Summary of transmission events.

<p>NA, not applicable; h, hours; hpi, hour post inoculation of donor.</p

Respiratory droplet transmission of influenza virus, occurred before but not during clinical signs.

<p>Four ferrets were inoculated with 10⁴ PFU of E195-I219K. For 30 hours between days 1 and 2 pi a naive sentinel (exposed group 7) was housed in an adjacent cage to each inoculated donor. A different group of sentinels (exposed group 8) were exposed to air from the inoculated animals for 30 hours between days 5 and 6 pi. The number of sneezes (A) and coughs (B) in a one hour observation of the inoculated donors were recorded. Viral titres in daily nasal wash were determined by plaque assay: inoculated (C), exposed group 6 (D) and exposed group 7 (E). Ferrets in adjacent cages are indicated (red, blue and black). Exposure periods are indicated by the open bars.</p

Transmission of influenza virus between co-housed ferrets, at both early and late periods during infection.

<p>Three ferrets were inoculated with 10⁴ PFU of E195. For 30 hours between days 1 and 2 pi each inoculated donor was co-housed with a naive sentinel (exposed group 2). A different group of naive animals were co-housed with the inoculated donors for 30 hours between days 5 and 6 pi (exposed group 3). (A) The core body temperatures of the inoculated donors were continuously monitored both before (above panel) and after (bottom panel) inoculation. The thin horizontal black line indicates baseline temperature (38.5°C) and the horizontal red line indicates fever (39.4°C). (B) The number of sneezes was recorded during a 15 minute observation period for the inoculated animals. Viral titres shed in nasal wash were determined by plaque assay: innoculated (C), exposed group 2 (D) and exposed group 3 (E). Ferrets in the same cage are indicated (red, blue and black). Exposure periods are indicated by the open bars.</p

Respiratory droplet transmission of influenza virus.

<p>Four ferrets were inoculated with 10⁴ PFU of E195-I219K and 1 day pi naive sentinels (Exposed Group 6) were housed in cages adjacent to each donor. (A) The core body temperature of two of the inoculated donors was monitored by telemetry. The thin horizontal black line indicates baseline temperature (38.5°C) and the horizontal red line indicates fever (39.4°C). Virus shed in nasal wash from inoculated (B) and exposed (C) animals was titrated by plaque assay. Ferrets in adjacent cages are indicated (red, blue, black and green).</p

The oligomeric states of wild-type BNP, BNP-Δ38 and BNP-Δ66 examined by election microscopy.

<p>Electron microscopy pictures of NP and its variants. <i>a)</i> wild-type BNP (<i>a1</i>), BNP-Δ38 (<i>a2)</i> and BNP-Δ66 (<i>a3</i>) in 100 mM sodium phosphate, 100 mM NaCl, pH6.8. b) Wild-type BNP (<i>b1</i>), BNP-Δ38 (<i>b2)</i> and BNP-Δ66 (<i>b3</i>) in PBS when RNA was added at 0h. c) Wild-type BNP (<i>c1</i>), BNP-Δ38 (<i>c2)</i> and BNP-Δ66 (<i>c3</i>) in PBS 16hr after RNA was added. The red arrow in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137802#pone.0137802.g003" target="_blank">Fig 3B</a>-c1 indicates the RNA-BNP complexes that displays a double layered structure.</p

Static light scattering analysis of purified wild-type NP and variants.

<p>Horizontal lines on top of the curves illustrate the calculated molecular weights.</p

The viral RNP activity of wild-type BNP and variants.

<p>Viral RNP activity of wild-type and BNP N-terminal deletion variants. For each mutants triplicate wells were set and recorded. Experiments are repeated twice. P values are calculated by T-test algorithm. *, <i>P</i> < 0.01; <i>**</i>, <i>P</i> < 0.001.</p

Wild-type BNP and BNP-Δ66 have comparable RNA binding activities.

<p>Indicated amount of purified BNP and BNP-Δ66 were incubated with 10 μM of 24 nt 2-O-methylated RNA and analyzed by agarose gel electrophoresis. Band intensities were calculated by ImageJ (National Institutes of Health)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137802#pone.0137802.ref019" target="_blank">19</a>], using the RNA without the protein as a control (100%).</p

The construction and growth curves of influenza B virus with BNP variants.

<p>(A) Construction of influenza B NP mutants by systematically deleting the N-terminal coding region of NP. (B) Growth curves of influenza B wild-type and NP N-terminal deletion mutants after virus rescue. The growth curves are averages from three independent replicates.</p

Nucleotide sequence changes required to switch H5 HA receptor binding preference.

<p>(A) Analysis of codon usage by 1374 H5 influenza viruses indicates that codon 228 is GGA in 93% strains and GGG in 7%. The table illustrates transversions and transitions by which coding capacity at this residue might change from glycine to serine. The ability of the two intermediate mutants 228R (B) and 228A (E) recombinant HA proteins to bind synthetic receptor ligands 3SLN (avian receptor) and 6SLN (human receptor) in a solid phase assay was assessed. The binding phenotype on HAE (C,F) or human tracheal epithelium (D,G) by recombinant proteins with each amino acid sequence changed at residue 228 are shown (C,D) G228R and (F,G) G228A.</p