Whole-genome sequence and genesis of an avian influenza virus H5N1 isolated from a healthy chicken in a live bird market in Indonesia: accumulation of mammalian adaptation markers in avian hosts

Abstract

Background: Influenza A viruses are a major pathogen that causes significant clinical and economic harm to many animals. In Indonesia, the highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in poultry since 2003 and has caused sporadic deadly infections in humans. The genetic bases that determine host range have not yet been fully elucidated. We analyzed the whole-genome sequence of a recent H5 isolate to reveal the evolution toward its mammalian adaptation.

Methods: We determined the whole-genome sequence of A/chicken/East Java/Av1955/2022 (hereafter, "Av1955") from a healthy chicken in April 2022 and conducted phylogenetic and mutational analysis.

Results: Phylogenetic analysis revealed that Av1955 belonged to the H5N1 clade 2.3.2.1c (Eurasian lineage). The six gene segments (PB1, PB2, HA, NP, NA, and NS) out of the eight segments derived from viruses of H5N1 Eurasian lineage, one (PB2) from the H3N6 subtype and the remaining one (M) from the H5N1 clade 2.1.3.2b (Indonesian lineage). The donor of the PB2 segment was a reassortant among three viruses of H5N1 Eurasian and Indonesian lineages and the H3N6 subtype. The HA amino acid sequence contained multiple basic amino acids at the cleavage site. Mutation analysis revealed that Av1955 possessed the maximal number of mammalian adaptation marker mutations.

Conclusions: Av1955 was a virus of H5N1 Eurasian lineage. The HA protein contains an HPAI H5N1-type cleavage site sequence, while the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. The virus has increased mammalian adaptation markers by mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among previously circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets.

Keywords: Clade 2.3.2.1c Eurasian lineage; Highly pathogenic avian influenza H5N1; Human health; Inter-subtype reassortment; Intra-subtype reassortment; Low pathogenic avian influenza H3N6; Mammalian adaptation marker mutation; Whole-genome sequence.

Figure 1. Phylogenetic analysis of the eight gene segments of Av1955.

The genetic information processing software Genetyx v14 (Genetyx Co., Tokyo, Japan) generated the phylogenetic trees using the RAxML with 100 bootstrap replicates. The tree is rooted in the A/Brevig Mission/1/1918 or A/South Carolina/1/1918 (H1N1). Four viruses with the highest identity to the nucleotide sequence of one or more gene segments of A/ck/EJ/Av1955/2022 (bold and pink color) were included in the phylogenies; these were A/eg/JT/20616-206-III/2016 (H5N1), A/ck/EJ/Av1210c/2017 (H5N1), A/ck/EJ/Spg119/2018 (H5N1) and A/ck/EJ/Av1534/2019 (H5N1). Three viruses isolated from poultry in East Java in 2013–2014 were also included; A/dk/EJ/Av39ac/2013 (H3N6) (bold and green), A/ck/EJ/Av240/2014 (H5N1-Ind) (bold and brown), and A/tk/EJ/Av154/2013 (H5N1-Eur) (bold and blue). Bold and black highlighted the virus at the nearest position to A/ck/EJ/Av1955c/2022 in each of the eight genome segments. “ck”: chicken, “eg”: eagle, “dk”: duck, “tk”: turkey, “EJ”: East Java, “JT”: Jakarta Timur, “–Ind”: Indonesian lineage (HA clade 2.1.3.2b), and “-Eur”: Eurasian lineage (HA clade 2.3.2.1c).

Figure 2. The genesis of Av1955 by multiple reassortments.

Av39: A/duck/East Java/Av39/2013 (H3N6), Av240: A/chicken/East Java/Av240/2014 H5N1 Indonesian lineage, Av154: A/turkey/East Java/Av154/2013 (H5N1 Eurasian lineage), Eg20616: A/eagle/Jakarta Timur/20616-206-III/2016 (H5N1 Eurasian lineage), Av1210: A/chicken/East Java/Av1210/2017 (H5N1 Eurasian lineage), Spg119: A/chicken/East Java/Spg119/2018 (H5N1 Eurasian lineage), and Av1534: A/chicken/East Java/Av1534/2019 (H5N1 Eurasian lineage). Isolate ID (EPI_ISL) in the GISAID database of the viruses was noted in Materials and Methods.