Naturally occurring swine influenza A virus PB1-F2 phenotypes that contribute to superinfection with Gram-positive respiratory pathogens

Abstract

A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.

Fig 1

C-terminal sequences of PB1-F2 from selected swine flu isolates. Amino acids that have been previously identified and characterized (1) are in red (virulent) and blue (avirulent).

Fig 2

Lung viral titers after secondary challenge. Viral load from groups of mice infected with swine isolates of influenza A virus, followed 5 days later with sublethal doses of bacteria (S. pyogenes or S. aureus) or PBS for controls were determined using MDCK monolayers. All groups contained 5 mice, except PR8-S. pyogenes at 48 h (n = 4 mice), CO77-PBS at 0 h (n = 4 mice), IA85-PBS at 48 h (n = 4 mice), and IA85-S. aureus at 24 h (n = 4 mice). Data are presented as box-and-whisker plots, where the whisker represents the 25th to 75th percentile and standard deviation, and the horizontal line indicates the mean. (A) Viruses from group 1: virulent PB1-F2; (B) viruses from group 2: avirulent PB1-F2; (C) viruses from group 3: truncated PB1-F2. *, P < 0.05 by ANOVA (with Dunn's correction) versus the corresponding PBS group (each time point examined individually). ND indicates not done, as no animals remained in this group at sampling.

Fig 3

Lung bacterial titers after secondary challenge. Bacterial loads were assessed from groups of 5 mice infected with swine influenza A virus isolates followed 5 days later with bacteria (S. pneumoniae, S. aureus, or S. pyogenes). All groups contained 5 mice, except PR8-S. pyogenes at 48 h (n = 4 mice) and IA85-S. pyogenes at 24 h (n = 4 mice). Data are presented as box-and-whisker plots, where the whisker represents the 25th to 75th percentile and standard deviation, while the horizontal line indicates the mean. (A) Viruses from group 1: virulent PB1-F2; (B) viruses from group 2: avirulent PB1-F2; (C) viruses from group 3: truncated PB1-F2. The PBS columns are identical for each pathogen in each panel (A, B, and C) and were included in each panel for ease of comparison. *, P < 0.05 by ANOVA (with Dunn's correction) versus the corresponding PBS group (each time point examined individually); **, P < 0.05 by ANOVA (with Dunn's correction) versus corresponding TX07 group; †, P < 0.05 by ANOVA (with Dunn's correction) versus corresponding TX98 group. ND indicates not done, as no animals remained in this group at sampling.

Fig 4

Survival after secondary challenge. Mice were infected intranasally with 0.25 LD₅₀ of influenza virus and followed 5 days later with a sublethal dose of bacteria (S. pneumoniae, S. aureus, or S. pyogenes) and monitored for survival for 9 days postsecondary challenge. All groups contained 5 mice, except PR8-S. pneumoniae (n = 10 mice). (A) Mice that received PBS at day 0 followed by the individual bacterial species at day 5; (B) viruses from group 1: virulent PB1-F2; (C) viruses from group 2: avirulent PB1-F2; (D) viruses from group 3: truncated PB1-F2. *, P < 0.05 by log rank test on Kaplan-Meier data versus S. aureus group; **, P < 0.05 by log rank test on Kaplan-Meier data versus both groups.

Fig 5

Contribution of the number of inflammatory PB1-F2 amino acids toward survival after secondary bacterial infection. Data presented in Fig. 4 are grouped based on survival after inoculation with swine influenza virus isolates that express either 0, 2, 3, or 4 provirulence amino acids, regardless of the secondary bacterial species delivered (S. pneumoniae, S. aureus, or S. pyogenes).