Pathogenesis and Immunity

Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

Sander Herfst, Salin Chutinimitkul, Jianqiang Ye, Emmie de Wit, Vincent J. Munster, Eefje J. A. Schrauwen, Theo M. Bestebroer, Marcel Jonges, Adam Meijer, Marion Koopmans, Guus F. Rimmelzwaan, Albert D. M. E. Osterhaus, Daniel R. Perez, Ron A. M. Fouchier
DOI: 10.1128/JVI.02634-09

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Activity of the NL602 polymerase complex with wild type (wt) PB2, PB2-E627K, PB2-E677G, and PB2-D701N in minigenome assays. Firefly luciferase was expressed from a synthetic viral RNA when the influenza virus polymerase complex was present. The luminescence of the firefly luciferase reporter was standardized using a plasmid constitutively expressing Renilla luciferase protein to correct for differences in transfection efficiencies and sample processing. Relative luminescence was calculated as the percent relative light units (firefly luciferase/Renilla luciferase), and the fold difference in reporter protein expression was calculated with the wild-type polymerase complex as a reference. Averages and standard deviations from two independent experiments performed in duplicate are shown. Neg, negative control.

  • FIG. 2.
    FIG. 2.

    Replication kinetics of NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses in MDCK cells. MDCK cells were inoculated with 0.01 TCID50/cell of the NL602 (closed circles), NL602/PB2-627K (open circles), NL602/PB2-677G (closed squares), and NL602/PB2-701N (open squares) viruses, and supernatant samples were harvested 6, 12, 24, and 48 h later. The supernatant samples were titrated in MDCK cells. Geometric mean titers and standard deviations were calculated from two independent experiments.

  • FIG. 3.
    FIG. 3.

    Weight loss of BALB/c mice inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses. Mean bodyweights and standard deviations were calculated as percentages of bodyweight compared to bodyweight at the time of inoculation for each group inoculated with NL602 (closed circles), NL602/PB2-627K (open circles), NL602/PB2-677G (closed squares), and NL602/PB2-701N (open squares) viruses.

  • FIG. 4.
    FIG. 4.

    Weight loss of ferrets inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses. Bodyweight is depicted as percentage of bodyweight at time of inoculation. Data are shown for individual animals until the animals were euthanized at 3 or 7 days p.i.

  • FIG. 5.
    FIG. 5.

    Virus shedding from the upper respiratory tract of ferrets inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses. Virus detection in throat swabs (A) and nose swabs (B) is indicated for NL602 (black bars), NL602/PB2-627K (white bars), NL602/PB2-677G (dark-gray bars), and NL602/PB2-701N (light-gray bars). Geometric mean titers for positive samples are displayed, and the error bars indicate the standard deviations. The lower limits of detection are indicated by the dotted lines.

  • FIG. 6.
    FIG. 6.

    Transmission of NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses by aerosol or respiratory droplets in ferrets. Virus titers in throat (black bars) and nose (white bars) swabs are displayed for inoculated (A to D) and exposed (E to H) ferrets. The geometric mean titers of positive samples are displayed, and the error bars indicate the standard deviations. The number of positive contact animals per day is depicted. The asterisk indicates that one ferret was negative for virus shedding but seroconverted to influenza A virus nucleoprotein in the course of the experiment. The lower limits of detection are indicated by the dotted lines.

Tables

  • TABLE 1.

    Virus titers in the lungs of BALB/c mice inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses

    Time after inoculation (days)Virus titer (log10 TCID50/g of tissue)a
    NL602NL602/PB2-627KNL602/PB2-677GNL602/PB2-701N
    36.0 ± 0.36.4 ± 0.56.5 ± 0.46.0 ± 0.3
    64.9 ± 0.34.9 ± 0.25.6 ± 0.85.3 ± 0.4

    • ↵ a Geometric mean titers ± standard deviations are given.

  • TABLE 2.

    Virus titers in the respiratory tracts of ferrets inoculated with NL602, NL602/PB2-627K, NL602/PB2-677G, and NL602/PB2-701N viruses

    TissueVirus titer (log10 TCID50/g tissue)a
    3 days p.i.7 days p.i.
    NL602NL602 627KNL602 677GNL602 701NNL602NL602 627KNL602 677GNL602 701N
    NT6.9 ± 0.3 (3/3)7.2 ± 0.6 (3/3)6.8 ± 0.5 (3/3)6.7 ± 0.6 (3/3)4.4 ± 0.7 (2/3)ND6.8 (1/3)4.9 ± 1.3 (3/3)
    Trachea5.0 ± 0.7 (3/3)4.4 ± 1.0 (3/3)5.3 ± 0.5 (2/3)5.1 ± 1.0 (3/3)2.4 (1/3)NDND2.1 (1/3)
    Lung5.6 ± 0.9 (3/3)5.4 ± 0.7 (2/3)4.8 ± 0.3 (2/3)4.9 ± 1.1 (3/3)1.5 (1/3)NDNDND

    • ↵ a Geometric mean titers of positive samples ± standard deviations are given, and the numbers of positive animals are indicated in parentheses. ND, not detected. The detection levels for nasal turbinates (NT), trachea, and lung were <1.5, <1.5, and <1 log10 TCID50/g tissue, respectively.

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KEYWORDS

amino acid substitution
Influenza A Virus, H1N1 Subtype
Mutation, Missense
Orthomyxoviridae Infections
Viral Proteins
virulence factors

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