Non-Avian Animal Reservoirs Present a Source of Influenza A PB1-F2 Proteins with Novel Virulence-Enhancing Markers

<div><p>PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.</p></div

Genetic markers of virulence in the PB1-F2 proteins from influenza viruses of domestic mammals.

<p>The IAV isolates of swine, horses, and dogs were categorized into the lineage (outer ring), PB1-F2 length (middle ring), and combination of inflammatory (indicated as “I”) and cytotoxic (indicated as “C”) residues (inner ring). Within the outer ring, the percentage of each grouping is shown relative to the total number of isolates. Within the middle and inner rings, the percentage of each grouping is shown relative to the preceding grouping. The numbers of inflammatory (indicated as “I1” through “I4”) and cytotoxic (indicated as “C1” and “C2”) residues were determined for PB1-F2 proteins with length of 62 or more amino acids (e.g. capable of encoding either inflammatory or cytotoxic residues).</p

Phylogeny overview of swine influenza A PB1 gene.

<p>Phylogenetic analysis for 789 H1N1, 529 H3N2, and 329 H1N2 SIVs are shown. 1, 2, 3, and 4 inflammatory residues are indicated in blue, green, fuchsia, and red, respectively. The presence of a cytotoxic residue is indicated by an asterisk. Cyan indicates PB1-F2 proteins truncated before residue 62. More detail, including strain names and bootstrap values, is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111603#pone.0111603.s001" target="_blank">Figure S1</a>.</p

Phylogeny overview of equine and canine influenza A PB1 genes.

<p>Maximum likelihood phylogenetic trees for 96 equine and 63 canine H3N8, 11 equine H7N7, and 19 canine H3N2 viruses were generated. 1, 2, 3, and 4 inflammatory residues are indicated in blue, green, fuchsia, and red, respectively. The presence of one or two cytotoxic residues is indicated by one or two asterisks, respectively. Cyan indicates PB1-F2 protein truncated before residue 62. More detail, including strain names and bootstrap values, is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111603#pone.0111603.s002" target="_blank">Figure S2</a>.</p

Glycosylation Analysis of Engineered H3N2 Influenza A Virus Hemagglutinins with Sequentially Added Historically Relevant Glycosylation Sites

The influenza virus surface glycoprotein hemagglutinin (HA) is the major target of host neutralizing antibodies. The oligosaccharides of HA can contribute to HA’s antigenic characteristics. After a leap to humans from a zoonotic host, influenza can gain <i>N</i>-glycosylation sequons over time as part of its fitness strategy. This glycosylation expansion has not been studied at the structural level. Here we examine HA <i>N</i>-glycosylation of H3N2 virus strains that we have engineered to closely mimic glycosylation sites gained between 1968 through 2002 starting with pandemic A/Hong Kong/1/68 (H3N2: HK68). HAs studied include HK68 and engineered forms with 1, 2, and 4 added sites. We have used: nano-LC–MS^E for glycopeptide composition, sequence and site occupancy analysis, and MALDI-TOF MS permethylation profiling for characterization of released glycans. Our study reveals that 1) the majority of <i>N</i>-sequons are occupied at ≥90%, 2) the class and complexity of the glycans varies by region over the landscape of the proteins, 3) Asn 165 and Asn 246, which are associated with interactions between HA and SP-D lung collectin, are exclusively high mannose type. Based on this study and previous reports we provide structural insight as to how the immune system responses may differ depending on HA glycosylation

Kinetics of Coinfection with Influenza A Virus and <em>Streptococcus pneumoniae</em>

<div><p>Secondary bacterial infections are a leading cause of illness and death during epidemic and pandemic influenza. Experimental studies suggest a lethal synergism between influenza and certain bacteria, particularly <i>Streptococcus pneumoniae</i>, but the precise processes involved are unclear. To address the mechanisms and determine the influences of pathogen dose and strain on disease, we infected groups of mice with either the H1N1 subtype influenza A virus A/Puerto Rico/8/34 (PR8) or a version expressing the 1918 PB1-F2 protein (PR8-PB1-F2(1918)), followed seven days later with one of two <i>S. pneumoniae</i> strains, type 2 D39 or type 3 A66.1. We determined that, following bacterial infection, viral titers initially rebound and then decline slowly. Bacterial titers rapidly rise to high levels and remain elevated. We used a kinetic model to explore the coupled interactions and study the dominant controlling mechanisms. We hypothesize that viral titers rebound in the presence of bacteria due to enhanced viral release from infected cells, and that bacterial titers increase due to alveolar macrophage impairment. Dynamics are affected by initial bacterial dose but not by the expression of the influenza 1918 PB1-F2 protein. Our model provides a framework to investigate pathogen interaction during coinfections and to uncover dynamical differences based on inoculum size and strain.</p> </div

Summary of the coinfection model hypotheses, results and possible experiments to confirm each hypothesis.

<p>Summary of the coinfection model hypotheses, results and possible experiments to confirm each hypothesis.</p

Parameter ensembles from bootstrap fits of the viral kinetic model.

<p>Plots of the parameters, in the form of two parameter projections of each fit, and the constraints (bottom left) from bootstrap fits of the viral kinetic model (Equations (4)–(7)) to lung titers from mice infected with PR8 (red) or PR8-PB1-F2(1918) (blue).</p

Maximum likelihood estimates of parameter values for influenza infection with PR8 and PR8-PB1-F2(1918).

<p>For each virus strain, PR8 and PR8-PB1-F2(1918), the MLE initial viral titer (), infection rate constant (), death rate of productively infected cells (), viral release rate per infected cell (), and viral clearance rate (). Initial number of target cells () is fixed at , and the transition rate for infected cells to produce virus () is fixed at .</p

Coinfection model fit to lung titers of mice coinfected with PR8 and 1000 CFU D39.

<p>Fit of the coinfection model (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003238#ppat.1003238.e183" target="_blank">Equations (6)</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003238#ppat.1003238.e187" target="_blank">(10)</a>) to viral (panel A) and bacterial (panel B) lung titers from individual mice infected with PR8 virus followed 7 days later by 1000 CFU <i>S. pneumoniae</i> strain D39. Parameters for the model curves are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003238#ppat-1003238-t001" target="_blank">Tables 1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003238#ppat-1003238-t002" target="_blank">2</a>.</p