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

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.

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.

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.

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.

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.

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]).