Infection with human metapneumovirus predisposes mice to severe pneumococcal pneumonia

Abstract

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes respiratory tract infections. Prior clinical studies have highlighted the importance of respiratory viruses, such as influenza virus, in facilitating secondary bacterial infections and increasing host immunopathology. The objective of the present work was to evaluate the effects of initial viral infection with hMPV or influenza A virus followed by Streptococcus pneumoniae superinfection 5 days later in a murine model. Both groups of superinfected mice demonstrated significant weight loss (mean of 15%) and higher levels of airway obstruction (mean enhanced pause value of 2.7) compared to those of mice infected with hMPV, influenza virus, or pneumococcus alone. Bacterial counts increased from 5 x 10(2) CFU/lung in mice infected with pneumococcus only to 10(7) and 10(9) CFU/lung in mice with prior infections with hMPV and influenza A virus, respectively. A more pronounced interstitial and alveolar inflammation correlated with higher levels of inflammatory cytokines and chemokines such as interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-12, monocyte chemotactic protein 1, macrophage inflammatory protein 1alpha, KC, and granulocyte colony-stimulating factor, as well as greater expression of Toll-like receptor 2 (TLR2), TLR6, TLR7, and TLR13 in the lungs of superinfected animals compared to results for single infections, with similar immunological effects seen in both coinfection models. Prior infection with either hMPV or influenza A virus predisposes mice to severe pneumococcus infection.

FIG. 1.

Weight loss in groups of mice with viral, bacterial, or dual infection. Groups include mice with hMPV-only infection (⧫), hMPV-pneumococcus coinfection (▪), influenza A virus-only (Flu) infection (▴), influenza A virus-pneumococcus coinfection (•), pneumococcus-only infection (□), and sham (PBS) infection (○). *, P ≤ 0.05 for the comparison between single infections and coinfections (unpaired t test).

FIG. 2.

Airway obstruction in groups of mice with viral, bacterial, or dual infection. Six mice/group were evaluated at different time points (days 6, 7, and 8 after virus infection, corresponding to 24, 48, and 72 h following bacterial superinfection, respectively) after sham infection, single infections, and virus-bacterium coinfections. Flu, influenza virus. Airway obstruction (reported as P_enh values) was determined using whole-body unrestrained plethysmography. P ≤ 0.05 for the comparison to results for sham-infected mice (#) and to virus-only infections (*) (unpaired t test).

FIG. 3.

Viral and bacterial loads in groups of mice with viral, bacterial, or dual infection. Six mice per group were infected with either hMPV, influenza virus (Flu), or PBS (sham), challenged 5 days later with pneumococcus or PBS, and then sacrificed for quantitative viral (A) or bacterial (B) pulmonary titers on days 6, 7, and 8 after virus infection (corresponding to 24, 48, and 72 h following bacterial superinfection, respectively). *, P ≤ 0.05 for the comparison between single infections and coinfections (Mann-Whitney test).

FIG. 4.

Lung histopathological evaluation of mice with viral, bacterial, or dual infection. Six mice per group were euthanized at different time points after infection, and their lungs were removed and fixed with 3.7% buffered formalin, cut, and stained with hematoxylin and eosin. (A) Representative sections (magnification, ×10) are shown for days 6, 7, and 8 after virus infection (corresponding to 24, 48, and 72 h after bacterial superinfection, respectively) for mice with sham, single, or dual infection. Flu, influenza virus. (B) Corresponding histopathological scores also are reported for the same groups of mice. The degree of lung inflammation (mean histopathological score) was evaluated for peribronchial, perivascular, interstitial, and alveolar areas.

FIG. 5.

Cytokine and chemokine levels in the lungs of mice with single-virus-only or dual viral-bacterial infections. Six mice per group were euthanized at different time points after infection (on days 6, 7, and 8 after virus infection, corresponding to 24, 48, and 72 h following bacterial superinfection, respectively) for the evaluation of 23 pulmonary cytokines and chemokines by the use of a Luminex 100 instrument system and the Bio-Plex mouse cytokine 23-plex panel. Values are expressed as changes (n-fold) in pulmonary levels for mice with coinfections compared to those of mice with hMPV-only infection (A) or influenza A virus-only (Flu) infection (B). *, P ≤ 0.05 for the comparison between single infections and coinfections (unpaired t test).

FIG. 6.

TLR transcript levels in the lungs of mice with single-virus-only or dual viral-bacterial infections. RNA was isolated from the lung tissues of infected animals on days 6, 7, and 8 after virus infections (corresponding to 24, 48, and 72 h following bacterial superinfection, respectively) for TLR quantification using real-time PCR. Values represent changes (n-fold) in pulmonary expression levels for mice with coinfections compared to those of mice with single infection with hMPV (A) or with influenza A virus (Flu) (B). *, P ≤ 0.05 for the comparison between single infections and coinfections (unpaired t test).