Genetic Characterization of Avian Influenza Viruses Isolated from the Izumi Plain, Japan in 2019/20 Winter Season

Abstract

The Izumi plain in the Kagoshima Prefecture, Japan, is known as an overwintering site for more than 30,000 migratory waterfowl, including endangered crane species. We previously reported that environmental water samples, from artificial wet paddies created as crane roost sites on the Izumi plain, are useful for avian influenza virus (AIV) surveillance. During the 2019/20 winter season, we collected 238 water samples from the crane roost sites and isolated 22 AIVs of six subtypes: one H1N1, one H3N2, seven H3N8, four H4N6, nine H6N6, and one H11N2 subtypes. Genetic analyses revealed that AIVs of the same subtype isolated from the Izumi plain during a single winter season exhibited multiple genetic constellations. Furthermore, phylogenetic analyses suggested that our H3N2 isolate may be a genetic reassortant between close relatives to our H3N8 and H11N2 isolates. Our study highlighted the importance of monitoring AIV circulation to better understand AIV ecology in migratory waterfowl populations.

Newly emerged genotypes of highly pathogenic H5N8 avian influenza viruses in Kagoshima prefecture, Japan during winter 2020/21

“…Genetic analyses of the H3 HA genes revealed that the H3 AIVs could be classified into three genetic clades: H3-I to H3-III (). Genetic clade H3-I included H3N8 AIVs isolated during the 2019/20 winter season [18]. All the H3N6 and H3N8 AIVs, with the exception of A/environment/Kagoshima/KU-C12/2020 (H3N6) and A/environment/Kagoshima/KU-J6/2020 (H3N6) that belonged to clade H3-II, were classified under genetic clade H3-I.…”

“…In this study, we demonstrated the viral transition of AIVs among wild waterfowl overwintering in Kagoshima prefecture during the 2020/21 winter season. We isolated the highest number of AIVs in the 2020/21 winter season in our AIV surveillance since the 2012/13 winter season [13,14,18,21,22]. Due to the high number of AIV-positive samples found in crane roost water during the 2020/21 winter season, we developed real-time RT-PCR methods to distinguish samples containing multiple HA subtypes of AIVs (Table 1).…”

“…RNA was extracted from allantoic fluids that showed hemagglutination activity using an InnuPREP virus DNA/RNA Kit (Analytik Jena AG, Jena, Germany) and subjected to real-time reverse transcription (RT)-PCR-based influenza A viral gene detection using the iTaq Universal SYBR Green One-Step Kit (BioRad, Hercules, CA, USA) and primer sets specific for a conserved region of influenza viral M genes [14]. To select samples that were possibly single isolates, M gene-positive samples were subjected to realtime RT-PCR using primer sets specific for the H1, H3, H4, H5, and H6 HA genes (Table 1), which have been frequently detected in the past [14,18,[21][22][23]. RNA samples that reacted with only one HA subtype-specific primer set were reverse transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) for complementary DNA (cDNA) synthesis and used as templates for conventional PCR-based NA subtyping using Tks Gflex DNA Polymerase (TaKaRa Bio Inc., Otsu, Japan) with a panel of subtype-specific primer sets [24].…”

“…Thus, AIVs could possibly have invaded the Izumi plain. Various AIVs, including HPAIVs, have been isolated from water samples from the Izumi plain almost every winter season [13][14][15][16][17][18].…”

“…In this study, we attempted to isolate AIVs from crane roost water in the Izumi plain and wild birds collected in Kagoshima prefecture during the 2020/21 winter season. Notably, we isolated the highest number of AIVs from the crane roost water in the 2020/21 winter season in our AIV surveillance since the 2012/13 winter season [13, 14, 18, 21, 22]. We found that H5 HPAIVs show various genotypes in wild birds, and genetic reassortment might have actively occurred in Kagoshima prefecture during the 2020/21 winter season.…”