1. Introduction
Influenza A virus (IAV) is an important respiratory pathogen that continually impacts human public health and the animal industry. The wild waterfowl has been thought to be the natural reservoir for IAV, however, the viruses frequently jump species barriers and infect humans and other mammals, such as pigs, cats, horses, and whales(Zhu et al., 2019a). Swine is of particular significance due to its susceptibility to avian, swine, and human influenza viruses and has been regarded as “gene mixing vessels” to generate virus with pandemic potential(Ma et al., 2008, Ito et al., 1998). Systematic surveillance of swine influenza viruses (SIVs) is essential for early warning and preparedness for the next potential pandemic. In China, pigs are not vaccinated against influenza virus, and distinct lineages of SIVs, such as classical swine H1N1 (CS H1N1), Eurasian avian-like H1N1 (EA H1N1), and triple reassortant H3N2 (TR H3N2), have been co-circulating in pig herds(Zhu et al., 2019b). EA H1N1 SIVs were firstly transmitted from waterfowl to pigs in 1979 in Europe(Pensaert et al., 1981) and gradually became the predominant lineage in China(Yang et al., 2016). Since 2009, the 2009 pandemic (pdm/09) H1N1 in humans quickly transmitted to pigs(Weingartl et al., 2010, Pereda et al., 2010), and genetic reassortants between EA H1N1 SIVs and pdm/09 H1N1 have been frequently reported among pigs(Zhu et al., 2011, Cao et al., 2019). Notably, sporadic human infections of EA H1N1 and other reassortant SIVs highlight the importance of influenza surveillance in pigs and humans(Yang et al., 2012, Zhu et al., 2016b, Xie et al., 2018).
As a multifunctional protein, a single amino acid mutation in hemagglutinin (HA) can largely alter the biological property of virus. For example, G225E mutation in HA has been confirmed to significantly improve the respiratory droplet transmission of EA H1N1 SIVs in guinea pigs(Wang et al., 2017). Neutralizing antibodies stimulated by HA protein can protect humans or animals against influenza virus infection. HA protein consists of two polypeptides, HA1 and HA2, and HA1 plays much important roles than HA2 in triggering host immune response(Chi et al., 2005). Accumulation of amino acid mutations in HA1 usually cause antigenic drift, making the vaccine unable to offer effective protection against antigenically mismatched circulating strains. When the prevalent viruses show a greater than 4-fold difference in hemagglutinin inhibition (HI) assay titer from the vaccine strain, the immunity induced by the vaccine does not prevent the circulation of such viruses in population(Smith et al., 2001). Therefore, identification of the genetic determinants for antigenic variation will be undoubtedly invaluable for vaccine development and prevention of influenza virus outbreaks.
Currently, EA H1N1 SIVs have been frequently recombining with other influenza viruses, such as pdm/09 H1N1 and TR H3N2(He et al., 2018). Importantly, the overwhelming majority of the reassortants preserved the HA and NA genes of EA H1N1 SIVs(Sun et al., 2020), indicating that studying the genetic basis for antigenic drift of EA H1N1 SIVs is essential to evaluate the antigenic properties of SIVs. Our previous study demonstrated that EA H1N1 SIVs formed two distinct antigenic groups, and A/swine/Guangdong/104/2013 (GD/104) virus exhibited 32~64-fold lower antigenic cross-reactivity with antibodies against A/swine/Guangxi/18/2011 (GX/18) virus(Yang et al., 2016). In this study, we used GX/18 and GD/104 as models to explore the underlying mechanism of this difference in antigenicity.