PB2 and NP of North American H5N1 virus drive immune cell replication and systemic infections.

Young-Il Kim¹ Seung-Gyu Jang^2,3 Woohyun Kwon^1,3 Jaemoo Kim¹ Dongbin Park¹ Isaac Choi¹ Jeong Ho Choi³ Juryeon Gil^1,3 Mina Yu^1,3 Boyeong Jeong¹ Eun-Ha Kim² Se-Mi Kim¹ Hyunjoon Kim¹ Jae-Woo Ahn¹ Suhee Hwang^1,4 Seo-Young Heo^1,4 Mark Anthony B Casel^1,3 Rare Rollon^1,3 Thomas Fabrizio⁵ Richard J Webby⁵ Young Ki Choi^1,2,3,4

Affiliations 5 institutions

Center for Study of Emerging and Re-emerging Viruses, Korea Virus Research Institute, Institute for Basic Science, Daejeon 34126, Republic of Korea.
Virus Research Resource Center, Korea Virus Research Institute, Institute for Basic Science, Daejeon 34126, Republic of Korea.
College of Medicine and Medical Research Institute, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju 28644, Republic of Korea.
Department of Metabiohealth, Sungkyunkwan University, Suwon 16419, Republic of Korea.
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN, USA.

PMID41004584 DOI10.1126/sciadv.ady1208 Year2025 Journal Science advances Sci Adv Languageeng Statusppublish

Abstract

The 2022 North American outbreak of 2.3.4.4b H5N1 avian influenza virus revealed substantial mammalian adaptation and pathogenicity, yet mechanisms remain unclear. To address this knowledge gap, we investigated the North American H5N1 strain (GA/W22-145E/22), which demonstrated unique immune cell-mediated systemic dissemination, neuroinvasion, and 100% mortality in ferrets, unlike the nonlethal Eurasian strain (KR/W811/21). Genomic and reverse genetics studies identified PB2_478I and NP_450N mutations as key determinants of enhanced polymerase activity, immune cell tropism, and pathogenicity. Mutant GA/W22-145E/22 virus carrying PB2_478V/NP_450S showed complete survival without systemic dissemination. Furthermore, GA/W22-145E/22 demonstrated robust replication in human peripheral blood mononuclear cells and bovine mammary gland organoids, raising concerns about zoonotic spillover. These findings underscore PB2_478I and NP_450N as pivotal markers of pathogenicity, emphasizing the urgent need for enhanced surveillance and targeted interventions.

Supporting text Virus Host Location

Influenza A Virus, H5N1 Subtype 300 Influenza, Human 286 Orthomyxoviridae Infections 228 Viral Proteins 152 Virus Replication 191 Animals 1948 Cattle 126 Ferrets 79 Humans 1440 Leukocytes, Mononuclear 3 Mutation 209

Evidence records

4 total

Experimental Infection

3 records · 2 evidence types

Evidence type Pathogenicity Experiment

2 records

OVE9921 Pathogenicity Experiment

Key finding

The North American H5N1 strain GA/W22-145E/22 caused systemic dissemination, neuroinvasion, and 100% mortality in ferrets, whereas the Eurasian KR/W811/21 strain was nonlethal.

Virus

Host

Location

Supporting text

The North American H5N1 strain (GA/W22-145E/22), which demonstrated unique immune cell-mediated systemic dissemination, neuroinvasion, and 100% mortality in ferrets, unlike the nonlethal Eurasian strain (KR/W811/21).

Method: controlled experimental infection in ferrets | clinical observation of mortality | pathological assessment of dissemination and neuroinvasion
Experimental system: ferret pathogenicity model

OVE9922 Pathogenicity Experiment

Key finding

A mutant GA/W22-145E/22 virus carrying PB2_478V and NP_450S substitutions produced complete survival and no systemic dissemination in ferrets.

Virus

Host

Location

Supporting text

Mutant GA/W22-145E/22 virus carrying PB2_478V/NP_450S showed complete survival without systemic dissemination.

Method: reverse genetics mutant generation | experimental infection in ferrets | clinical observation for survival | assessment of tissue dissemination
Experimental system: ferret pathogenicity model

Evidence type Host Range Experiment

1 records

OVE9924 Host Range Experiment

Key finding

The North American H5N1 strain GA/W22-145E/22 replicated in human peripheral blood mononuclear cells and bovine mammary gland organoids, indicating cross-species cellular susceptibility.

Virus

Host

Location

Supporting text

GA/W22-145E/22 demonstrated robust replication in human peripheral blood mononuclear cells and bovine mammary gland organoids.

Method: in vitro infection | viral replication assay
Sample type: peripheral blood mononuclear cells | mammary gland organoids
Experimental system: cell and organoid infection assay

Functional Mechanism

1 records · 1 evidence types

Evidence type Molecular Adaptation

1 records

OVE9923 Molecular Adaptation

Key finding

PB2_478I and NP_450N mutations in North American H5N1 enhance polymerase activity, immune cell tropism, and pathogenicity.

Virus

Host

Location

Supporting text

Genomic and reverse genetics studies identified PB2_478I and NP_450N mutations as key determinants of enhanced polymerase activity, immune cell tropism, and pathogenicity.

Genes or proteins: PB2 | NP
Mutations: PB2_478I | NP_450N
Mechanism types: replication adaptation | tissue tropism | virulence adaptation

References

54 references

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