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

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

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

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

Experimental protocol.

<p>Male Fitch ferrets (Mustela putorius furo), approximately 6 months of age were used in this study. Groups of 8 ferrets were infected intranasally with 1 x 10⁶ pfu of A/Perth/16/09, or groups of 4 to 6 ferrets were mock infected. Baseline weights and temperatures were obtained for the three consecutive days prior to challenge and on day 0 (the day of challenge). Following challenge, ferrets were monitored for change in body weight and temperature as well as clinical signs of illness on a daily basis for 5 days. Blood samples were collected for Flow Cytometry (days 0–5) and serum collected (days 0, 2 and 5) to asses antibody responses (HI assays). All ferrets were euthanized on day 2 (4 Perth/16-infected ferrets) or day 5 post-challenge (4 Perth/16-infected and all mock-infected ferrets). Viral titers were determined from nasal washes (days 0–5) nasal turbinates (days 2 and 5), BALF (days 2 and 5) and trachea (upper and lower regions; days 2 and 5). Leukocyte purification was performed from peripheral blood (days 0–5), from lymph nodes (days 2 and 5) and from BALF (days 2 and 5).</p

Lymph node leukocyte subsets following infection.

<p>For screening of immune cell migration in response to influenza infection, MRLN, MdLN and MsLN were collected. Purified cell subsets from MRLN (A-B), MdLN (C) and MsLN (D) were stained and analyzed by flow cytometry. In MRLN, percentage (A) and absolute number (B) of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured. In MdLN and MsLN, only percentages (C-D) of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured. For mock infected animals (M), LNs were screened at day 5 post-challenge and for Perth/16 infected animals at days 2 and 5 post-challenge. A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

BALF and Spleen leukocyte subsets following infection.

<p>For screening of immune cell migration in response to influenza infection, BALF and Spleen were collected. Purified cell subsets from BALF (A-B) and Spleen (C-D) were stained and analyzed by flow cytometry. Percentages (A, C) and absolute number (B, D) of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured. For mock infected animals (M), BALF and spleen were screened at day 5 post-challenge and for Perth/16 infected animals, at days 2 and 5 post-challenge. A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Clinical responses to infection.

<p>To measure morbidity the temperature (A) and body weight (B) readings of infected and control ferrets were recorded daily (days -3 to 5). Data shown are normalized to the individual animals’ weight or temperature on the day of challenge (day 0), and group averages are reported. “Mock” ferrets were infected intranasally with sterile egg allantoic fluid; “Perth/16” ferrets infected intranasally with 1 x 10⁶ pfu of A/Perth/16/09. The nasal cavities of all ferrets were washed daily with 1ml of PBS on days 0–5; and viral titers in the nasal washes (C) were determined. In addition, viral titers were determined in tissues (D); BALF, nasal turbinates (NT), upper trachea (UTr), and lower trachea (LTr). A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Peripheral blood leukocyte subsets following infection.

<p>Ferrets were bled on days 0–5 relative to the day of viral challenge, and cells were stained and analyzed by flow cytometry as described in the text. Percentage and absolute number of T_H (CD3+ and CD4+), Tc lymphocytes (CD3+ and CD8+), B cells (CD79a+) and CD11b-positive cells were measured (A-H). For each animal the frequencies were normalized to the ferret’s values on day 0; the Y axis represents percent of values on day 0. Group averages are reported here. “Mock” ferrets were infected intranasally with sterile egg allantoic fluid; “Perth/16” ferrets infected intranasally with 1 x 10⁶ pfu of A/Perth/16/09. For mock animals (M), absolute number of same cell subsets were measured at day 5 post-challenge and for the infected animals at days 2 and 5 post-challenge. A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p

Tissue leukocyte cell counts following infection.

<p>For evaluation of cellularity, the total cell numbers were calculated in spleen, MRLN, BALF (A), and in peripheral blood (PB) (B). Group averages are reported. For the mock-infected animals (M), all data are collected on day 5 post-challenge, and for the virus-infected animals on days 2 and 5 post-challenge (d2 and d5). A p value of 0.05 was used as the cutoff for statistical significance (* p ≤ 0.05; ** p ≤ 0.01; † p ≤ 0.001). Error bars represent SEM.</p