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Figure 1.

Differences between attenuated mengovirus and HRV in the kinetics of lung viral titers.

Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0 [A (n = 7 mice per group), B (n = 6 mice per group)], or 5×106 PFU of HRV-A01a (C; n = 4 mice per group). Lungs were harvested at the indicated times, and viral titers in lung homogenates were determined by plaque assays. Data are the total amount of virus present in the lung homogenates (virus concentrations were multiplied by lung homogenate volumes). No virus was detected in lungs from vehicle-inoculated mice. Data are presented as box plots. For one HRV-A01a-inoculated mouse at 3 h postinoculation, a value of 1 PFU was assigned for graphing purposes because virus was undetectable. ND, not detectable.

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Figure 2.

Induction of type I and III IFN expression in response to inhalation of attenuated mengovirus.

Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Levels of (A) IFN-α, (B) IFN-β, and (C) IFN-λ protein in BAL fluid on days 1 and 2 postinoculation were determined by ELISA. Data are the total amount of IFN recovered (ELISA values were multiplied by the BAL fluid volume). IFN-λ protein levels below the limit of detection (dotted line) were assigned a value of 5 pg for graphing purposes. Data are presented as box plots. Veh, vehicle; ND, not detectable. * P≤0.01 (vMC0 vs. vehicle or UV-inactivated vMC0).

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Figure 3.

Increased pulmonary expression of viral RNA sensors in response to inhalation of attenuated mengovirus.

(A–C) Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). (D–F) Mice were treated with anti-neutrophil mAb or control mAb before receiving intranasal inoculations of 106 PFU of vMC0 or vehicle (n = 5–6 mice per group). Levels of (A, D) TLR3, (B, E) TLR7, and (C, F) NOD2 mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. * P<0.01 (vMC0 vs. vehicle or UV-inactivated vMC0), ** P<0.05 (anti-neutrophil mAb/Vehicle vs. control mAb/Vehicle), † P = 0.01 (anti-neutrophil mAb/vMC0 vs. control mAb/vMC0).

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Figure 4.

Body weight reduction in response to inhalation of attenuated mengovirus.

Percent change in body weight on days 1 (n = 26–30 mice per group), 2 (n = 18–21 mice per group), 3 (n = 14–16 mice per group), and 5 (n = 9 mice per group) after intranasal inoculation with 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle. Data are presented as box plots. * P<0.001 (vMC0 vs. vehicle); ** P<0.0001, *** P<0.01 (vMC0 vs. vehicle or UV-inactivated vMC0); † P<0.01 (vMC0 vs. UV-inactivated vMC0).

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Figure 5.

Recruitment of inflammatory cells to the lungs in response to inhalation of attenuated mengovirus.

Numbers of (A) total cells, (B) neutrophils, (C) lymphocytes, and (D) macrophages in the BAL fluid harvested at the indicated times from the lungs of mice inoculated with 106 PFU of vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Data are presented as box plots. † P<0.05, * P<0.01, ** P<0.001, *** P<0.0001 (vMC0 vs. vehicle or UV-inactivated vMC0).

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Figure 6.

Recruitment of inflammatory cell infiltrates to the lungs in response to inhalation of attenuated mengovirus.

Giemsa-stained sections of the lungs from mice intransasally inoculated with (A) vehicle, (B) vMC0 (106 PFU) or (C) an equivalent amount of UV-inactivated vMC0. Lungs were harvested on day 2 postinoculation. Magnification, 20X.

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Figure 7.

Neutrophilic inflammation in the lungs in response to inhalation of attenuated mengovirus.

Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle. (A) Lung-associated MPO levels on day 1 postinoculation. MPO levels in lung tissue homogenates from mice inoculated with vehicle or vMC0 were determined by ELISA and normalized to total protein levels (n = 4 mice per group). (B) MPO release into airway fluids. BAL fluid was harvested on the indicated days, and MPO levels were determined by ELISA (n = 6 mice per group). Data are the total amount of MPO recovered (ELISA values were multiplied by the BAL fluid volume). MPO levels below the limit of detection were assigned a value of 1 ng for graphing purposes. (C) CXCR2 and (D) IL-17A expression in the lungs on day 1 postinoculation. Levels of CXCR2 and IL-17A mRNA were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels (n = 6 mice per group). Data are presented as box plots. * P<0.05 (vMC0 vs. vehicle). ** P<0.01 (vMC0 vs. vehicle or UV-inactivated vMC0).

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Figure 8.

Increased pulmonary expression of CXCR2 ligands in response to inhalation of attenuated mengovirus.

Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Levels of (A) CXCL1 (KC), (B) CXCL2 (MIP-2), and (C) CXCL5 (LIX) mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. Levels of (D) CXCL1, (E) CXCL2, and (F) CXCL5 protein in BAL fluid on day 2 postinoculation were determined by ELISA. Data are the total amount of chemokine recovered (ELISA values were multiplied by the BAL fluid volume). CXCL2 protein levels below the limit of detection (dotted line) were assigned a value of 4 pg for graphing purposes. Data are presented as box plots. * P≤0.01, ** P<0.05 (vMC0 vs. vehicle or UV-inactivated vMC0).

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Figure 9.

Increased pulmonary expression of CXCL10 and CCL2 in response to inhalation of attenuated mengovirus.

Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Levels of (A) CXCL10 (IP-10) and (B) CCL2 (MCP-1) mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. (C) CCL2 protein levels in BAL fluid on day 2 postinoculation were determined by ELISA. Data are the total amount of CCL2 recovered (ELISA values were multiplied by the BAL fluid volume). ND, not detectable. Data are presented as box plots. * P<0.01 (vMC0 vs. vehicle or UV-inactivated vMC0).

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Figure 10.

Mucin expression in the lungs in response to inhalation of attenuated mengovirus.

Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, or vehicle (n = 5 mice per group). Levels of (A) MUC5B and (B) MUC5AC mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. * P<0.01 (vMC0 vs. vehicle).

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Figure 11.

Lung edema in response to inhalation of attenuated mengovirus.

Wet:dry lung weight ratios were measured for lungs harvested on day 2 after intranasal inoculation with 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 9 mice per group). Data are presented as box plots; whiskers indicate the 10th and 90th percentiles. * P<0.01 (vMC0 vs. vehicle or UV-inactivated vMC0).

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Figure 12.

Effect of inhalation of attenuated mengovirus on pulmonary physiology.

Mice received intranasal inoculations of vehicle, 106 PFU of attenuated mengovirus, vMC0 (n = 6–7 mice per group), or an equivalent amount of UV-inactivated vMC0 (n = 4 mice), and on day 2 postinoculation, pulmonary physiology measurements were obtained after exposure to aerosols of normal saline followed by escalating concentrations of methacholine. Values for respiratory system resistance (Rrs) are presented as the group means ± the standard error. There were no significant differences among the groups.

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