A single-residue deletion in H9N2 hemagglutinin uncouples viral burden from disease severity
- 1State 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
- 2Jilin Research and Development Center of Biomedical Engineering, Changchun University, Changchun, China
- 3Institute of Animal Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, China
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.
Related
- Genomic Epidemiology of Canine Distemper Virus (CDV) in Neotropical Primates in Brazil Reveals Multiple Spillover Events and Convergent Evolution at Hemagglutinin Residue 519
- Breaking the brown bridge for residue-borne fungal disease control in broadacre agricultural systems. A review
- Melatonin Uncouples Ripening Promotion from Softening and Enhances the Polyphenol Profile in Postharvest ‘Reineta’ Apples (Malus domestica)
- Influenza H1N1 Imprinting Uncouples Serological and B-Cell Responses in Mice
- Partial sleep deprivation diminishes and uncouples neuronal activity and energy metabolism in the live mouse hippocampus
BibTeX
@article{sun2026singleresidue,
title = {{A single-residue deletion in H9N2 hemagglutinin uncouples viral burden from disease severity}},
author = {Yixue Sun and Yanting Zhu and Pengju Zhang and Shouzhi Sheng and Lili Gai and Siqi Li and Fangyu Zhang and Peige Cao and Ao He and Weixia Wang and Xintao Li and Yanlong Cong},
journal = {PLOS Pathogens},
year = {2026},
month = nov,
doi = {10.1371/journal.ppat.1014776},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014776},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A single-residue deletion in H9N2 hemagglutinin uncouples viral burden from disease severity
AU - Yixue Sun
AU - Yanting Zhu
AU - Pengju Zhang
AU - Shouzhi Sheng
AU - Lili Gai
AU - Siqi Li
AU - Fangyu Zhang
AU - Peige Cao
AU - Ao He
AU - Weixia Wang
AU - Xintao Li
AU - Yanlong Cong
PY - 2026
DA - 2026/11/05
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014776
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014776
AB - 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.
SN - 1553-7374
ER -