Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia

Abstract

Secondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic.

Figure 1

Secondary bacterial pneumonia following influenza. (A) Groups of 4–6 mice infected with either wt influenza virus PR8 (WT) or an isogenic mut virus that does not express PB1-F2 (PB1-F2-) were euthanized 1, 2, 3, 5, and 7 days after infection for determination of viral lung load. Groups of 6 mice infected with either WT or PB1-F2- were challenged with pneumococcus on d7 post-infection. (B) weight loss and (C) survival are plotted until 7 days after secondary challenge. Error bars represent standard deviation and an asterisk (*) denotes a significant difference (p < 0.05) compared to the group infected with PB1-F2- virus. (D) Pictures of anesthetized mice taken 36–48 hours after bacterial infection with luciferase-expressing pneumococci show bioluminescence indicative of pneumonia. The scale on the right indicates the number of relative light units per shaded pixel.

Figure 2

Titers from mice with pneumonia. Groups of mice were infected with either PR8 wt (WT) or an isogenic mut strain of PR8 unable to express PB1-F2 (PB1-F2-) and challenged 7 days later with pneumococcus. Four mice per group were assayed when they developed early pneumonia (> 10,000 relative light units (RLU)/min from the thorax) and late pneumonia (24 h of visual bioluminescence and > 1,000,000 RLU/min from the thorax) for A) mean flux of RLU/min and B) bacterial lung titers (CFU/ml lung homogenate). There were no significant differences between mice infected with PR8 wt and PR8 mut at any time point for any measurement (p > 0.1).

Figure 3

Cell counts from broncho-alveolar fluid (BALF) of secondarily infected mice with pneumonia. Mice were infected with PR8 wt (WT), an isogenic mut strain of PR8 unable to express PB1-F2 (PB1-F2-), or PBS, followed 7 days later by pneumococcus. Selected mice that developed early pneumonia (6–10 per group) were assayed for number of (A) neutrophils, (B) macrophages, (C) T-cells, and (D) B-cells from BALF samples. The 25^th – 75^th percentiles are represented by the shaded box-plots with the horizontal bar indicating the mean value. Error bars indicate the standard deviation of the measurements. A double asterisk (**) indicates a significant difference (p < 0.05) compared to all other groups, an asterisk (*) compared to the mut PR8 group. In a separate experiment, lungs were taken from mice 3 days after infection with either PR8 wt (E, F) or PR8 mut (G, H). Pictured are representative sections at either 10X (E, G) or 40X (F, H) magnification. Necrosis of the airway epithelium, manifest as killed cells with disruptions in the epithelial layer and deposition of debris, and inflammatory infiltration with neutrophils are more prominent in the mice infected with the wt virus.

Figure 4

Peptide from the C-terminal portion of PB1-F2 elicits an inflammatory response and primes for secondary bacterial pneumonia. Groups of 5 mice were given peptides or PBS as a control intranasally on day -1 then challenged with 100 CFU of bacteria 24 hours later. (A) Weight loss and (B) mortality are compared. The peptide from the C-terminal region of the PB1-F2 protein caused more weight loss prior to and after bacterial challenge (* p < 0.05 by Student’s t-test) and less survival (* p < 0.01 by log-rank test on the Kaplan-Meier survival data) than did a peptide from the N-terminal region or PBS. Groups of 5 mice were assayed for number of (C) neutrophils and (D) macrophages from broncho-alveolar lavage fluid samples 3 days after exposure to peptide alone. The 25^th – 75^th percentiles are represented by the shaded box-plots with the horizontal bar indicating the mean value. Error bars indicate the standard deviation of the measurements. An asterisk (*) indicates a significant difference (p < 0.05) by ANOVA compared to both controls at that timepoint.

Figure 5

Growth and plaque size of a virus expressing the 1918 PB1-F2. An otherwise isogenic virus engineered to express the PB1-F2 protein from the 1918 pandemic strain (1918) was compared to the wt PR8 strain (WT). (A) HA titer is compared for a virus expressing the 1918 PB1-F2 to wt PR8. Error bars indicate standard deviation of the measurements and an asterisk (*) indicates a significant difference at that timepoint by the paired Student’s t-test (p < 0.05). (B) Plaque size was measured for a virus expressing the 1918 PB1-F2 (1918) compared to wt PR8 (WT) and a virus that does not express the PB1-F2 protein (PB1-F2-). An asterisk (*) indicates a significant difference (P < 0.00001) compared to both other groups.

Figure 6

Virulence of and inflammatory response to a virus expressing the 1918 PB1-F2. An otherwise isogenic virus engineered to express the PB1-F2 protein from the 1918 pandemic strain (1918) was compared to the wt PR8 strain (WT). (A) Survival of groups of 4 mice infected with 500 or 1000 TCID₅₀ of wt PR8 or PR8-PB1-F21918. (B) Viral titers from lung homogenates taken from groups of 6–12 mice at multiple timepoints after infection with wt PR8 or PR8-PB1-F21918. Error bars indicate standard deviation of the measurements and an asterisk (*) indicates a significant difference at that timepoint by ANOVA compared to the wt PR8. In a separate experiment, mice were infected with PR8 wt or PR8-PB1-F21918 and were assayed for number of (C) neutrophils, (D) macrophages, (E) T-cells, and (F) B-cells from broncho-alveolar lavage fluid samples 1, 3, and 7 days after infection. The 25^th – 75^th percentiles are represented by the shaded box-plots with the horizontal bar indicating the mean value. Error bars indicate the standard deviation of the measurements. An asterisk (*) indicates a significant difference (p < 0.05) by ANOVA compared to wt PR8 at that timepoint.

Figure 7

1918 PB1-F2 and secondary bacterial pneumonia. Groups of 10 mice were infected with 50 TCID₅₀ of wt PR8 or PR8-PB1-F21918 followed 7 days later by bacterial challenge. (A) Survival following bacterial challenge is plotted, an asterisk (*) indicates a significant difference in survival by the log-rank test on the Kaplan-Meier data. (B) Bacterial lung loads from 5 mice per group and timepoint were determined 24, 48, and 72 hours after bacterial challenge. The 25^th – 75^th percentiles are represented by the shaded box-plots with the horizontal bar indicating the mean value. Error bars indicate the standard deviation of the measurements. An asterisk (*) indicates a significant difference (p < 0.05) by ANOVA compared to wt PR8 at that timepoint. Lungs were taken from 5 mice per group with evidence of pneumonia by bioluminescence 3 days after infection with either wt PR8 (data not shown) or PB8-PB1-F21918 (C, D). Pictured are representative sections at either 10X (C) or 40X (D) magnification. Massive, lobar, coagulative necrosis with pleuritis and leukocytic infiltration are evident.

Figure 8

Cytokines and chemokines in secondary bacterial pneumonia. Groups of 10 mice were infected with wt PR8 or PR8-PB1-F21918 followed 7 days later by bacterial challenge. Levels of (A) TNF-α, (B) IL-1α, (C) IL-6, (D), IL-10, (KC), and (F) MIP-1α from lung homogenates taken from mice 3 and 7 days after viral infection (prior to bacterial challenge) and 72 hours after bacterial challenge (10 days after viral infection) are presented. The 25^th – 75^th percentiles are represented by the shaded box-plots with the horizontal bar indicating the mean value. Error bars indicate the standard deviation of the measurements. An asterisk (*) indicates a significant difference by ANOVA compared to the wt PR8 group (p < 0.05).