Respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: an experimental murine model

Abstract

Background: Respiratory syncytial virus (RSV) is the leading respiratory viral pathogen in young children worldwide. RSV disease is associated with acute airway obstruction (AO), long-term airway hyperresponsiveness (AHR), and chronic lung inflammation. Using two different mouse strains, this study was designed to determine whether RSV disease patterns are host-dependent. C57BL/6 and BALB/c mice were inoculated with RSV and followed for 77 days. RSV loads were measured by plaque assay and polymerase chain reaction (PCR) in bronchoalveolar lavage (BAL) and whole lung samples; cytokines were measured in BAL samples. Lung inflammation was evaluated with a histopathologic score (HPS), and AO and AHR were determined by plethysmography.

Results: Viral load dynamics, histopathologic score (HPS), cytokine concentrations, AO and long-term AHR were similar in both strains of RSV-infected mice, although RSV-infected C57BL/6 mice developed significantly greater AO compared with RSV-infected BALB/c mice on day 5. PCR detected RSV RNA in BAL samples of RSV infected mice until day 42, and in whole lung samples through day 77. BAL concentrations of cytokines TNF-alpha, IFN-gamma, and chemokines MIG, RANTES and MIP-1alpha were significantly elevated in both strains of RSV-infected mice compared with their respective controls. Viral load measured by PCR significantly correlated with disease severity on days 14 and 21.

Conclusion: RSV-induced acute and chronic airway disease is independent of genetic background.

Figure 1

Effect of RSV on airway obstruction (AO) in two mouse strains. BALB/c (△) and C57BL/6 (●) mice were inoculated with sterile 10% EMEM (control) and were compared with RSV A2 infected BALB/c (□) and C57BL/6 (◆) mice to evaluate differences in airway obstruction (AO), by measuring Penh via whole-body plethysmography. Penh values are presented as means ± SEM. Comparisons were made by t-test when data normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed.

Figure 2

Airway Hyperresponsiveness (AHR) in BALB/c and C57BL/6 Mice. Data presented as Delta Penh values which is the difference between pre-and post methacholine Penh for each group of mice, in sham inoculated () and RSV inoculated () BALB/c mice, and sham inoculated () and RSV inoculated () C57BL/6 mice from days 14 to 77. Values represent the mean SEM from 10–30 mice per group. Data shown are the result of four separate experiments. p < 0.05, comparison by t-test when data normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed.

Figure 3

Comparison of acute and long-term histopathologic scores after RSV inoculation. BALB/c (△) and C57BL/6 (●) mice were inoculated with sterile 10% EMEM (control) and were compared with RSV A2 infected BALB/c (□) and C57BL/6 (◆) mice. Serial formalin fixed lung samples were obtained between day 0 (+2 hours) and day 77 after inoculation. HPS scores are represented as means ± SEM. p < .05, by t-test when data normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed.

Figure 4

RSV induced histopathology. Lung specimens were harvested on days 5 and 77 from groups of C57BL/6 and BALB/c mice inoculated with sterile medium (control) or RSV. Sections from control C57BL/6 and BALB/c mice above (4A and 4B, respectively), show rare, scattered, small lymphocytic infiltrates on day 5 after inoculation with medium, similar to sections of control mice harvested 77 days after (not shown). Acute and chronic inflammatory infiltrates, surrounding airways and vessels are demonstrated in RSV-inoculated mice of both strains, on days 5 and 77 after inoculation (4C to F).

Figure 5

Cytokine and chemokine concentrations in bronchoalveolar lavage (BAL) samples after RSV inoculation. BAL samples were obtained from BALB/c (△) and C57BL/6 (●) mice inoculated with sterile 10% EMEM (control), and RSV A2 infected BALB/c (□) and C57BL/6 (◆) mice, to measure concentrations of pro-inflammatory cytokines (A) IFN-γ and (B)TNF-α; and the chemokines (C) RANTES, (D) MIP-1α, and (E) MIG. Values presented in means ± SEM pg/ml. p < .05, by t-test when data were normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed.

Figure 6

RSV loads in BAL samples measured by the plaque assay method. Groups of 4–16 BALB/c (△) and C57BL/6 (●) mice per group per time point were inoculated intranasally with sterile 10% EMEM (control), and were compared with RSV A2 infected BALB/c (□) and C57BL/6 (◆) mice. Viral load was determined by HEp-2 plaque assay in BAL samples. Data are presented as mean ± SEM Log10 PFU/ml of BAL. p < .05, by t-test when data normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed.

Figure 7

RSV loads in RSV infected BALB/c mice BAL and lung supernatant samples measured by PCR vs. plaque assay. RSV loads measured by PCR in BAL samples (▽) remain positive up to 42 days after inoculation, while viral loads measured by plaque assay (□) become negative on day 7 post-inoculation (upper panel). Viral load measured in lung supernatants by the plaque assay also become undetectable by day 7 after inoculation, whereas RSV loads measured by PCR in lung supernatants remain detectable throughout all the time points evaluated (lower panel). All pair-wise multiple comparisons made by One-Way ANOVA. † p < 0.01 between D1 and D5 and *p < 0.05 comparing D1 with D3 and D5.

Figure 8

Comparison of RSV loads measured by PCR in lung supernatants of BALB/c and C57BL/6 mice. Groups of 2–12 BALB/c (△) and C57BL/6 (●) mice per group per time point were inoculated intranasally with sterile 10% EMEM (control), and were compared with RSV A2 infected Balb/c (□) and C57Bl/6 (◆) mice. Viral load was determined by PCR to detect RSV N gene. Data are presented as mean ± SEM Log10 PFU/ml of BAL. p < .05, by t-test when data normally distributed, or by Mann-Whitney Rank sum test when data were not normally distributed.