Abstract
Live-attenuated vaccines (LAVs) represent a promising approach for flavivirus vaccine development. In the present study, we demonstrated a method for generating flavivirus LAVs based on breaking spatially and temporally regulated C-prM cleavage to disturb the viral assembly process, using an avian flavivirus (Tembusu virus) as the model. Using reverse genetics technology, we successfully generated two recombinant viruses (CQW1-IRES-mC and CQW1-MINI-mC) with bicistronic genomic RNA in which native capsid genes were deleted and instead expressed in the 3'UTR under the control of an internal ribosome entry site (IRES) or minimum IRES. Both viruses showed a significantly attenuated phenotype in vitro due to impaired viral assembly, and the engineered mutations were genetically stable in vitro within ten passages. Importantly, their virulence was also highly attenuated in ducklings and suckling mice and did not cause any overt clinical symptoms or mortality. In addition, a single dose of immunization with any of these mutant viruses could completely protect ducklings from a lethal challenge, and no viremia was detected after immunization and challenge, even though the viruses induced a relatively moderate immune response in terms of the T-lymphocytes proliferative response and the level of neutralization antibodies compared with that obtained with the wild-type virus. Besides, a recombinant virus ectopically expressing the prM-E protein was also generated in the present study, but this virus was too attenuated with severely decreased proliferation. Our results indicated that the use of a recombinant flavivirus that ectopically expresses structural proteins could be an effective and universal method for flavivirus LAVs development.