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
ABSTRACT
A combination of viral, bacterial, and host factors contribute to the
severity and overall mortality associated with influenza virus:bacteria
super-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.
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 flu
isolates. 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).