Contribution of naturally occurring swine influenza A virus PB1-F2 phenotypes toward secondary complications with Gram-positive respiratory pathogens

Jenni N. Weeks1,Heather R. Hurtig2 Amy R. Iverson1,Margaret J. Schuneman2, Richard J. Webby1, Jonathan A. McCullers1, Victor C. Huber2

1Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN2Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069

ACKNOWLEDGMENTSAcknowledgments: The authors acknowledge Michael S. Chaussee for providing the MGAS315 strain of Streptococcus pyogenes bacteria that were used in this study. Funding was provided by the University of South Dakota (USD) Foundation, the Division of Basic Biomedical Sciences, The U. Discover Program (MS), the SSOM Faculty Research Program (VCH), the USD Inside TRACK program (VCH), and the American Lebanese Syrian Associated Charities (ALSAC).

ABSTRACT

A combination of viral, bacterial, and host factors contribute to the severity and overall mortality associated with influenza virus:bacteriasuper-infections. To date, the virulence associated with the recently-identified influenza protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae super-infection. We hypothesized that virally-expressed proteins, like PB1-F2, dictate the severity of secondary bacterial infections, which can vary based on the bacterial species to which the host is exposed. To test this hypothesis, we selected naturally occurring viruses expressing variants in PB1-F2 and evaluated outcomes from super-infection 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 are molecular signatures of PB1-F2 virulence for swine influenza viruses in the setting of bacterial super-infection, and that truncated PB1-F2 proteins can preferentially increase mortality when associated with S. pyogenes super-infection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of super-infections with specific Gram-positive respiratory pathogens.

Figure 2. Lung viral titers after secondary challenge.Viral load from groups of 5 mice infected with swine isolates of influenza A, followed 5 days later with sublethal doses of bacteria (S. pyogenes or S. aureus) or PBS for control were determined using MDCK monolayers. 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) vs. corresponding PBS group (each timepoint examined individually).

Table 1. Characteristics of PB1-F2-expressing viruses used in this study �

Group 1: Virulent PB1-F2

Virus Name Abbreviation 50% Tissue Culture Infectious Dose

(TCID50)1

50% Mouse Lethal Dose (MLD50)2

A/Puerto Rico/8/34-H1N1 PR8 9.2 2.5

A/swine/Germany/2/81-H1N1 GE81 9.0 5.5

A/swine/Texas/042995-27/2007-H1N2 TX07 7.1 4.3

A/swine/Colorado/1/77-H3N2 CO77 7.5 6.0

Group 2: Avirulent PB1-F2

Virus Name Abbreviation 50% Tissue Culture Infectious Dose

(TCID50)1

50% Mouse Lethal Dose (MLD50)2

A/swine/Texas/4199-2/98-H3N2 TX98 6.67 4.8

A/swine/Wisconsin/194/80-H3N2 WI80 6.7 4.2

Group 3: truncated PB1-F2

Virus Name Abbreviation 50% Tissue Culture Infectious Dose

(TCID50)1

50% Mouse Lethal Dose (MLD50)2

A/swine/North Carolina/057225/2008-H1N2

NC08 7.4 6.0

A/swine/Iowa/1/85-H1N1 IA85 5.5 3.0

1Values are reported as log10 TCID50/mL. 2Values are reported as log10 TCID50/0.1 mL.

Figure 1. C-terminal sequences of PB1-F2 from selected swine flu isolates.Amino acids that have been previously identified and characterized (5) are in red (virulent) and blue (avirulent).

Figure 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). 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 column is identical for each pathogen in each panel (A, B, and C), and was included in each panel for ease of comparison. *p<0.05 by ANOVA (with Dunn’s correction) vs. corresponding PBS group (each timepoint examined individually). **p<0.05 by ANOVA (with Dunn’s correction) vs. corresponding TX07 group. †p<0.05 by ANOVA (with Dunn’s correction) vs. corresponding TX98 group..

Figure 4. Survival after secondary challenge.Mice were infected intranasally with 0.25 LD50 of influenza virus and followed 5 days later with a sub-lethal dose of bacteria (S. pnuemoniae, S. aureus, or S. pyogenes) and monitored for survival for 9 days post-secondary challenge. 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 vs. S. aureus group. **p<0.05 by log-rank test on Kaplan-Meier data vs. both groups.

Figure 5. Contribution of the number of inflammatory PB1-F2 amino acids toward survival after secondary bacterial infection.Data presented in Figure 4 are grouped based on survival after inoculation with swine influenza virus isolates that express either 0, 2, 3, or 4 pro-virulence amino acids, regardless of the secondary bacterial species delivered (S. pnuemoniae, S. aureus, or S. pyogenes).