Pathogenic and Antigenic Analyses of H5N1 High Pathogenicity Avian Influenza Virus Isolated in the 2022/2023 Season From Poultry Farms in Izumi City, Japan

Abstract

During the winter of 2022/2023, Japan experienced its largest outbreak of high pathogenicity avian influenza (HPAI), affecting 84 poultry premises. In this study, we investigated the pathogenicity and antigenicity of A/chicken/Kagoshima/22A1T/2022 (Kagoshima/22A1T), a clade 2.3.4.4b H5N1 virus belonging to the G2b group. It was isolated from a poultry farm in Izumi City, where the largest number of consecutive cases was recorded. The 50% lethal dose, mean death time (MDT), amount of virus shed, and transmissibility in chickens of Kagoshima/22A1T were similar to those of A/chicken/Kagoshima/21A6T/2022 (Kagoshima/21A6T), the previous season's isolate of the same group, indicating that their pathogenicities were comparable. However, the antigenicity of these isolates differed according to the hemagglutination inhibition (HI) test results. We found that the amino acid substitutions in residues 189 and 193, corresponding to antigenic site B in the H3 virus of the HA1 subunit, could have an impact on the HI cross-reactivity of Kagoshima/21A6T. This study provides important insights into the factors contributing to the consecutive HPAI outbreaks on poultry farms in Izumi City during the 2022/2023 season and the prediction of antigenic changes in G2b group HPAI viruses.

Figure 1

The recent trend of high pathogenicity avian influenza virus (HPAIV) groups in Japan. In the 2020/2021 season, there were only two groups, G1 and G2a, derived from the European isolates. In the 2021/2022 season, two new groups, G2d and G2b, were identified in addition to the G2a group. In the following season, in addition to the outbreaks of the G2d-0 and G2b-1 genotypes, three genotypes of G2d and G2b-3 and 11 genotypes of a new group, G2c, were confirmed.

Figure 2

Location of the farms where the high pathogenicity avian influenza (HPAI) outbreak occurred during the 2022/2023 season. The white area in the northwest of Izumi City in the close-up map corresponds to the Izumi Plain. The nine sites (a red star and red circles) from which G2b-1 group viruses were isolated are located within an 8 km radius. A red star represents the farm where Kagoshima/22A1T was isolated. The remaining eight sites are in hilly areas.

Figure 3

Survival rates of chickens inoculated Kagoshima/22A1T. The survival rates of chickens inoculated with 10², 10³, 10⁴, 10⁵, and 10⁶ EID₅₀ of each virus are shown by rhombuses, crosses, triangles, squares, and circles, respectively.

Figure 4

Boxplot of viral titer in tracheal and cloacal swabs of the carcasses of chickens inoculated with the 10⁶ EID₅₀ of Kagoshima/22A1T. Chickens (n = 5) were nasally inoculated with 10⁶ EID₅₀ of Kagoshima/22A1T. ⁣^∗p < 0.05; Wilcoxon signed-rank test.

Figure 5

Kinetics of viral titers in each tracheal and cloacal swab. Black and white circles represent the viral titer in the tracheal and cloacal swabs, respectively, from the chicken inoculated with the 10⁶ EID₅₀ of Kagoshima/22A1T. Black triangles, squares, and rhombuses represent the viral titer in the tracheal swabs of the cohabiting chickens, while white triangles, squares, and rhombuses represent the viral titer in the cloacal swabs of the cohabiting chickens.

Figure 6

Main chains of modeled influenza hemagglutinin (HA) molecules (Kagoshima/22A1T, green; Kagoshima/21A1T, magenta; Kagoshima/21A6T, cyan; and Hiroshima/21A10C, orange). Root mean square distance (RMSD) of α-carbons among the four models was 0.749 Å.

Figure 7

Surface environment of hemagglutinin (HA) of four isolates: (a) Kagoshima/22A1T, (b) Kagoshima/21A1T, (c) Kagoshima/21A6T, and (d) Hiroshiima/21A10C. The left panel shows the structure from P/S185 to T200, and the right panel shows the molecular surface in each figure. Colored residues are K/E189 and D/A193. K/E189 and D/A193 are shown as stick models of the same color in Figure 6, and the other residues from P185 to T200 are drawn as line models. Red, white, and blue areas on each electrostatic surface mean positive, neutral, and negative charge, respectively.