1. 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, TN 2Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069 ABSTRACTA combination of viral, bacterial, and host factors contribute to theseverity 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 modelsof primary influenza virus infection, with only limited assessment inmodels of Streptococcus pneumoniae super-infection. We hypothesizedthat virally-expressed proteins, like PB1-F2, dictate the severity ofsecondary bacterial infections, which can vary based on the bacterialspecies to which the host is exposed. To test this hypothesis, we selectednaturally occurring viruses expressing variants in PB1-F2 and evaluatedoutcomes from super-infection with three distinct Gram-positiverespiratory pathogens: Streptococcus pneumoniae, Staphylococcusaureus, and Streptococcus pyogenes. Our results demonstrate that theamino acid residues 62L, 66S, 75R, 79R, and 82L are molecular signaturesof PB1-F2 virulence for swine influenza viruses in the setting of bacterialsuper-infection, and that truncated PB1-F2 proteins can preferentiallyincrease mortality when associated with S. pyogenes super-infection.These findings support efforts to increase influenza virus surveillance toconsider viral genotypes that could be used to predict increased severityof super-infections with specific Gram-positive respiratory pathogens. Table 1. Characteristics of PB1-F2-expressing viruses used in this study Group 1: Virulent PB1-F2 Virus Name Abbreviation 50% Tissue Culture 50% Mouse Lethal Infectious Dose Dose (MLD50)2 (TCID50)1 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 Figure 2. Lung viral titers after secondary challenge. Figure 3. Lung bacterial titers after secondary challenge. Figure 4. Survival after secondary challenge. Virus Name Abbreviation 50% Tissue Culture 50% Mouse Lethal Infectious Dose Dose (MLD50)2 Viral load from groups of 5 mice infected with swine isolates of Bacterial loads were assessed from groups of 5 mice infected with swine influenza A Mice were infected intranasally with 0.25 LD50 of (TCID50)1 influenza A, followed 5 days later with sublethal doses of bacteria (S. virus isolates followed 5 days later with bacteria (S. pneumoniae, S. aureus, or S. influenza virus and followed 5 days later with a A/swine/Texas/4199-2/98-H3N2 TX98 6.67 4.8 pyogenes or S. aureus) or PBS for control were determined using pyogenes). A) Viruses from Group 1: virulent PB1-F2. B) Viruses from Group 2: sub-lethal dose of bacteria (S. pnuemoniae, S. MDCK monolayers. A) Viruses from Group 1: virulent PB1-F2. B) avirulent PB1-F2. C) Viruses from Group 3: truncated PB1-F2. The PBS column is aureus, or S. pyogenes) and monitored for A/swine/Wisconsin/194/80-H3N2 WI80 6.7 4.2 Viruses from Group 2: avirulent PB1-F2. C) Viruses from Group 3: identical for each pathogen in each panel (A, B, and C), and was included in each survival for 9 days post-secondary challenge. A) Group 3: truncated PB1-F2 truncated PB1-F2. *p<0.05 by ANOVA (with Dunn’s correction) vs. panel for ease of comparison. *p<0.05 by ANOVA (with Dunn’s correction) vs. Mice that received PBS at day 0 followed by the Virus Name Abbreviation 50% Tissue Culture 50% Mouse Lethal corresponding PBS group (each timepoint examined individually). corresponding PBS group (each timepoint examined individually). **p<0.05 by individual bacterial species at day 5. B) Viruses Infectious Dose Dose (MLD50)2 ANOVA (with Dunn’s correction) vs. corresponding TX07 group. †p<0.05 by ANOVA from Group 1: virulent PB1-F2. C) Viruses from (TCID50)1 Group 2: avirulent PB1-F2. D) Viruses from Group (with Dunn’s correction) vs. corresponding TX98 group. A/swine/North NC08 7.4 6.0 . 3: truncated PB1-F2. *p<0.05 by log-rank test on Carolina/057225/2008-H1N2 Kaplan-Meier data vs. S. aureus group. **p<0.05 A/swine/Iowa/1/85-H1N1 IA85 5.5 3.0 by log-rank test on Kaplan-Meier data vs. both groups. 1 Values are reported as log10 TCID50/mL. 2 Values are reported as log10 TCID50/0.1 mL. ACKNOWLEDGMENTS Acknowledgments: 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).Figure 1. C-terminal sequences of PB1-F2 from selected swine fluisolates. Figure 5. Contribution of the number of inflammatory PB1-F2 amino acids toward survival after secondary bacterial infection.Amino acids that have been previously identified and characterized (5)are in red (virulent) and blue (avirulent). 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).