Detection of Clinical Specimens by mRT-PCR.

<p>Detection of Clinical Specimens by mRT-PCR.</p

Influenza viruses used in this study.

<p>Influenza viruses used in this study.</p

Specificity of the mRT-PCR assay.

<p>Lane M: molecular marker. Lane 1: a mixture containing equine-origin H3N8 (A/canine/Colorado/6723-8/2008; 148 bp predicted size), hH3N2 (A/Jiangxi/262/05; 303 bp predicted size), H1N1/2009 CIV (A/canine/Beijing/cau2/2009; 407 bp predicted size), and cH3N2 (A/canine/Beijing/364/2009; 544 bp predicted size) influenza viruses. Lane 2: equine-origin H3N8 CIV (A/canine/Colorado/6723-8/2008). Lane 3: hH3N2 influenza virus (A/Jiangxi/262/2005). Lane 4: H1N1/2009 CIV (A/canine/Beijing/cau2/2009). Lane 5: cH3N2 influenza virus (A/canine/Beijing/364/2009). Lane 6: avian-origin H9N2 influenza virus (A/chicken/Jiangsu/TS/2010). Line 7: avian-origin H5N1 influenza virus (A/chicken/Sheny/0606/2008). Lane 8: CDV (CDV-WZ). Lane 9: CPIV. Lane 10: CAV-2. Lane 11: negative control allantoic fluid.</p

Weight loss and lung virus titration in mice inoculated with H4N6 AIVs.

<p>Six-week-old female BALB/c mice (n = 3 mice/group) were inoculated i.n. with 10⁶ EID₅₀ of virus. The body weights of inoculated mice were measured daily and are represented as percentages of weight on the day of inoculation (day 0). The averages for each group are shown (A).The lungs of each mouse in each group were collected at 3 and 5 dpi, respectively, for virus titration (B).</p

The genotypic evolution of H4N6 AIVs in China from 2000 to 2016.

<p>The eight gene segments listed are (top to bottom): PB2, PB1, PA, HA, NP, NA, M, and NS genes. The colors of the eight gene segments of the first isolate (A/duck/Nanchang/4-165/2000) were defined to be the same, and a new color represent a different lineage from this strain. The abbreviation of virus which possessed the corresponding genotype are listed below the genotype. The gene constellation for different genotypes, the viruses that possessed these, and the abbreviation of virus name are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184437#pone.0184437.s002" target="_blank">S1 Table</a>.</p

Contact transmission of H4N6 AIVs in guinea pigs.

<p>Three guinea pigs were intranasally inoculated with 10⁶ EID₅₀ of A/mallard/Beijing/10/2016 (A) or A/mallard/Beijing/16/2016 (B) viruses. At 24 hpi, the inoculated guinea pigs were placed in a cage with three naive guinea pigs. Nasal wash titers are plotted as a function of time post-inoculation. Titers of intranasally inoculated animals are represented by solid lines and filled squares; titers of exposed guinea pigs are shown with dashed lines and filled triangles.</p

Schematic representation of putative genomic compositions of the four H4N6 AIVs isolated in this study and their possible parent viruses.

<p>The eight gene segments (from top to bottom) in each virus are PB2, PB1, PA, HA, NP, M, and NS. Each color represents a separate virus background. The simplified schematic illustration is based on nucleotide-distance comparison and phylogenetic analysis.</p

Arterial blood gas analysis.

<p>Mice were inoculated i.n. with 10^2.5 pfu SD/09 viruses. Arterial blood samples were collected at indicated times p.i.</p><p>Values are expressed as mean ± SD.</p><p>*p<0.05,</p><p>**p<0.01, comparison between virus-infected and control groups.</p

White blood cell (WBC) summary and differential counts in BALF.

<p>Mice were inoculated i.n. with 10^2.5 pfu SD/09 viruses, and BALF was prepared at indicated times p.i.</p><p>Values are expressed as mean ± SD.</p><p>*p<0.05.</p><p>**, p<0.01,</p><p>***p<0.001, comparison between virus-infected and control groups.</p

Cytokine levels in virus-infected mouse lungs.

<p>Mice were euthanized at indicated times p.i. The collected lungs were weighed, and 10% homogenates were prepared in cold PBS. Cytokine levels from lung homogenates were measured using the Cytokine Bead Array system. *p<0.05, **p<0.01, ***p<0.001, comparison between virus-infected and control groups.</p