Amino acid residues 253 and 591 of the PB2 protein of avian influenza virus A H9N2 contribute to mammalian pathogenesis

Abstract

We investigated the tropism, host responses, and virulence of two variants of A/Quail/Hong Kong/G1/1997 (H9N2) (H9N2/G1) with D253N and Q591K in the PB2 protein in primary human macrophages and bronchial epithelium in vitro and in mice in vivo. Virus with PB2 D253N and Q591K had greater polymerase activity in minireplicon assays, induced more tumor necrosis factor alpha (TNF-α) in human macrophages, replicated better in differentiated normal human bronchial epithelial (NHBE) cells, and was more pathogenic for mice. Taken together, our studies help define the viral genetic determinants that contribute to pathogenicity of H9N2 viruses.

Fig. 1.

Induction of cytokine in primary human macrophages infected by two plaque-purified H9N2/G1 virus variants. Primary human macrophages were infected at MOI of 2 with the two H9N2 virus variants and human seasonal H1N1 virus. The mRNA was collected at 3 h and 6 h postinfection, while the supernatants were collected at 8 h and 24 h postinfection. The gene copy numbers of TNF-α (A), interferon-β (B), and influenza virus matrix 1 (M1) (D) mRNA were analyzed by real-time RT-PCR. (C) The amount of TNF-α protein was quantified by ELISA. Results are expressed as means ± standard deviations (SD) from three independent experiments. Mock, uninfected cells. *, P < 0.05.

Fig. 2.

Replication kinetics of plaque-purified H9N2/G1 virus variants in MDCK cells, embryonated chicken eggs, and well-differentiated NHBE cells. MDCK cells (A) and chicken embryonic eggs (B) were each infected with 1,000 PFU virus. The virus yield was observed for 2 consecutive days, and the amounts of infectious viruses in the culture supernatant and the allantoic fluid were determined by plaque-forming assay. (C) The well-differentiated NHBE cells were infected with the two H9N2/G1 isolates at 37°C and 33°C at an MOI of 0.01. The supernatant was collected 6, 24, 48, and 72 h postinfection, and the virus titers were measured by TCID₅₀ assay in MDCK cells. The values were statistically analyzed using the two-tailed paired t test. Results are expressed as means ± SD from three independent experiments. *, P < 0.05.

Fig. 3.

Effect of the PB2 mutations on polymerase activity, virus replication, and cytokine induction. (A) 293T cells were transfected with plasmids containing H9N2/G1 PB2, PB1, PA, and NP genes plus a control luciferase reporter plasmid and a viral untranslated region (UTR)-driven luciferase reporter plasmid. After transfected cells were cultured at 37°C for 24 h, luciferase activity was then assayed in cell extracts. Results are the average of triplicate transfections. (B) MDCK cells were infected by different recombinant H9N2/G1 viruses at an MOI of 0.01, and the virus yield was observed for 2 consecutive days. The amount of infectious viruses in the culture supernatant was determined by plaque-forming assay. (C) Primary human macrophages were infected by the recombinant viruses at an MOI of 2. The amount of TNF-α protein was quantified by ELISA. The values were statistically analyzed by two-tailed, paired t test. Mock, uninfected cells. *, P < 0.05.

Fig. 4.

Weight change, virus replication, and cytokine responses of the mice infected with the H9N2/G1 variants. Female BALB/c mice were infected intranasally with 1.5 × 10⁵ PFU of the H9N2/G1 isolates. (A) Weights of the infected mice (8 mice per group) were determined every 2 days. Lungs (B) and nasal wash (C) were harvested for virus titration at various times postinoculation. Lungs were homogenized in 2 ml of PBS, and 200 μl of PBS was used as a nasal wash. The virus titers were determined by TCID₅₀ in MDCK cells. The TNF-α (D), MCP-1 (E), and MIP1-α (F) cytokine levels from infected lungs (n = 3 mice per virus group) on days 3, 5, and 10 postinoculation were measured individually by the FlowCytomix system. Baseline cytokine levels from mock-infected mice are shown in each cytokine graph. Results from each time point are expressed as means ± SD of ≥3 infected mice. *, P < 0.05.