Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts

Abstract

A novel avian-origin influenza A/H7N9 virus emerged in 2013 to cause more than 130 cases of zoonotic human disease, with an overall case fatality rate of around 30% in cases detected. It has been shown that an E-to-K amino acid change at residue 627 of polymerase basic protein 2 (PB2) occurred frequently in the H7N9 isolates obtained from humans but not in viruses isolated from poultry. Although this mutation has been reported to confer increased mammalian pathogenicity in other avian influenza subtypes, it has not been experimentally investigated in the H7N9 virus. In this study, we determined the contribution of PB2-E627K in H7N9 virus to its pathogenicity in mammalian hosts. In addition, the compensatory role of the PB2 mutations T271A, Q591K, and D701N in H7N9 virus was investigated. We characterized the activity of polymerase complexes with these PB2 mutations and found that they enhance the polymerase activity in human 293T cells. The rescued mutants enhanced growth in mammalian cells in vitro. Mice infected with the H7N9 mutant containing the avian signature protein PB2-627E showed a marked decrease in disease severity (weight loss) and pathology compared to mice infected with the wild-type strain (PB2-627K) or other PB2 mutants. Also, mutants with PB2-627E showed lower virus replication and proinflammatory cytokine responses in the lungs of the virus-infected mice, which may contribute to pathogenicity. Our results suggest that these amino acid substitutions contribute to mouse pathogenicity and mammalian adaptation.

Importance: A novel avian H7N9 influenza A virus emerged in east China in 2013 to cause zoonotic human disease associated with significant mortality. It is important to understand the viral genetic markers of mammalian adaptation and disease severity in this H7N9 virus. Since many human (but not avian) H7N9 virus isolates have an amino acid substitution at position E627K in the polymerase basic protein 2 (PB2) gene, we investigated the role of this and other functionally related mutations for polymerase activity in vitro, virus replication competence, and pathogenicity in the mouse model. We found that E627K and functionally related mutations are associated with increased polymerase activity, increased viral replication competence, and increased disease severity in mice.

FIG 1

Polymerase activity of the Sh2/H7N9 and its PB2 mutants. 293T or DF-1 cells were transfected with plasmids containing Sh2/H7N9 PB2, PB1, PA, and NP genes plus a control luciferase reporter plasmid and a viral untranslated-region (UTR)-driven luciferase reporter plasmid. After transfection, the 293T cells were cultured at 37°C (A) and 33°C (B) and the DF-1 cells were cultured at 37°C (C) and 39°C (D) for 24 h. Luciferase activity was then assayed from the cell extracts. Results are the averages from three independent experiments. The values were statistically analyzed by two-tailed paired t test. *, P < 0.05.

FIG 2

Replication kinetics of the H7N9 variants on MDCK and A549 cells. MDCK (A) and A549 (B) cells were infected with the indicated viruses at an MOI of 0.01 and cultured at 37°C in the presence of TPCK-trypsin at 1 μg/ml and 0.2 μg/ml, respectively. Culture supernatants were harvested at the indicated times, and virus titers were determined by TCID₅₀ assay. Results are the averages from three independent experiments. The viral titers of PB2 mutants were compared to the those of the recombinant wild-type virus using the two-tailed paired t test. *, P < 0.05.

FIG 3

Weight change in mice infected with the Sh2/H7N9 virus and its PB2 mutants. Female BALB/c mice were infected intranasally with 1 × 10⁵ PFU of the indicated viruses. The virus-infected mice were monitored for 14 days, and their weights were determined daily. Results are means ± standard deviations (SD) for six infected mice. The values were statistically analyzed by two-tailed paired t test. *, P < 0.05.

FIG 4

Histology of the mice infected with the Sh2/H7N9 and its PB2 mutants. The histology of lung sections was determined in samples stained by hematoxylin-eosin from mice infected with Sh2/H7N9 (A), Sh2/H7N9-PB2-K627E (B), Sh2/H7N9-PB2-K627E/D701N (C), Sh2/H7N9-PB2-K627E/Q591K (D), or Sh2/H7N9-PB2-K627E/T271A (E) or mock infected (F) at 5 days postinfection. Magnification, ×10.

FIG 5

Immunohistochemistry of mice infected with the Sh2/H7N9 virus and its PB2 mutants. Immunohistochemical staining of NP antigens in the lung sections were determined from the samples of the mice infected with Sh2/H7N9 (A), Sh2/H7N9-PB2-K627E (B), Sh2/H7N9-PB2-K627E/D701N (C), Sh2/H7N9-PB2-K627E/Q591K (D), or Sh2/H7N9-PB2-K627E/T271A (E) at 5 days postinfection. Arrowheads indicate influenza virus nucleoprotein-positive cells. Magnification, ×10.

FIG 6

Lung virus titers of mice infected with Sh2/H7N9 and its PB2 mutants. BALB/c mice were infected with the indicated viruses at 1 × 10⁵ PFU. Infected mice were sacrificed on 3 and 5 days postinfection, and virus titers in lung homogenates were measured in MDCK cells. Results from each group and each time point are expressed as means ± SD from four infected mice. The dotted line represents the detection limit at 10^−0.5 TCID₅₀/100 μl. The values were statistically compared with those for the PB2-K627E mutant using the two-tailed paired t test. *, P < 0.05.

FIG 7

Cytokine responses in the lungs of mice infected with Sh2/H7N9 and its PB2 mutants. Cytokine levels (A) IP-10, (B) MCP-3, (C) KC, (D) RANTES, (E) MCP-1, (F) MIP-1α, and (G) TNF-α from virus-infected lungs (4 mice per virus group, days 3 and 5 postinoculation) were measured individually by the FlowCytomix system (eBioscience). Results are means ± SD for four infected mice (n = 4). The values were statistically compared using the two-tailed, paired t test. *, P < 0.05.