A single-amino-acid substitution in a polymerase protein of an H5N1 influenza virus is associated with systemic infection and impaired T-cell activation in mice

Abstract

The transmission of H5N1 influenza viruses from birds to humans poses a significant public health threat. A substitution of glutamic acid for lysine at position 627 of the PB2 protein of H5N1 viruses has been identified as a virulence determinant. We utilized the BALB/c mouse model of H5N1 infection to examine how this substitution affects virus-host interactions and leads to systemic infection. Mice infected with H5N1 viruses containing lysine at amino acid 627 in the PB2 protein exhibited an increased severity of lesions in the lung parenchyma and the spleen, increased apoptosis in the lungs, and a decrease in oxygen saturation. Gene expression profiling revealed that T-cell receptor activation was impaired at 2 days postinfection (dpi) in the lungs of mice infected with these viruses. The inflammatory response was highly activated in the lungs of mice infected with these viruses and was sustained at 4 dpi. In the spleen, immune-related processes including NK cell cytotoxicity and antigen presentation were highly activated by 2 dpi. These differences are not attributable solely to differences in viral replication in the lungs but to an inefficient immune response early in infection as well. The timing and magnitude of the immune response to highly pathogenic influenza viruses is critical in determining the outcome of infection. The disruption of these factors by a single-amino-acid substitution in a polymerase protein of an influenza virus is associated with severe disease and correlates with the spread of the virus to extrapulmonary sites.

FIG. 1.

Replication and pathogenicity of H5N1 viruses in mice. Groups of four mice were inoculated intranasally with 10⁵ TCID₅₀ of HK486, HK486PB2 MT, and HK483. (A) Lungs, brain, and spleen were harvested on 2 and 4 dpi, and virus titers were determined in MDCK cells. Each dot represents one animal, and black bars represent the mean viral titers for the four mice in each group. The limit of detection (10^1.8) is denoted by the dotted line. (B) Groups of eight mice were weighed daily for 16 dpi and were sacrificed if a weight loss of ≥ 20% from the original weight was observed. (C) Oxygen saturation levels of groups of H5N1-infected mice were measured daily for 5 dpi using a pulse oximeter. Data points represent the means ± standard deviations for eight mice. Differences between HK486PB2 MT- and HK483-infected mice and HK486-infected mice were statistically significant at 4 dpi (*, P < 0.005).

FIG. 2.

Lung and splenic lesions associated with H5N1 virus infection. (A) Histopathology of lungs from mice infected with HK486, HK486PB2 MT, and HK483 at 4 dpi were stained with H&E (left), and IHC was performed for H5N1 viral antigen (brown color on the right). Viral antigen is found in the bronchioles (B) and alveoli (A) of mice in each group except the mock infection group. More alveolar inflammation and viral antigen is present in the lungs of mice given HK486PB2 MT and HK483 strains. (B to E) Spleen of HK486-, HK486PB2 MT-, and HK483-infected mice at 2 dpi were stained with H&E (B to D), and IHC was performed for H5N1 viral antigen (E). Note the large pale GCs in panels C, D, and E but not in panel B and viral antigen (brown color) in panel E. The magnification for lung is ×200 and for spleen is ×400. The splenic section in panel E was taken from an experiment separate from those in panels B to D.

FIG. 3.

Examination of early apoptosis and apoptosis-related gene expression in the lungs of H5N1-infected mice. (A) Lungs were isolated from mice infected with HK486, HK486PB2 MT, and HK483 at 4 dpi and were subjected to annexin V staining to determine the percentage of cells in early apoptosis. As a positive control, cells from uninfected mice were treated with 100 nM staurosporine overnight at 37°C. The means and standard errors for groups of four mice are presented. Statistical significance was determined using the unpaired t test. *, P < 0.05. (B) RNA was isolated from the lungs of mice infected with the H5N1 viruses mentioned in panel A and subjected to gene expression profiling. Represented is the expression of genes related to the caspase cascade as analyzed using MetaCore software (GeneGo, Inc.) at 4 dpi. Thresholds for inclusion in analysis were a change (n-fold) of 1.5 or greater (log ratio = 0.18) relative to levels for the mock infection group in at least one experiment at P ≤ 0.01. Expression bars represent HK486 on day 4 (bar 1), HK486PB2 MT on day 4 (bar 2), and HK483 on day 4 (bar 3). Red bars and blue bars represent genes whose expression was up- or downregulated, respectively, by at least 1.5-fold (log ratio = 0.18) at P ≤ 0.01 relative to results for the mock infection group. The extent of shading within each bar represents the magnitude of up- or downregulation. Red, green, and blue lines and hexagons represent negative, positive, and unspecified effects, respectively. Lettering within hexagons is as follows: B, binding; C, cleavage; +P, phosphorylation; and Tn, transport. TNF, tumor necrosis factor.

FIG. 4.

Impaired TCR activation in the lungs of HK483- and HK486PB2 MT-infected mice at 2 and 4 dpi. (A) Hierarchical clustering of selected genes related to TCR signaling in the lungs of HK486-, HK486PB2 MT-, and HK483-infected mice at 2 and 4 dpi based on analysis using MetaCore software (GeneGo, Inc.). Thresholds for inclusion in analysis were a change (n-fold) of 1.5-fold or greater (log ratio = 0.18) relative to results for the mock infection group in at least one experiment at P ≤ 0.01. Green and red shading represent genes whose expression was down- or upregulated, respectively. (B) Antigen presentation by MHCI using MetaCore software (GeneGo, Inc.). Expression bars represent HK486 on days 2 (bar 1) and 4 (bar 4), HK486PB2 MT on days 2 (bar 2) and 4 (bar 5), and HK483 on days 2 (bar 3) and 4 (bar 6). Red bars and blue bars represent genes whose expression was up- or downregulated, respectively, by at least 1.5-fold (log ratio = 0.18) at P ≤ 0.01 relative to results for the mock infection group. The extent of shading within each bar represents the magnitude of up- or downregulation. Red, green, and blue lines and hexagons represent negative, positive, and unspecified effects, respectively. Lettering within hexagons is as follows: B, binding, Tn, transport; and CS, complex subunit.

FIG. 5.

Key processes related to the innate and early adaptive immune responses are preferentially upregulated in the spleen of HK483- and HK486-infected mice early during infection. The hierarchical clustering of selected genes related to (A) NK cell cytotoxicity, (B) antigen presentation, and (C) interferon signaling in the spleen of HK486-, HK486PB2 MT-, and HK483-infected mice at 2 and 4 dpi. Thresholds for inclusion in analysis were a change (n-fold) of twofold or greater (log ratio = 0.3) in at least one experiment at P ≤ 0.01. Green and red shading represent genes whose expression was down- or upregulated, respectively. Graphs beneath each heat map represent genes whose expression was twofold (log ratio = 0.3) upregulated (UP) at P ≤ 0.01 in an experimental group at a given time point postinfection based on analysis using MetaCore software (GeneGo).