Efficacy of a high-yield attenuated vaccine strain wholly derived from avian influenza viruses by use of reverse genetics

The preparation of high-yield influenza H5N1 vaccine strains is challenging for researchers and manufacturers. Here, we used reverse genetics to generate a high-yield avian influenza vaccine strain based on a novel avian influenza virus. A high-yield attenuated recombinant H5N3 virus (rH5N3-DL) was prepared from the HA gene of A/Goose/Anhui/08 (H5N1), modified by deletion of the multiple basic amino acids at the cleavage site, the NA gene from A/Duck/Germany/1215/73 (H2N3), and the six internal genes from the high-yield A/Goose/Dalian/3/01 (H9N2) virus. rH5N3-DL grew to high HA titers (1:2048) in eggs, eight times those of the parental H5N1 virus, and four times higher than that of rH5N3-PR8 (six internal genes from the high-yield PR8). Infection tests demonstrated that rH5N3-DL was avirulent in chickens, chicken embryos, and mice. rH5N3-DL-vaccinated chickens were fully protected against the morbidity and mortality of a lethal challenge with homologous A/Goose/Anhui/08, but only 80% of chickens were protected after challenge with heterologous A/Goose/Guangdong/1/96. The N3 neuraminidase marker distinguishes rH5N3-DL-vaccinated from H5N1-infected animals. rH5N3-DL is thus a promising vaccine candidate to combat highly pathogenic avian influenza virus infections. The A/Goose/Dalian/3/01 virus could be a promising candidate as providing internal genes donors with high-yield properties in reverse-genetics system and might be applicable for future avian influenza vaccine development.