4. Discussion
EA H1N1 SIVs have been circulating in pig herbs in Europe and Asia for decades. Continual mutations and recombination with other viruses have entitled EA H1N1 SIVs to form distinct antigenic groups, with markedly different antigenicity from those of the pdm/09 H1N1 viruses. And antigenic variants escaping pre-existing immunity might cause outbreaks in pigs and pose greater risks to human health. In this study, we used A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013 as models to explore the genetic basis of antigenic drift of EA H1N1 SIVs, and found that a single amino acid at position 158 in Sa antigenic site substantially contributed to the antigenic variation.
Five major antigenic sites, Sa, Sb, Ca1, Ca2 and Cb, have been mapped onto the HA1 protein and antibodies targeting each of which can neutralize the infectivity of influenza virus. The immunodominant site Sa is located proximal to the receptor binding site and elicits high potency neutralizing antibodies(Shembekar et al., 2013). In the past decades, amino acid mutations in Sa site have induced antigenic variability of different subtype influenza viruses. The pdm/09 H1N1 continues to circulate worldwide and cause periodic outbreaks in human population. Boivin et al . previously reported that A/California/07/09 vaccine cannot protect mice against lethal infection of rescued virus with G158E and N159D mutations in the Sa antigenic site(Retamal et al., 2017). Smith et al . created an antigenic evolution map using HI assay data and demonstrated that amino acid changes in Sa site repeatedly involved in antigenic variations of H3N2 influenza viruses(Smith et al., 2004). In the present study, G158E mutation considerably decreased the reactivity of GX/18 virus with the homologous antibodies, further suggesting Sa antigenic site played a pivotal role in antigenic drift of EA H1N1 SIVs.
Amino acid mutations can affect influenza virus antigenicity in a variety of ways, such as altering glycosylation of HA protein and the binding affinity between epitope and antibody. Previous study demonstrated that E131N mutation changed the antigenic properties of H5N1 avian influenza viruses through forming a new N-linked glycosylation site at positions 131-133(Gu et al., 2019). Here, we found that G158E substitution contributed to the antigenic drift of EA H1N1 SIVs via decreasing the binding affinity between HA and antibody. Accompanied with G158E mutation, the R group changed from -H to -(CH2)2-COOH, resulting in the alteration of spatial structure and hydrophobicity. The umbrella-like sidechain of E and its improved hydrophilicity might jointly block the interaction between HA protein and antibody.
Notably, G158E mutation substantially decreased, rather than eliminated, the reactivity of GX/18 with mAb102-95, suggesting other amino acid(s) acts as assistant to unitedly cause antigenic variation. Moreover, Sunet al . identified 77 EA H1N1 SIVs, isolated in slaughterhouse in 10 provinces in China from 2011 to 2018, and found these viruses could be classified into two antigenic groups based on the HI assays(Sun et al., 2020). However, sequence analysis showed that 158G, not 158E, were conserved in all the 77 viruses (data not shown), indicating that the 158G-related viruses are still the predominant strains in China currently. The underlying mechanism of G158E altering the antigenic property of EA H1N1 SIVs remains ambiguous and the other amino acids behind antigenic drift still needs to be further investigated. These data revealed that EA H1N1 SIVs have been evolving in a complex direction and highlight the importance of active surveillance and evaluating the virus antigenic properties.