{ "abstract": "H9N2 avian influenza viruses are characterized by significant genetic diversity. Deletions within the hemagglutinin (HA) receptor-binding domain represent a notable, albeit rare, evolutionary trajectory. Here, we systematically characterized the multifaceted impact of a single-amino-acid deletion at residue 227 of HA (designated HAΔ227), a naturally occurring deletion within the 220-loop that overlaps a key antigenic epitope, using isogenic reverse genetics H9N2 viruses. Paradoxically, the HAΔ227 imposed a fitness cost and was associated with enhanced HA receptor-binding avidity, reduced apparent virion-associated NA activity, altered HA/NA functional balance, attenuated viral replication in vitro, and reduced respiratory shedding in vivo. Notably, despite this compromised replicative fitness, infection with the mutant virus was associated with an altered host immune response in chickens, characterized by elevated levels of interferon-γ (IFN-γ), interleukin-10 (IL-10), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1). These altered immune responses were associated with more severe pulmonary lesions, indicating a dissociation between disease severity and viral RNA load. Furthermore, the deletion increased the pH threshold for HA-mediated membrane fusion and substantially altered antigenic relationships. Collectively, our findings identify HA residue 227 as a functionally important site at which a single-residue deletion is associated with a phenotypic trade-off between replicative capacity and disease severity: reduced replicative fitness accompanied by altered inflammatory responses and greater tissue injury. This study highlights the potential phenotypic significance of a rare deletion in influenza virus HA and demonstrates that viral burden alone may not adequately predict disease severity. Author summary: H9N2 avian influenza viruses present a persistent challenge to poultry health and a significant zoonotic threat. This study identifies a rare but potentially consequential evolutionary event—a single amino acid deletion at residue 227 of the viral hemagglutinin (HA) protein—and characterizes its complex and apparently contrasting biological effects. We demonstrate that this deletion imposes a cost on replicative fitness by disrupting the functional balance between HA and neuraminidase, thereby impairing viral replication. Paradoxically, the same deletion alters antigenic recognition and is associated with more severe disease and an inflammatory profile consistent with greater tissue injury. Our findings illustrate that a low-prevalence deletion in influenza virus HA may nevertheless be associated with notable phenotypic effects and that viral burden alone may not adequately predict disease severity. They also support complementing conventional measures of viral burden with immunopathological assessments when evaluating influenza variants with atypical phenotypes.", "abstract_shown": true, "affiliations": [ "State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, and College of Veterinary Medicine, Jilin University, Changchun, China", "Jilin Research and Development Center of Biomedical Engineering, Changchun University, Changchun, China", "Institute of Animal Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, China" ], "article_type": "Research Article", "arxiv": null, "author_total": 12, "authors": [ { "affs": [ 0 ], "name": "Yixue Sun" }, { "affs": [ 1 ], "name": "Yanting Zhu" }, { "affs": [ 2 ], "name": "Pengju Zhang" }, { "name": "Shouzhi Sheng" }, { "name": "Lili Gai" }, { "name": "Siqi Li" }, { "name": "Fangyu Zhang" }, { "name": "Peige Cao" }, { "name": "Ao He" }, { "name": "Weixia Wang" }, { "name": "Xintao Li" }, { "name": "Yanlong Cong" } ], "bibtex": "https://research.draconitic.com/paper/7zkghhngwjgk3.bib", "cited_by": 0, "doi": "10.1371/journal.ppat.1014776", "fulltext_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014776", "id": "7zkghhngwjgk3", "issns": [ "1553-7374" ], "landing_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014776", "language": null, "licence": "CC-BY-4.0", "licence_label": "CC BY 4.0", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "plos", "/Biology and life sciences/Cell biology/Cellular structures and organelles/Cell membranes/Membrane fusion" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular types/Animal cells/Blood cells/Red blood cells" ], [ "plos", "/Biology and life sciences/Cell biology/Cellular types/Animal cells/Blood cells/Red blood cells/Hemagglutination" ], [ "plos", "/Biology and life sciences/Immunology/Immune response/Inflammation" ], [ "plos", "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Viral pathogens/Orthomyxoviruses/Influenza viruses/Influenza A virus" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral replication" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral structure/Virions" ], [ "plos", "/Biology and life sciences/Organisms/Eukaryota/Animals/Vertebrates/Amniotes/Birds/Fowl/Gamefowl/Chickens" ], [ "plos", "/Biology and life sciences/Organisms/Eukaryota/Animals/Vertebrates/Amniotes/Birds/Poultry/Chickens" ], [ "plos", "/Biology and life sciences/Organisms/Viruses/RNA viruses/Orthomyxoviruses/Influenza viruses/Influenza A virus" ], [ "plos", "/Biology and life sciences/Organisms/Viruses/Viral pathogens/Orthomyxoviruses/Influenza viruses/Influenza A virus" ], [ "plos", "/Biology and life sciences/Zoology/Animals/Vertebrates/Amniotes/Birds/Fowl/Gamefowl/Chickens" ], [ "plos", "/Biology and life sciences/Zoology/Animals/Vertebrates/Amniotes/Birds/Poultry/Chickens" ], [ "plos", "/Medicine and health sciences/Clinical medicine/Signs and symptoms/Inflammation" ], [ "plos", "/Medicine and health sciences/Immunology/Immune response/Inflammation" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Viral pathogens/Orthomyxoviruses/Influenza viruses/Influenza A virus" ] ], "openalex": null, "pdf_url": "https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1014776&type=printable", "pmcid": null, "pmid": null, "published": "2026-11-05", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/7zkghhngwjgk3.ris", "sources": [ { "first_seen": "2026-11-06T12:20:42Z", "native_id": "10.1371/journal.ppat.1014776", "source": "plos", "url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014776" } ], "subjects": [ { "field": "biology", "origin": "plos:cell biology", "subfield": "cell-biology" }, { "field": "biology", "origin": "plos:immunology", "subfield": "immunology" }, { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" } ], "title": "A single-residue deletion in H9N2 hemagglutinin uncouples viral burden from disease severity", "title_text": "A single-residue deletion in H9N2 hemagglutinin uncouples viral burden from disease severity", "type": "peer-reviewed", "updated_at": "2026-11-07T12:20:24Z", "url": "https://research.draconitic.com/paper/7zkghhngwjgk3", "venue": "PLOS Pathogens", "versions": [] }