Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention.

From 2013 onwards, the spread of novel H5N6 highly pathogenic avian influenza (HPAI) viruses in China has posed great threats to not only poultry industry but also human health. Since late-2016 in particular, frequent outbreaks of clade 2.3.4.4 H5N6 HPAI viruses among wild birds have promoted viral dissemination in South Korea, Japan, and European countries. In response to those trends, we conducted molecular genetic analysis of global clade 2.3.4.4 H5N6 viruses in order to characterize spatio-temporal patterns of viral diffusion and genetic diversity among wild birds and poultry. The clade 2.3.4.4 H5N6 viruses were classified into three groups (Group B, C, and D). During the cocirculation of Group C/D H5N6 viruses from 2013 to 2017, viral movements occurred between close or adjacent regions of China, Vietnam, South Korea, and Japan. In addition, viral migration rates from Guangdong and Hunan to multiple adjacent provinces seemed to have been highly supported by transmission routes (Bayes Factors >100), suggesting that southern China was an epicentre for the spread of H5N6 viruses in poultry during that period. Since the introduction of H5N6 viruses originating in wild birds in late-2016, evolving H5N6 viruses have lost most previous genotypes (e.g. G1, G2, and G1.2), whereas some prevailing genotypes (e.g. G1.1, G1.1.b, and G3) in aquatic birds have been dominated, and in particular, the effective population size of H5N6 originating in wild birds dramatically increased; however, the population size of poultry-origin H5N6 viruses declined during the same period, indicating that wild bird migration might accelerate the genetic diversity of H5N6 viruses. Phylogeographic approaches revealed that two independent paths of H5N6 viruses into South Korea and Japan from 2016 to 2018 and provided evidence of Group B and Group C H5N6 viruses were originated from Europe and China, respectively, as regions located in the East Asia–Australian migration flyway, which accelerated the genetic variability and dissemination. Altogether, our study provides insights to examine time of origin, evolutionary rate, diversification patterns, and phylogeographical approach of global clade 2.3.4.4 H5N6 HPAI viruses for assessing their evolutionary process and dissemination pathways.