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.