Simultaneous coinfection with influenza virus and an arbovirus impedes influenza-specific but not Semliki Forest virus-specific responses

Abstract

Outbreaks of respiratory virus infections and arbovirus infections both pose a substantial threat to global public health. Clinically, both types of infection range from mild to severe and coinfections may occur more commonly than supposed. Our previous experimental coinfection study in mice demonstrated that prior infection with the arbovirus Semliki Forest virus (SFV) negatively impacted immune responses to influenza A virus (IAV). Here, we investigate whether simultaneous coinfection impacts the outcome of immune responses or disease. Simultaneous SFV and IAV infection did not lead to exacerbated or attenuated disease compared with the single virus infection control groups. SFV brain virus titers and brain pathology, including inflammation and immune responses, were comparable in the coinfection and single infection groups. By contrast, there was enhanced IAV replication, but no exacerbated lung pathology in coinfected mice. The magnitude of IAV-specific CD8⁺ T-cell responses in the lungs was lower compared with IAV-only infection. Considered along with our previous study, this study provides evidence that the timing of viral coinfection is pivotal in determining effects on immune responses, pathological changes and disease outcome.

Keywords: CD8+ T cells; Semliki Forest virus; central nervous system; encephalitis; immunity to viral coinfections; influenza virus.

Figure 1

Simultaneous coinfection of Semliki Forest virus (SFV) + influenza A virus (IAV) leads to higher lung IAV virus titers. (a) Mice were infected at the same time with 10⁴ plaque‐forming units (pfu) A/HKx31 [intranasally (i.n.)] and 5 × 10³ pfu A7(74) SFV [intraperitoneally (i.p.)]. The SFV‐only and IAV‐only groups were included as controls. Brain, lungs and spleens were analyzed at 3, 7 and 10 days postinfection (dpi). (b) Weight loss and (c) survival of SFV + IAV–, IAV‐ and SFV‐infected mice monitored for 10 days (n = 10, two independent experiments, error bars represent standard deviation). IAV infectivity titers in the (d) lungs and (e) brain were determined by plaque assay on Madin–Darby canine kidney (MDCK) cells. Each symbol denotes an individual mouse (n = 10, two independent experiments, error bars represent mean). Significance was determined by the Student's unpaired t‐test. SFV infectivity titers in the (f) brain and (g) lungs were determined by a plaque assay on Vero cells. Each symbol denotes an individual mouse (n = 7–10, two independent experiments, error bars represent mean). Histopathological changes in the (h) lungs and (j) brains of SFV‐, IAV‐ and SFV + IAV–infected mice on 7 dpi. Brains and lungs from uninfected mice are included as controls. Tissue sections were stained with hematoxylin and eosin; representative images are shown. Bar = 200 μm for 5×; 100 μm for 15×; 50 μm for 20× and 20 μm for 40× magnification. Yellow arrowheads point to areas of inflammation and damage as described in the text, and 15× images are the magnified areas outlined in yellow at 5× magnification. Quantification of the extent of inflammation and damage in the SFV, IAV and SFV + IAV groups in the (i) lungs and (k) brains (n = 3, error bars represent standard error of the mean). Significance was determined by Tukey's multiple comparison test. LOD, limit of detection. *P < 0.05, **P < 0.005.

Figure 2

Cytokine and chemokine profiles in the brains and lungs of Semliki Forest virus (SFV)–, influenza A virus (IAV)– and SFV + IAV–infected mice. (a) Heatmaps summarizing the differences in cytokine and chemokine concentrations in lung homogenates across time points for each infection type (n = 10 in each group and time point, two independent experiments). (b, c) Comparison of significantly different chemokines in the lungs (n = 10 per time point, two independent experiments, error bars represent standard deviation). (d) Heatmaps summarizing the differences in cytokine and chemokine concentrations in brain homogenates across time points in each infection type (n = 10 in each group and time point, two independent experiments). (e) Comparison of significantly different cytokines and chemokines in the brain (n = 5–10 per time point, two independent experiments, error bars represent standard deviation). Each symbol denotes an individual mouse. Significance was determined by Tukey's multiple comparison test. CCL, C–C motif chemokine ligand; CXCL, C–X–C motif chemokine ligand 1; GM‐CSF, granulocyte–macrophage colony‐stimulating factor; IFN, interferon; IL, interleukin; IP‐10, interferon gamma–induced protein 10; KC, keratinocyte‐derived chemokine; MCP‐1, monocyte chemoattractant protein‐1; RANTES, regulated upon activation, normal T‐cell expressed and secreted; TNF, tumor necrosis factor.

