Exacerbation of Influenza A Virus Disease Severity by Respiratory Syncytial Virus Co-Infection in a Mouse Model

Abstract

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are leading causes of childhood infections. RSV and influenza are competitive in vitro. In this study, the in vivo effects of RSV and IAV co-infection were investigated. Mice were intranasally inoculated with RSV, with IAV, or with both viruses (RSV+IAV and IAV+RSV) administered sequentially, 24 h apart. On days 3 and 7 post-infection, lung tissues were processed for viral loads and immune cell populations. Lung functions were also evaluated. Mortality was observed only in the IAV+RSV group (50% of mice did not survive beyond 7 days). On day 3, the viral loads in single-infected and co-infected mice were not significantly different. However, on day 7, the IAV titer was much higher in the IAV+RSV group, and the RSV viral load was reduced. CD4 T cells were reduced in all groups on day 7 except in single-infected mice. CD8 T cells were higher in all experimental groups except the RSV-alone group. Increased airway resistance and reduced thoracic compliance were demonstrated in both co-infected groups. This model indicates that, among all the infection types we studied, infection with IAV followed by RSV is associated with the highest IAV viral loads and the most morbidity and mortality.

Keywords: IAV; RSV; co-infection; mortality; viral titer.

Figure 1

A schematic representation of the experimental strategy for the RSV and IAV co-infection model. Mice were intranasally infected with RSV A2, IAV PR8 or both, one after the other, after a 24 h interval (RSV+IAV or IAV+RSV). One group of mice was intranasally inoculated with PBS and served as the control group. All groups were monitored for up to 7 days (daily weight and survival); on selected days post-infection (d3 and d7), mice were sacrificed for experimental analyses that included organ (lung, spleen and thymus) weights, viral titers, immunophenotyping and lung function.

Figure 2

Body weight loss was more severe in mice infected with IAV 24 h prior to RSV inoculation. Mice were infected with IAV PR8, RSV A2 or one after the other, 24 h apart (IAV+RSV and RSV+IAV) and monitored for (A) survival and (B) body weight changes for 7 days. Mice inoculated with PBS served as the control. Values are mean ± SEM (in percentages) of the daily weight divided by the starting weight for each mouse. Survival data were derived from two independent experiments (n = 6 mice per group). Body weight data were derived from five independent experiments (n = 18 mice per group). Asterisks denote significant differences between the control and experimental groups; (** p < 0.005). Survival analysis was performed by Kaplan–Meier survival curves and a log-rank test, using GraphPad Prism.

Figure 3

IAV and RSV lung viral loads in co-infection mouse model. On day 3 or 7 post-infection, mRNA was extracted from lungs and processed for real time PCR analysis of (A) IAV and (B) RSV genes. Results represent mean pooled values +/− SEM from three independent experiments, (n = 5–9 mice per group). Asterisks denote significant differences between experimental groups; * p < 0.05; ns = not significant. Comparison among groups was performed using the nonparametric test (2 independent variables: Mann–Whitney).

Figure 4

Co-infection leads to significant atrophy of the spleen and thymus, and an increase in lung weight. On day 3 or 7 post-infection, mice were sacrificed, and lung (A,D), spleen (B,E) and thymus (C,F) were excised and weighed. Results represent mean pooled values +/− SEM from three independent experiments. Asterisks denote significant differences among the control, co-infected and singly-infected groups; (* p < 0.05, ** p < 0.005, *** p < 0.0005); ns = not significant. Comparison among groups was performed using the nonparametric test (two independent variables: Mann–Whitney).

Figure 5

The effects of co-infection on immune-cell populations in the lungs. On day 7 post-infection, mice were sacrificed, and lung immune cells were stained with anti-CD4 (A), anti-CD8 (B), anti-CD19 (C) and anti-CD11B (D) antibodies and analyzed by flow cytometry. Results represent mean pooled values +/− SEM from two independent experiments. Asterisks denote significant differences among the control, co-infected and singly-infected groups (* p < 0.05, ** p < 0.005, *** p < 0.0005); ns = not significant. Comparison among groups was performed using the nonparametric test (two independent variables: Mann–Whitney).

Figure 6

Lung function assessment on day 3 post-infection. Upper panels: Baseline airway reactivity expressed as thoracic resistance (Rrs), thoracic compliance (Crs) and large airway resistance (Rn). Lower panels: Methacholine responsiveness shown as “area under the curve” (AUC) of Rrs, Crs and Rn against methacholine concentration. The values are mean ± SEM. Two separate experiments (n = 6) were performed. Asterisks denote statistically significant differences between experimental groups and the control group (* p < 0.05, ** p  <  0.005, *** p < 0.0005). Two separate experiments (n = 6) were performed; ns = not significant. Comparison among groups was performed using the nonparametric test (two independent variables: Mann–Whitney).