Abstract: Virology Journal
Kim et al. Virology Journal
(2025) 22:183
https://doi.org/10.1186/s12985-025-02811-w
Open Access
BRIEF REPORT
Immediate PB2-E627K amino acid substitution
after single infection of highly pathogenic
avian influenza H5N1 clade 2.3.4.4b in mice
Deok-Hwan Kim1†, Dong-Yeop Lee2† , Yeram Seo1, Chang-Seon Song1* and Dong-Hun Lee2*
Abstract
The highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b has rapidly disseminated globally, with
mammalian infections reported in multiple species. Recent evidence of mammal-to-mammal transmission has
heightened concerns about the virus’s potential adaptation to mammals. The polymerase basic 2 (PB2) protein
E627K mutation appears to be of key importance for mammalian adaptation. We isolated an HPAI H5N1 clade
2.3.4.4b virus from wild birds in Korea with 96% E and 4% K at amino acid position 627 of PB2. To investigate the
genomic characteristics of this clade regarding mammalian adaptation, we studied the replication and transmission
of the H5N1 virus in mice. Two experiments with different challenge-to-contact ratios were conducted to assess
transmission dynamics and mutation development. In experiment 1, a 4:1 challenge-to-contact ratio resulted in
100% transmission among direct-contact mice, with all mice succumbing to the infection. In experiment 2, a 1:1
ratio yielded 50% transmission, with all challenged mice also succumbing. High viral loads were observed in the
lungs and brains in both experiments, with viral titers increasing over time. Notably, the PB2-E627K variant, initially
present at 4% in the virus stock, was selected and reached near-fixation (~ 100%) in the lungs and brains by 6
days post-challenge and was subsequently transmitted. No other mammalian-adaptive mutations were identified,
emphasizing the pivotal role of PB2-E627K in early stages of mammalian adaptation. These findings highlight
the need for continuous genomic monitoring to detect mammalian adaptation markers and assess interspecies
transmission risks.
Keywords Highly pathogenic avian influenza, H5N1, Clade 2.3.4.4b, Transmission, PB2 E627K, Deep sequencing
†
Deok-Hwan Kim and Dong-Yeop Lee contributed equally to this
work.
*Correspondence:
Chang-Seon Song
songcs@konkuk.ac.kr
Dong-Hun Lee
donghunlee@konkuk.ac.kr
1
Avian Disease Laboratory, College of Veterinary Medicine, Konkuk
University, Seoul, Republic of Korea
2
Wildlife Health Laboratory, College of Veterinary Medicine, Konkuk
University, Seoul, Republic of Korea
The H5N1 subtype of highly pathogenic avian influenza
(HPAI) virus, first identified in a goose in Guangdong,
China, in 1996 (Gs/GD), has since spread globally, infecting various domestic and wild bird species and occasionally crossing the species barrier to infect mammals,
including humans [1]. In autumn 2020, novel reassortant
clade 2.3.4.4b H5N1 HPAI viruses were detected and
became predominant among poultry and wild birds in
Europe, subsequently spread to Africa, the Middle East,
and Asia [2]. Since then, numerous mammalian infections
have been reported worldwide in species such as black
bears, bobcats, coyotes, and ferrets. Most cases were
© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
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