Construction of a Vero cell-based high-yield H7N9 influenza vaccine candidate strain

Abstract

Objective To construct an H7N9 vaccine strain by using a previously obtained Vero cell-based high-yield influenza A virus as the donor strain. Methods The recombinant virus strains, 4mut-H7N9 and PR8-H7N9, were respectively rescued with reverse genetics technique by combining the genes encoding hemagglutinin (HA) and neuraminidase (NA) of H7N9 virus with the 6 internal genes of PR8-4mut or PR8 virus strains. The growth feature of 4mut-H7N9 virus strain was compared with that of PR8-H7N9 virus strain with growth curves and plaque assays. The viral proteins produced by 4mut-H7N9 and PR8-H7N9 virus strains were measured by Western blot and Coomassie blue staining. Results The PR8-H7N9 and 4mut-H7N9 virus strains were successfully rescued. The virus titer of 4mut-H7N9 strain was about 3000 times higher than that of PR8-H7N9 strain at 72 hours after infecting Vero cells. The 4mut-H7N9 virus strain formed plaques of about 1 mm in diameter on Vero cells, while the PR8-H7N9 virus strain only formed pinpoint plaques on Vero cells. The levels of viral proteins encoded by purified 4mut-H7N9 virus strain were significantly higher than that of the PR8-H7N9 virus strain as indicated by both Western blot and Coomassie blue staining. Moreover, the 4mut-H7N9 virus strain was less pathogenic than PR8-H7N9 strain in mice, and retained the trypsin dependence for infecting cells. Conclusion The reassortant 4mut-H7N9 vaccine strain as established by reverse genetics technique grew faster and better in Vero cells, suggesting the possibility of using it as a candidate vaccine strain whenever facing a potential epidemic of H7N9 virus. Key words: H7N9; Vero cell; High-yield vaccine candidate strain