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Pathogenesis and Immunity

Genetic Compatibility of Reassortants between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity in Mammals

Yasuha Arai, Madiha S. Ibrahim, Emad M. Elgendy, Tomo Daidoji, Takao Ono, Yasuo Suzuki, Takaaki Nakaya, Kazuhiko Matsumoto, Yohei Watanabe
Stacey Schultz-Cherry, Editor
Yasuha Arai
Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, JapanCREST, Japan Science and Technology Agency, Saitama, Japan
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Madiha S. Ibrahim
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt
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Emad M. Elgendy
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt
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Tomo Daidoji
Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, Japan
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Takao Ono
The Institute of Scientific and Industrial Research, Osaka University, Osaka, JapanCREST, Japan Science and Technology Agency, Saitama, Japan
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Yasuo Suzuki
College of Life and Health Sciences, Chubu University, Aichi, JapanCREST, Japan Science and Technology Agency, Saitama, Japan
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Takaaki Nakaya
Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, Japan
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Kazuhiko Matsumoto
The Institute of Scientific and Industrial Research, Osaka University, Osaka, JapanCREST, Japan Science and Technology Agency, Saitama, Japan
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Yohei Watanabe
Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, JapanCREST, Japan Science and Technology Agency, Saitama, Japan
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Stacey Schultz-Cherry
St. Jude Children’s Research Hospital
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DOI: 10.1128/JVI.01969-18
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    FIG 1

    Infectivity and virulence of H5N1-wt and H9N2-wt viruses. (A and B) Binding of H5N1-wt (A) and H9N2-wt (B) to α2,3 sialylglycopolymers (blue) and α2,6 sialylglycopolymers (red). A/duck/Egypt/D1Br/2007 and A/turkey/Wisconsin/1/1966 are ancestral H5N1 clade 2.2.1 and classical H9N2 strains, respectively. Each data point reflects the mean ± the SD of three independent experiments. (C and D) Virulence in mice infected with the H5N1-wt (C) and H9N2-wt viruses (D). Five-week-old BALB/c mice (five mice per group) were inoculated intranasally with serial 10-fold dilutions of the viruses. The body weights of infected mice were monitored for 14 dpi. The mean ± the SD of the percentage of the initial body weight for each group of mice is shown. Survival was calculated, including mice that were sacrificed after they had lost more than 30% of their body weight.

  • FIG 2
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    FIG 2

    Properties of reassortants with H5N1 HA and NA surface genes and six internal genes from H5N1 and H9N2 viruses. The eight gene segments are indicated at the top of each column. Blue denotes an H5N1 gene segment and white denotes an H9N2 gene segment. The mean death time (MDT) was calculated based on egg death after inoculation with cell culture supernatants at 96 h posttransfection with plasmid DNAs. Virus stocks were prepared after one or two passages, and their titers were assayed as HAU by hemagglutination assays.

  • FIG 3
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    FIG 3

    Replication of reassortants with H5N1 HA and NA surface genes and six internal genes from H5N1 and H9N2 viruses in avian and human cells. DF-1 (A) and Calu-3 (B and C) cells were infected with the indicated viruses at MOIs of 0.003 and 0.03, respectively, and cultured at 37°C for 48 hpi (A and B) or 33°C for 72 hpi (C). Progeny viruses were collected, and virus titers were determined by focus-forming assays. The titers were calculated relative to that of H5N1-wt. Each data point indicates the mean ± the SD of three independent experiments.

  • FIG 4
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    FIG 4

    Viral polymerase activity in human cells infected with H5N1-H9N2 reassortants. 293T cells were transfected with plasmids expressing a combination of the PB2, PB1, PA, and NP genes from H5N1 or H9N2 viruses, a human polymerase I-driven plasmid expressing a reporter gene, and a Renilla luciferase-expressing plasmid as an internal control. After 24 h of incubation at 33 or 37°C, the luciferase activity of each sample was measured and normalized to the internal Renilla luciferase activity. The data were expressed relative to the results for H5N1-wt. Each data point represents the mean ± the SD of three independent experiments. Asterisks indicate relative polymerase activity significantly lower than that of a polymerase complex consisting of PB2, PB1, PA, and NP genes from the H5N1 virus (ANOVA with Tukey’s multiple-comparison test [*, P < 0.01]). The number of the virus containing each corresponding gene combination is shown below the appropriate column, with the number of the viruses selected for further characterization underlined.

