Genetic and antigenic analyses of H5N8 and H5N1 subtypes high pathogenicity avian influenza viruses isolated from wild birds and poultry farms in Japan in the winter of 2021–2022

Abstract

In the winter of 2021–2022, multiple subtypes (H5N8 and H5N1) of high pathogenicity avian influenza viruses (HPAIVs) were confirmed to be circulating simultaneously in Japan. Here, we phylogenetically and antigenically analyzed HPAIVs that were isolated from infected wild birds, an epidemiological investigation of affected poultry farms, and our own active surveillance study. H5 subtype hemagglutinin (HA) genes of 32 representative HPAIV isolates were classified into clade 2.3.4.4b lineage and subsequently divided into three groups (G2a, G2b, and G2d). All H5N8 HPAIVs were isolated in early winter and had HA genes belonging to the G2a group. H5N1 HPAIVs belong to the G2b and G2d groups. Although G2b viruses were widespread throughout the season, G2d viruses endemically circulated in Northeast Japan after January 2022. Deep sequence analysis showed that the four HPAIVs isolated at the beginning of winter had both N8 and N1 subtypes of neuraminidase genes. Environmental water-derived G2a HPAIV, A/water/Tottori/NK1201-2/2021 (H5N8), has unique polymerase basic protein 1 and nucleoprotein genes, similar to those of low pathogenicity avian influenza viruses (LPAIVs). These results indicate that multiple H5 HPAIVs and LPAIVs disseminated to Japan via transboundary winter migration of wild birds, and HPAIVs with novel gene constellations could emerge in these populations. Cross-neutralization test revealed that G2a H5N8 HPAIVs were antigenically distinct from a G2b H5N1 HPAIV, suggesting that antibody pressure in wild birds was involved in the transition of the HPAIV groups during the season.

Novel Genotype of HA Clade 2.3.4.4b H5N8 Subtype High Pathogenicity Avian Influenza Virus Emerged at a Wintering Site of Migratory Birds in Japan, 2021/22 Winter

“…The influenza virus closely related to NK1201 was cross-referenced with sequences from public databases (Tables 2 and S1). Six genes (PB2, PA, HA, NA, M, and NS) of the NK1201 isolated in the present study had high similarities with the HPAIVs A/environment/ Akita/TU1-49/2021 (H5N8), A/chicken/Akita/7C/2021 (H5N8), A/chicken/Kagoshima/ B3T/2021 (H5N8) and A/chicken/Kagoshima/TU3-46.47/2021 (H5N8), which were responsible for outbreaks in two poultry farms in Japan in November 2021 [19,25]. Two internal genes, PB1 and NP, of NK1201 had high nucleotide identity with the PB1 and NP LPAIV genes of A/eurasian coot/Shandong/W22/2022 (H8N4) and A/Eurasian wigeon/Shanghai/NH112330/2021 (H9N2) obtained from public databases, respectively, and had a higher nucleotide and amino acid identity with genes of the LPAIVs isolated in this study.…”

“…Moreover, representative isolates from the G2a, G2b, and G2d groups were found to have different infectivities in chickens [21]. The H5N8 viruses belonged to the G2a group and caused HPAI in two poultry farms in Akita and Kagoshima prefectures in November 2021 [19,25]. The origin of the NK1201 HPAIV isolated on 1 December in our surveillance is unknown.…”

“…Six of the eight genes of NK1201 were closely related to those of H5N8 HPAIVs that caused outbreaks in poultry farms in Japan in November 2021. These viruses were of the E2 genotype H5N8 virus lineage, which were prevalent in the previous 2020/21 winter season in Japan, South Korea, China and Europe [19], but not detected in the 2021 summer in Japan. Based on the public database, the PB1 and NP genes of NK1201 showed high similarity to those of Chinese LPAIVs, A/eurasian coot/Shandong/W22/2022 (H8N4) and A/Eurasian wigeon/Shanghai/NH112330/2021 (H9N2), which were isolated in 2021/22 winter; this implies that the LPAIVs, along with HPAIVs, might have been brought to Japan by an autumn migration.…”

“…In this study, we surveyed environmental water and fecal samples for AIVs at a wintering site of migratory waterfowls in Tottori, Japan, during the winter and spring migration [18] of one season (December 2021 to March 2022) and isolated HPAIV A/water/Tottori/NK1201-2/2021 (H5N8) (NK1201) [19]. Recently, various genotypes of H5Nx clade 2.3.4.4b HPAIVs [19][20][21][22][23][24] and H7N7 LPAIVs [24] have been detected in Japan. H5N8 and H5N1 HPAIVs isolated in the 20221-2022 winter season in Japan were divided into three groups (G2a, G2b, and G2d) based on their HA gene sequences [19].…”

“…Recently, various genotypes of H5Nx clade 2.3.4.4b HPAIVs [19][20][21][22][23][24] and H7N7 LPAIVs [24] have been detected in Japan. H5N8 and H5N1 HPAIVs isolated in the 20221-2022 winter season in Japan were divided into three groups (G2a, G2b, and G2d) based on their HA gene sequences [19]. Moreover, representative isolates from the G2a, G2b, and G2d groups were found to have different infectivities in chickens [21].…”