Figure 3

Analysis of total immune cell numbers in Semliki Forest virus (SFV)–, influenza A virus (IAV)– and SFV + IAV–infected mice at 7 and 10 days postinfection (dpi). (a) Absolute numbers of leukocytes in the brain, lungs and spleen of IAV‐, SFV‐ and SFV + IAV–infected mice at 3, 7 and 10 dpi; (n = 5–10, two independent experiments, error bars represent standard deviation). (b) Representative fluorescence‐activated cell sorting (FACS) plots show the proportion of CD8⁺ and CD4⁺ T cells across different tissues in SFV‐, IAV‐ and SFV + IAV–infected mice at 7 and 10 dpi. (c) Absolute numbers of CD8⁺ (top row) and CD4⁺ (bottom row) T cells across different anatomical sites in SFV‐, IAV‐ and SFV + IAV–infected mice at 7 and 10 dpi (n = 5–10, two independent experiments error bars represent standard deviation). (d) Absolute numbers of activated (CD44⁺ CD62L^lo) CD8⁺ (top row) and CD4⁺ (bottom row) T cells across different anatomical sites in IAV‐, SFV‐ and SFV + IAV–infected mice at 7 and 10 dpi (n = 5–10, two independent experiments, error bars represent standard deviation). Each symbol denotes an individual mouse. Significance was determined by Tukey's multiple comparison test.

Figure 4

Analysis of virus‐specific CD8⁺ T cells across different anatomical sites in each infection type at 7 and 10 days postinfection (dpi). (a) Representative fluorescence‐activated cell sorting (FACS) plots show the proportion of influenza A virus (IAV)–specific CD8⁺ T cells of D^bNP₃₆₆ and D^bPA₂₂₄ specificities across different tissues in IAV‐ and Semliki Forest virus (SFV) + IAV–infected mice at 7 and 10 dpi. (b) Absolute numbers of IAV‐specific CD8⁺ T cells of D^bNP₃₆₆ and D^bPA₂₂₄ specificities across different anatomical sites in IAV‐ and SFV + IAV–infected mice at 7 and 10 dpi. (n = 5–10, error bars represent mean). (c) Representative FACS plots show the proportion of SFV‐specific CD8⁺ T cells directed at K^bE1₁₅₉ epitope across different tissues of SFV‐ and SFV + IAV–infected mice at 7 and 10 dpi. (d) Absolute numbers of SFV‐specific CD8⁺ T cells directed at K^bE1₁₅₉ epitope across different anatomical sites of SFV‐ and SFV + IAV–infected mice at 7 and 10 dpi (n = 5–10, two independent experiments, error bars represent mean). Each symbol denotes an individual mouse. Significance was determined by the Student's unpaired t‐test.

Figure 5

Activation profiles of effector CD4⁺ T cells and CD8⁺ T cells across anatomical sites in influenza A virus (IAV)–, Semliki Forest virus (SFV)– and SFV + IAV–infected mice at 7 days postinfection (dpi). Stacked bar graphs depicting frequencies of activation marker combinations on (a) CD8⁺ T cells and (e) CD4⁺ T cells on 7 dpi in IAV‐, SFV‐ and SFV + IAV–infected mice; (n = 5, error bar represents standard deviation). Comparison of significantly different activation marker combinations in the brain, lungs and spleen on CD8⁺ T cells (b–d) and CD4⁺ T cells (f–h) on 7 dpi (n = 5, error bar represents standard deviation). Each symbol denotes an individual mouse. Significance was determined by Tukey's multiple comparisons test. PD‐1, programmed cell death protein 1.