  • FIG 5
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    FIG 5

    Growth kinetics of H5N1-H9N2 reassortants in avian and human cells. DF-1 (A) and Calu-3 cells (B and C) were infected with the indicated viruses at MOIs of 0.003 or 0.03, respectively, and incubated at 37 (A and B) or 33°C (C). At the indicated times postinfection, virus titers were determined by focus-forming assays. Each data point represents the mean ± the SD of three independent experiments. Asterisks indicate virus titers significantly higher than that of H5N1-wt virus (ANOVA with Tukey’s multiple-comparison test [*, P < 0.01]). NS, no statistical significance.

  • FIG 6
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    FIG 6

    Body weights and survival of mice infected with H5N1-H9N2 reassortants. Five-week-old BALB/c mice (five mice per group) were inoculated intranasally with serial 10-fold dilutions of the indicated viruses. (A) The body weights of the infected mice were monitored for 14 dpi. The mean ± the SD of the percentage of the initial body weight for each group of mice is shown. (B) Survival of the infected mice. Survival was calculated including mice that were sacrificed after they had lost more than 30% of their body weight. MLD50 values of the viruses are shown, with each value relative to H5N1-wt in parenthesis. (C and E) Virus titers in the lungs (C) and brains (E) of mice infected with 10 FFU of the indicated viruses at 3 and 6 dpi. Each symbol marks the titer in an individual mouse. Asterisks indicate virus titers significantly higher than those of H5N1-wt virus (ANOVA with Tukey’s multiple-comparison test [*, P < 0.01]). (D) Representative photomicrographs of hematoxylin and eosin (H&E)-stained (upper panel) and immunohistochemically (IHC) stained (lower panel) lung sections from mice infected with the indicated viruses at 3 dpi. In IHC-stained tissues, the viral antigen is stained deep brown on a hematoxylin-stained background.

  • FIG 7
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    FIG 7

    Chemokine/cytokine responses in the lungs of mice infected with 10 FFU of the indicated H5N1-H9N2 reassortants. The concentrations of the indicated chemokines and cytokines in the lungs of mice at 3 and 6 dpi were measured by ELISA. Each bar marks the concentration in an individual mouse. Asterisks indicate chemokine/cytokine levels significantly higher than those of H5N1-wt virus (ANOVA with Tukey’s multiple-comparison test [**, P < 0.01; *, P < 0.05]).

  • FIG 8
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    FIG 8

    Virion morphology of H5N1-H9N2 reassortants. (A) Representative transmission electron micrographs of thin sections of the indicated virions released from infected MDCK cells at low magnification. (B) Magnified views of panel A. (C) Additional views of the indicated virions in transverse sections, in which viral ribonucleoproteins were observed. (D) Representative transmission electron micrographs of the indicated virions budding from the surface of infected MDCK cells at low magnification. (E) Additional views of virions budding from the cell surface at high magnification.

Additional Files

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    • Supplemental file 1 -

      Fig. S1 (Phylogenetic trees of the genes of H5N1 viruses isolated in Egypt.)

      Fig. S2 (Phylogenetic trees of the genes of H9N2 viruses isolated in Egypt.)

      PDF, 241K

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Genetic Compatibility of Reassortants between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity in Mammals
Yasuha Arai, Madiha S. Ibrahim, Emad M. Elgendy, Tomo Daidoji, Takao Ono, Yasuo Suzuki, Takaaki Nakaya, Kazuhiko Matsumoto, Yohei Watanabe
Journal of Virology Feb 2019, 93 (4) e01969-18; DOI: 10.1128/JVI.01969-18

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Genetic Compatibility of Reassortants between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity in Mammals
Yasuha Arai, Madiha S. Ibrahim, Emad M. Elgendy, Tomo Daidoji, Takao Ono, Yasuo Suzuki, Takaaki Nakaya, Kazuhiko Matsumoto, Yohei Watanabe
Journal of Virology Feb 2019, 93 (4) e01969-18; DOI: 10.1128/JVI.01969-18
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KEYWORDS

H5N1
H9N2
influenza reassortants
pathogenicity
public health risk

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