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Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions


In this study, a novel multiple-gene reassortant H1N3 subtype avian influenza virus (AIV) (A/chicken/Zhejiang/81213/2017, CK81213) was isolated in Eastern China, whose genes were derived from H1 (H1N3), H7 (H7N3 and H7N9), and H10 (H10N3 and H10N8) AIVs. This AIV belongs to the avian Eurasian-lineage and exhibits low pathogenicity. Serial lung-to-lung passages of CK81213 in mice was performed to study the amino acid substitutions potentially related to the adaptation of H1 AIVs in mammals. And the mouse-adapted H1N3 virus showed greater virulence than wild-type H1N3 AIV in mice and the genomic analysis revealed a total of two amino acid substitutions in the PB2 (E627K) and HA (L67V) proteins. Additionally, the results of the animal study indicate that CK81213 could infect mice without prior adaption and become highly pathogenic to mice after continuous passage. Our findings show that routine surveillance of H1 AIVs is important for the prediction of influenza epidemics.

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This study was funded by grants from the National Science and Technology Major Project for the Control and Prevention of Major Infectious Diseases in China (2018ZX10711001, 2018ZX10102001 and 2020ZX10001016-004-002), Zhejiang Provincial Natural Science Foundation of China (LY19H260006), and the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (2022ZZ02).

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Correspondence to Haibo Wu.

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The animal experiment was approved by the First Affiliated Hospital, School of Medicine, Zhejiang University (No. 2019-39).

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Supplementary Figure S1. Phylogenetic trees of the eight genes of H1N3 avian influenza viruses. The A/chicken/Zhejiang/81213/2017 virus is indicated with a black dot. The scale bar is used to express the unit of distance between sequences. (DOCX 15 kb)

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Supplementary Figure S2. The representative gross pathology of each group. Red arrows showing typical areas of focal pneumonia. (PNG 724 kb)


Supplementary Figure S3. Positions of amino acid substitutions detected in the mouse-adapted CK81213 virus. The 3D crystal structure of the H1 hemagglutinin (PDB: 3HTO, Green) (A) and PB2 (PDB: 3KHW, Blue) (B) proteins is displayed. The receptor-binding sites and the receptor pocket of the H1 HA protein is indicated in purple and with a purple circle, respectively. The amino acid at position 67 in the HA protein is marked in orange, and the amino acid at position 627 in the PB2 protein is marked in yellow. (PNG 26905 kb)


Supplementary Figure S4. Histological analysis of lungs from mice infected with wide type (WT) and mouse-adapted (MA)-CK81213. (A & B) Histological images show the lungs of mice treated with PBS (mock). (C & D) Histological images show the lungs of mice infected with the wide-type avian influenza virus and (E & F) its mouse-adapted variant at 5 days post-infection. Alveolar edema with fibrin, erythrocytes, and inflammatory cells is indicated by the triangles. Arrows are used to indicate the positive staining of alveolar epithelial cells. A, C and E: H&E staining; B, D and F: IHC. (PNG 47124 kb)

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Yang, F., Zhang, X., Liu, F. et al. Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions. Virus Genes (2022).

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  • Avian influenza virus
  • H1N3
  • Mouse adaptation
  • Amino acid substitutions
  • Virulence