Construction of a Vero cell-based high-yield H7N9 influenza vaccine candidate strain / 中华微生物学和免疫学杂志

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 en-coding 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 vi-rus 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 pin-point 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 possi-bility of using it as a candidate vaccine strain whenever facing a potential epidemic of H7N9 virus.