Figure 6

Activation profiles of virus‐specific CD8⁺ T cells across anatomical sites in influenza A virus (IAV)–, Semliki Forest virus (SFV)– and SFV + IAV–infected mice at 7 days postinfection (dpi). Stacked bar graphs depicting frequencies of activation marker combinations on (a) D^bPA₂₂₄ ⁺CD8⁺ T cells (top row) and D^bNP₃₆₆ ⁺CD8⁺ T cells (bottom row) on 7 dpi in IAV‐ and SFV + IAV–infected mice; (n = 5, error bar represents standard deviation). Comparison of significantly different activation marker combinations in the (b) lungs and (c) spleen on D^bPA₂₂₄ ⁺CD8⁺ T cells and D^bNP₃₆₆ ⁺CD8⁺ T cells on 7 dpi (n = 5, error bar represents standard deviation). (d) Stacked bar graphs depicting frequencies of activation marker combinations on K^bE1₁₅₉ ⁺CD8⁺ T cells on 7 dpi in SFV‐ and SFV + IAV–infected mice; (n = 5, error bar represents standard deviation). Comparison of significantly different activation marker combinations in the (e) brain, (f) lungs and (g) spleen on K^bE1₁₅₉ ⁺CD8⁺ T cells at 7 dpi (n = 5, error bar represents standard deviation). Each symbol denotes an individual mouse. Significance was determined by the Student's unpaired t‐test.

Figure 7

Similar numbers of influenza A virus (IAV)–specific memory CD8⁺ T cells across different anatomical sites in IAV and Semliki Forest virus (SFV) + IAV. (a) Mice were infected at the same time with 10⁴ plaque‐forming units (pfu) A/HKx31 [intranasally (i.n.)] and 5 × 10³ pfu A7(74) SFV [intraperitoneally (i.p.)]. The IAV‐only group was included as control. Brain, lungs and spleens were harvested at 30 days postinfection (dpi). (b) Absolute numbers of IAV‐specific CD8⁺ T cells directed at the D^bPA₂₂₄ and D^bNP₃₆₆ epitopes are shown across different anatomical sites in IAV‐ and SFV + IAV–infected mice. Representative fluorescence‐activated cell sorting (FACS) plots are shown for each group and tissue at different time points (n = 10, error bars represent median). (c) Representative FACS plots are shown for each group and tissue. Absolute numbers of IAV‐specific (d) effector memory T cells (T_EM) and (e) central memory T cells (T_CM) CD8⁺ T cells are shown for D^bPA₂₂₄ ⁺CD8⁺ and D^bNP₃₆₆ ⁺CD8⁺ T‐cell specificities in the brain, lungs and spleen of IAV‐ and SFV + IAV–infected mice at 30 dpi; (n = 10, error bar represents standard deviation). (f) Absolute numbers and frequencies of IAV‐specific tissue‐resident memory T cells (T_RM) CD8⁺ T cells are shown for D^bPA₂₂₄ ⁺CD8⁺ and D^bNP₃₆₆ ⁺CD8⁺ T‐cell specificities in the brain and lungs of IAV‐ and SFV + IAV–infected mice at 30 dpi. Representative FACS plots are shown for each group and tissue (n = 10, two independent experiments, error bar represents standard deviation). Each symbol denotes an individual mouse. Significance was determined by the Student's unpaired t‐test.

Figure 8

Similar numbers of Semliki Forest virus (SFV)–specific memory CD8⁺ T cells across different anatomical sites in SFV and SFV + influenza A virus (IAV). (a) Mice were infected at the same time with 10⁴ plaque‐forming units (pfu) A/HKx31 [intranasally (i.n.)] and 5 × 10³ pfu A7(74) SFV [intraperitoneally (i.p.)]. The SFV‐only group was included as control. Brain, lungs and spleens were harvested at 30 days postinfection (dpi). (b) Absolute numbers of SFV‐specific CD8⁺ T cells directed at the K^bE1₁₅₉ epitope are shown across different anatomical sites in SFV‐ and SFV + IAV–infected mice. Representative fluorescence‐activated cell sorting (FACS) plots are shown for each group and tissue at different time points (n = 10, error bars represent median). (c) Representative FACS plots are shown for each group and tissue. Absolute numbers of SFV‐specific (d) effector memory T cells (T_EM) and (e) central memory T cells (T_CM) CD8⁺ T cells are shown for K^bE1₁₅₉ ⁺CD8⁺ T‐cell specificities in the brain, lungs and spleen of SFV‐ and IAV + SFV–infected mice at 30 and 90 dpi (n = 10, error bar represents standard deviation). (f) Absolute numbers and frequencies of SFV‐specific tissue‐resident memory T cells (T_RM) CD8⁺ T cells are shown for K^bE1₁₅₉ ⁺CD8⁺ T‐cell specificities in the brain and lungs of SFV‐ and SFV + IAV–infected mice at 30 dpi. Representative are plots shown for each group and tissue (n = 10, two independent experiments, error bar represents standard deviation). Each symbol denotes an individual mouse. Significance was determined by the Student's unpaired t‐test.