Assessment of the internal genes of influenza A (H7N9) virus contributing to high pathogenicity in mice

Abstract

The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans.

Importance: To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.

FIG 1

Multiple-cycle growth curves of the H7N9 and H9N2 recombinant viruses. The replication abilities of the rescue viruses were measured by multiple-cycle growth curve analysis on MDCK and A549 cells, as indicated. Data shown are the means ± SD of results from three independent experiments. The statistically significant differences between the rAH-H7N9 and rAH-H7N9/PB2-627E groups were analyzed by a paired-sample t test and two-way ANOVA, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The experiments were performed at the same time, and the growth curves of rAH-H7N9 in panels A and B were used for comparison with the results of rAH-H7N9/PB2-627E in panels C and D, respectively.

FIG 2

Polymerase activity of RNP combinations between the H9N2 and H7N9 viruses. The polymerase activity of the RNP combinations between CK/1023-H9N2 and AH-H7N9 viruses were evaluated by a dual-luciferase reporter system with the PB2, PB1, PA, and NP expression vectors on human 293T and chicken DF-1 cells, respectively. H and A represent the gene segments from AH-H7N9 and CK/1023-H9N2, respectively. Values shown are the means ± SD of results from three independent experiments and are standardized to values of AH-H7N9 measured on 293T and DF-1 cells, respectively (100%). The data were analyzed by a paired-sample t test and two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The red and blue asterisks represent the results between AH-H7N9 and other recombinants in 293T and DF-1 cells, respectively. The black asterisks represent the results of the same group between 293T and DF-1 cells.

FIG 3

Body weight changes, lung virus titers, and lung indexes of the mice infected by the H7N9 and H9N2 recombinant viruses. Mice in each group were inoculated i.n. with 10⁶ EID₅₀s of virus. Their body weights were monitored daily for a 14-day observation period and expressed as percentages of the initial values (A). Three mice from each group were euthanized at 3, 5, and 7 days p.i. The LVTs were measured in embryonated eggs and expressed as log₁₀ EID₅₀/0.1 ml (B). The lung weights were measured for lung index calculation (C). The data are presented as the means ± SD. The LVTs were compared to each other by two-way ANOVA. The undetected LVTs of the groups were expressed as negative numbers. The red, blue, and black asterisks represent the results for the rAH-H7N9, rCK/1023-H9N2, and PBS (mock) infection groups compared to the other groups, respectively. The lung indices were analyzed between the infection groups and PBS (mock) infection group by a paired-sample t test and two-way ANOVA, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 4

Pathological changes of mouse lung infected by the H7H9 and H9N2 recombinant viruses. Panels A, B, C, D, I, J, K, L, and Q show the H&E-stained lung sections (magnification, ×200) at 5 days p.i. of the rCK/1023-H9N2, rAH-H7N9, H9N2-AH/PB2, H9N2-AH/PB1, H9N2-AH/PA, H9N2-AH/NP, H9N2-AH/M, H9N2-AH/NS, and PBS (mock) infection groups, respectively. Panels E, F, G, H, M, N, O, P, and R show enlargements (600×) of the insets for the respective panels. Scale bar, 100 μm. Inflammatory cell infiltration, deciduous epithelium mucosae and inflammatory cells in the bronchial lumen, and hemorrhage are denoted with thick black arrows, thick white arrows, and black triangles, respectively.

FIG 5

Cytokines in mouse lung evoked by the H7N9 and H9N2 recombinant viruses. Three mice of each group were euthanized at 3, 5, and 7 days p.i., and the lung suspensions were used for cytokine detection, as indicated on the panels. The data are presented as the means ± SD and analyzed by a paired-sample t test and two-way ANOVA. The red, blue, and black asterisks represent the results of the AH-H7N9-, CK/1023-H9N2-, and PBS (mock)-infected groups compared with those of the other groups, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001).