Avian influenza virus (AIV) is classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes based on antigenic differences . They not only cause serious economic damage to the global poultry industry, but also occasionally pose substantial threats to public health . H9N2 is one of the most widespread subtypes of the AIV family and is found worldwide in wild birds and as an enzootic pathogen in poultry across much of Asia, the Middle East, and North Africa . H9N2 viruses not only infect different types of wild birds and terrestrial poultry, but also evolve to break through the interspecies barrier transmitting to pigs, dogs, horses, mink, pika, and bats [4–6]. Most noteworthily, the long-term prevalence favors the interspecies transmission of H9N2 viruses from avian species to humans [5, 6]. These findings indicate that H9N2 viruses have gained the capacity to overcome host restriction factors. The enzootic and zoonotic nature of H9N2 viruses is a great source of concern and emphasizes the importance of compressively understanding the fitness of this particular subtype of AIVs.
Although the molecular basis of host-range restrictions is not completely defined, the compatibility between the HA protein of virus and its corresponding receptor, sialic acid (SA), on the host cell is thought to contribute in part to the adaptation of virus to a specific host [5, 7]. The HAs of influenza viruses isolated from avian species typically preferentially bind to SAα2,3 glycans, which represent avian-type receptors. The rise in the ability of HAs to preferentially bind to SAα2,6 glycans, which represent human-type receptors, is proved to be one of the important factors for the human adaptation of AIVs . The receptor-binding property of influenza A viruses depends on the receptor-binding domain (RBD) in the globular head of HA . It has been well demonstrated that residue 226 within the 220-loop RBD is a major determinant of the HA binding preference for either “human-type” or “avian-type” receptors  and is thought to be associated with mammalian tropism of AIVs [11–14].
From July 1998 to August 2020, at least 69 people worldwide were infected with H9N2 viruses. Retrospective serosurveys also revealed high seropositivity rates for H9N2 antibodies among poultry workers [5, 6]. Once humans are infected with influenza virus, the usage of antiviral drugs is one of the important means to control the progression of the disease. Compared with the M2 inhibitors which have been abandoned, the NA inhibitors may be the only effective drugs available at present. However, these NA inhibitors also face severe challenges because of high variability of NA genes. In view of the questioned efficacy and adverse side effect of Oseltamivir, a NA inhibitor, the World Health Organization had reduced it from a core drug to an adjuvant drug in June 2017. In this case, the inhibitory effect of another NA inhibitor, Zanamivir, on influenza virus needs to be evaluated.
In the phylogenetic tree of HA genes, H9N2 AIVs comprise of two distinctive phylogenetic lineages: North American lineage (A/Turkey/Wisconsin/1/1966-like (Ty66-like)) and Eurasian lineage. Among the Eurasian lineage, it can be further classified into three sublineages: A/Duck/Hong Kong/Y439/1997-like (Y439-like), A/Quail/Hong Kong/G1/1997-like (G1-like), and A/Chicken/Beijing/1/1994-like (BJ94-like). Before 2010, the BJ94-like sublineage mainly includes three clades: BJ94-like, A/Duck/Hong Kong/Y280/1997-like (Y280-like), and A/Chicken/Shanghai/F/1998-like (SH98-like). Since 2010, a novel G57-like clade in the BJ94-like sublineage was identified, which was responsible for the H9N2 outbreaks during 2010–2013 and became the most predominant genotype recently . According to the HA phylogenetic tree of human H9N2 viruses registered in the NCBI influenza database (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go = database) and the GISAID EpiFlu database (http://platform.gisaid.org), we found that the potential of G57-like AIVs to infect humans was obviously higher than that of other clades of viruses (Fig. 1). Therefore, we evaluated the potential public health threat and drug susceptibility of the G57-like H9N2 viruses in this study.