Human metapneumovirus induces more severe disease and stronger innate immune response in BALB/c mice as compared with respiratory syncytial virus

Abstract

Background: Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are members of the Pneumovirinae subfamily of Paramyxoviridae and can cause severe respiratory disease, especially in infants and young children. Some differences in the clinical course of these infections have been described, but there are few comparative data on pathogenesis in humans and animal models. In this study, HMPV and RSV were compared for replication, pathogenesis and immune induction in BALB/c mice infected with equivalent inocula of either virus.

Methods: Viral titers in the lungs and in the nasal turbinates of mice were determined by plaque assay. Histopathological changes in the lungs as well as weight loss and levels of airway obstruction were monitored in the infected mice to record the severity of illness. Inflammatory cells recruited to the lungs were characterized by flow cytometry and by differential staining. In the case of natural killer cells, cytotoxic activity was also measured. Cytokine levels in the BAL were determined by cytometric bead array.

Results: RSV replicated to higher titers than HMPV in the lung and in the upper respiratory tract (URT), and virus elimination from the lungs was more rapid in HMPV-infected mice. Clinical illness as determined by airway obstruction, weight loss, and histopathology was significantly more severe after HMPV infection. A comparison of the cellular immune response revealed similar recruitment of T lymphocytes with a predominance of IFN-gamma-producing CD8+ T cells. By contrast, there were obvious differences in the innate immune response. After HMPV infection, more neutrophils could be detected in the airways and there were more activated NK cells than in RSV-infected mice. This correlated with higher levels of IL-6, TNF-alpha and MCP-1.

Conclusion: This study shows important differences in HMPV and RSV pathogenesis and suggests that the pronounced innate immune response observed after HMPV infection might be instrumental in the severe pathology.

Figure 1

Course of HMPV and RSV infection in the BALB/c mice. (A) Kinetics of HMPV replication in the respiratory tract. Animals were infected i.n. with 2 × 10⁵ PFU of HMPV and viral titers were determined in nasal turbinates and lungs at the indicated time points. (B) Comparison of HMPV and RSV titers measured on day 4 and 7 p.i. in nasal turbinates and lungs of mice infected with equivalent doses (2 × 10⁵ PFU) of either virus preparation. * P < 0.05, ** P < 0.01, (n = 5–7). (C) Weight curves of HMPV- or RSV-infected BALB/c mice. Four animals per group were infected i.n. with 2 × 10⁵ PFU of either HMPV (—) or RSV (----) and weight was recorded daily. The experiment was repeated three times with similar results. (D) Airway obstruction following HMPV, RSV or mock infection of BALB/c mice. Airway function was determined by measuring enhanced pause (Penh) via whole-body plethysmography on day 7 p.i.. *** P < 0.001, (n = 4).

Figure 2

Immunohistochemistry of lungs on day 7 after infection with HMPV or RSV. (A) Normal lung tissue from an uninfected animal. (B) Pulmonary section from an RSV-infected mouse showing mild bronchopneumonia with scattered macrophages and neutrophils in alveolar spaces. (C) Severe bronchopneumonia in a mouse inoculated with HMPV. Bronchioli and adjacent alveoli are densely infiltrated by macrophages and neutrophils admixed with fibrin. (D) Immunohistochemical staining for HMPV. Groups of intraalveolar macrophages and pneumocytes expressing HMPV antigens. (A to D): hematoxylin-eosin staining, original magnification × 20; D: immunostaining with anti-HMPV serum, (× 63). Representative sections from groups of 4 mice are shown.

Figure 3

Cellular infiltration of the lungs after HMPV or RSV infection. (A) Total BAL cells in HMPV-, RSV-, and mock-infected mice. (B) Differential count of BAL cells from HMPV- and RSV-infected mice after Giemsa staining. Ly, lymphocytes; Mac, macrophages; Neu, neutrophils. * P < 0.05, ** P < 0.01, ***P < 0.001 (n = 4–10)

Figure 4

Characterization and functional analysis of BAL cells after HMPV or RSV infection. Percentage of (A) CD8+, (B) CD4+, and (C) IFN-γ-producing CD8+ cells of total BAL lymphocytes as determined by FACS analysis of BAL cells from HMPV- or RSV-infected BALB/c mice. * P < 0.05, ** P < 0.01 (n = 6–11). (D) Percentage of NK cells (DX5+/CD3-) of total BAL lymphocytes in the BAL of HMPV- or RSV-infected BALB/C mice (2 × 105 PFU/mouse), and (E) NK cell-mediated cytotoxicity in mice infected with 2 × 105 PFU of HMPV or 2 × 105 and 106 PFU of RSV, respectively. The experiment was repeated three times with similar results. ***P < 0.001 (n = 5). (F) Cytokines in the BAL of HMPV- or RSV-infected mice. BALB/c mice were infected with 2 × 105 PFU of HMPV or RSV. BAL fluid was collected at different time points after infection and TNF-α, IFN-γ, IL-6, MCP-1, and IL-10 were measured by cytometric bead array. Values represent mean +/- SEM. ***P < 0.001 (n = 4).