the 627 position and this is known to enhance the viral replication efficiency and increase virulence in mice. Recombinant viruses with substitutions in HA and PB2 genes using A/Shanghai/2/2013 (H7N9) as backbone for loss of function studies were generated by reverse genetics for ex vivo infection of human bronchus and lung explant cultures. It is important to develop a comprehensive risk-assessment strategy to identify the key biological features of the influenza H7N9 viruses to assess the pandemic potential. Methods: Fresh biopsy of human respiratory tissues were obtained from patients undergoing surgical resection of lung tissues. Bronchus and lung tissue fragments were cultured in 24-well tissue culture plates with F12K medium incubated at 37oC. For viral infection experiments, Influenza viruses including a wildtype recombinant Sh2 virus, HA mutants, rgSh2-A160T, rgSh2- L226Q and rgSh2-A160T&L226Q and PB2 mutants, rgSh2-K627E, rgSh2-K627E&Q591K and rgSh2-K627E&D701N at a viral titer of 106 TCID50/ml were used for ex vivo culture infection. After 1 h infection at 37oC, the tissues were washed with warm PBS for three times, followed by the replenishment of 1ml culture medium. Supernatant was collected at 1, 24 and 48 hpi.10% formalin-fixed tissues were collected at 24, 48 hpi. Immunohistochemical staining was performed on the tissues embedded in parafilm. Conclusion: We showed that Q226L in HA gene and E627K in PB2 gene were important in viral replication and tissue tropism in the human respiratory tract. Identification of such viral genetic determinant improves our understanding of specific genetic markers that affect the human-adaptation of the H7N9 viruses, and other avian viruses. This information can serve as useful data for risk-assessing the pandemicity of the emerging H7N9 virus.