Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China

Abstract

H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010-2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605-4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.

Keywords: H9N2; HA gene; antigenic; genetic evolution; new designated clade.

Figure 1.

Phylogenetic analysis of HA genes of H9N2 subtype AIV from 2010 to 2020 in China. The maximum clade credibility (MCC) tree of HA gene of H9N2 subtype AIVs from 2010 to 2020 in China. Phylogenetic tree with tip names is depicted in Supplementary Figure S1. Detailed information and clades of viruses in the constructed phylogenetic trees are listed in Supplementary Table S2. Vertical lines with a number indicate the clade partitioned in this study. Different colours represented different clades.

Figure 2.

The bar plot indicated the proportion of H9N2 strains from clade15 and clade16 used in this study by year, respectively. The proportion of isolated strains from clade15 was coloured in blue, and the proportion of isolated strains from clade16 in red.

Figure 3.

The antigenic cartography of represented H9N2 strains. The represented strains, which were selected in different clades from 1998 to 2020, were coloured differently according to clades and labelled with isolation year and virus name. Clade5 was represented by purple. Clade12 was represented by orange. Clade14 was represented by yellow. Clade15 and Clade16 were represented by blue and red, respectively. The spacing of the grid lines of y axis was equivalent to antigenic unit distance, which corresponds to a two-fold HI difference. All represented strains were divided into 3 groups based on the K-means clustering algorithm and enriched in an oval coloured differently according to antigenic groups.

Figure 4.

Different amino acid sites in HA1 between clade15 and clade16. The first column represents the clade of each strain, where clade15 was coloured in blue and the clade16 was coloured in red. Other columns represented different amino acid identity with different colours in each site.

Figure 5.

Stacked bar chart within 11 potential antigenic amino acid residues of 590 HA sequences from Clade 15 and Clade 16 strains. The y-axis (count) represents the counts of amino acids at each position in the sequence alignment. The colour of letter represents type of specific amino acid at this position.