Replication of H9N2 viruses in lungs of mice and chickens.

a<p>Mice and chickens (n = 3) were infected i.n. with each of six H9N2 viruses at a dose of 10⁶ EID₅₀ (mice), or 10⁷ EID₅₀ (chickens). Clarified homogenates of lungs were titrated for virus infectivity in eggs from initial dilutions of 1∶10. Virus endpoint titers were expressed as mean log₁₀ EID₅₀ per 10 milligram wet tissues ± standard deviation (SD).</p>b<p><, no virus detectable at dilution of ≥10⁻¹.</p

Replication of H9N2 viruses in tissues of mice.

a<p>Mice (n = 3) were inoculated i.n., and tissues were taken on days 5 p.i. Clarified homogenates of tissues were titrated for virus infectivity in eggs from initial dilutions of 1∶10. Mean viral titers are expressed as log₁₀ EID₅₀ per 10 milligram wet tissues.</p>b<p>Average virus titers for each tissue (the number of positives/total number tested for each virus).</p>c<p><, no virus detectable at dilution of ≥10⁻¹.</p

Comparison of amino acid sequences of HA and NA of H9N2 viruses tested in this study

a<p>RBS, receptor binding site.</p>b<p>Amino acid sequence of residues 324–330 (numbering according to H3 HA) at the cleavage site between HA1 and HA2; aa, amino acid.</p

Phylogenetic trees for PB2 (<i>A</i>), PB1 (<i>B</i>), PA (<i>C</i>) and NP (<i>D</i>) genes of the H9N2 influenza viruses analyzed.

<p>Trees were generated on the basis of the following gene sequences: nucleotides 28–2307 (2279 bp, PB2), 40–1473 (1434 bp, PB1), 25–2175 (2151 bp, PA) and 46–1542 (1497 bp, NP). The PB2 and NP trees are rooted to A/Equine/Praque/1/56(H7N7), and the PB1 and PA trees to A/Equine/London/1416/73(H7N7). The other information was described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013063#pone-0013063-g001" target="_blank">Fig. 1</a>.</p

Replication of H9N2 viruses in tissues of the infected mice and chickens.

<p>Mice and chickens were inoculated i.n. with Ck/HB/4/08 virus at a dose of 10⁶ EID₅₀ (mice), or 10⁷ EID₅₀ (chickens). Tissues were collected on 5 days p.i., and the virus was titrated in embryonated eggs from initial dilutions of 1∶10. Mean viral titers based on three mice or chickens per group are expressed as log₁₀ EID₅₀ per 10 milligrams wet tissues ± SD. Virus is below the detectable level at dilution of ≥10⁻¹. <i>Open bars</i>, chicken group; <i>solid bars</i>, mouse group.</p

Phylogenetic trees for HA (<i>A</i>), NA (<i>B</i>), M (<i>C</i>) and NS (<i>D</i>) genes of the H9N2 influenza viruses analyzed.

<p>Trees were generated by the neighbor-joining method with the MEGA program (version 4.1). Nucleotides 80–1090 (1011 bp, HA), 20–1420 (1401 bp, NA), 26–984 (959 bp, M) and 41–859 (819 bp, NS) were used for phylogenetic analysis. The HA phylogenetic tree is rooted to A/Duck/Alberta/60/76(H12N5), and the trees of NA, M, and NS are rooted to A/Equine/Praque/1/56(H7N7). The length of the horizontal lines is proportional to the minimum number of nucleotide differences required to join nodes. Vertical lines are for spacing and labeling. Viruses characterized in this study are highlighted in bold, representative H9N2 viruses are in red, viruses isolated from swine and human are in green, and H5N1 influenza viruses are highlighted with asterisk. Abbreviations: Ck, chicken; Dk, duck; Sw, swine; Gf, guinea fowl; Pg, pigeon; Gs, goose; Qa, quail; Ty, turkey; Pf, peregrine falcon; WDk, wild duck; Cu, chukar; HK, Hong Kong; WI, Wisconsin, Bei, Beijing; CA, California; VNM; Viet Nam.</p

Survival percentage of mice after H9N2 viral infection.

<p>Groups of mice (n = 14) were inoculated i.n. with each of six H9N2 viruses at a dose of 10⁷ EID₅₀ (<i>A</i>), 10⁶ EID₅₀ (<i>B</i>), 10⁵ EID₅₀ (<i>C</i>) or 10⁴ EID₅₀ (<i>D</i>), respectively. Percent survival was observed daily for 14 days.</p