| H7N9 subtype influenza viruses caused sporadic human infections in China, and were evolving and reassorting with other influenza viruses. The H7N9 viruses not only caused great economical losses to poultry industry but also posed a threat to human health. As of 28 th Feburary 2016, there were 747 reported human infections with 297 deaths.In this study, we conducted system evolution analysis of 210 H7N9 viruses isolated in China since 2013. The viruses were divided into 4 waves by collection date. The gene homology of HA and NA were 96.9-100% and 97.3-100%, respectively; while for the six internal genes segments PB2, PB1, PA, NP, M and NS, the nucleotide homology were 95.1-100%, 95.3-100%、94.7-100%, 93.6-100%, 95.2-100% and 93.7-100%, respectively. The 2013-lineage H7N9 viruses were divided into 12 genotypes according to the phylogenetic tree of different gene segments. In the first wave of H7N9, there were only 3 genotypes, while in second and third wave of H7N9 viruses, there were 8 and 7 genotypes, respectively. Although the genotype numbers were different, the main genotype was G1. These results indicated that H7N9 viruses are reassorting with H9N2 viruses in the process of spreading. Further analysis of amino acids showed that all the M2 protein of 2013-lineage isolates harbored S31 N mutation, which indicated reduced susceptibility to amantadine and rimantadine, and all the isolates were susceptible to neuraminidase inhibitors. All the H7N9 isolates had one basic amino acid in cleavage site and all harbored PB2 627 E, both of them were low pathogenic molecular marker.The continued spread of the newly emerged H7N9 viruses among poultry in China, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. In this study, we generated a cold-adapted, live attenuated H7N9 vaccine(H7N9/AAca) that contains wild-type HA and NA genes from AH/1, and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus(AAca) using reverse genetics. We evaluated the attenuation phenotype, immunogenicity and protective efficacy of H7N9/AAca in mammal models. H7N9/AAca was attenuated in mice and ferrets, and induced robust neutralizing antibody responses in mice, ferrets, and guinea pigs immunized once or twice intranasally. The animals immunized once were not completely protected from H7N9 virus replication in three different animal models, but the replication level was significantly lower. The animals immunized twice were completely protected from virus challenge. Importantly, the animals vaccinated once were fully protected from infection when exposed to or in contact with the H7N9 virus-inoculated animals.In summary, the H7N9 viruses circulating in China are relatively stable. The dominant genotype is genotype 1. The H7N9/AAca was well-tolerated, immunogenic and efficacy, and one single dose of cold-adapted H7N9 vaccine can prevent H7N9 virus infection via natural route of transmission; they provide a compelling argument for further testing of this vaccine in human trials. |
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