Serological status (mean ± SD) of mallards before and after LPAIV H5N9 challenge using ELISA and HI tests.

*<p>Bold numbers indicate days after LPAIV H5N9 inoculation.</p>1<p>The ELISA scores represent the signal to noise (S/N) ratio where values ≤0.50 are considered seropositive.</p>2<p>The hemagglutination inhibition (HI) values represent the mean titer (log₂) of sera samples.</p>3<p>Before LPAIV H5N9 inoculation.</p>4<p>Mean (±1 SD) test scores include all infected birds in each treatment.</p

Breast condition score for wild mallard treatment groups at capture vs. LPAIV H5N9 challenge.

<p>Bars represent mean condition score ±1 standard error (n = 10), asterisks represent significant within group differences (paired t-test, alpha = 0.05).</p

Regression analysis for the calibration of the number of IAV matrix gene copies (circles) and plaque forming units (triangles).

<p>The standard curve was generated using a log₁₀ dilution series of quantified RNA runoff transcripts or known concentrations of LPAI H5N9 stock virus.</p

Body condition for wild mallard treatment groups throughout the study.

<p>Data points represent mean condition (±1 standard error), A = start of diet manipulation, B = LPAIV H5N9 inoculation.</p

Duration of viral shedding (A) and mean number of positive samples (B) for captive-bred and wild mallard treatment groups.

<p>Bars represent means ±1 standard error, letters identify significant differences among wild mallard treatment groups (Mann-Whitney rank sum, alpha = 0.05). The mean number of positive samples was calculated from the 4 sampling days between 1–5 dpi.</p

Viral shedding profiles (log₁₀ GEC/140 µl of swab sample fluid) for all infected mallards with detectable viral RNA using matrix gene RRT-PCR.

<p>A: captive-bred (n = 9), B: wild normal (n = 10), C: wild lean (n = 10), D: wild poor (n = 6).</p

Group mean daily (±1 SD) viral genome load (log₁₀ GEC/140 µl swab sample fluid) for mallards inoculated with LPAIV H5N9.

<p>Mean genome load is calculated using only infected mallards (birds that seroconverted or shed detectable viral RNA≥3 dpi). Minimum and maximum values are reported for the number of birds <i>(n)</i> with detectable viral RNA on the given day.</p

Birth death multitype model initialization file, PB2 segment wild birds

xml file created from the BEAUTI template for the birth-death multi type model (https://github.com/denisekuehnert/bdmm) to implement Bayesian estimation of phylodynamic parameters in BEAST v2.4.3 (available from www.BEAST2.org) from 1 chain of 10 million iterations. Set-up for analysis of 38 aligned whole segment sequences of the polymerase subunit (PB2) gene of isolates from wild birds during the 2014-2015 highly pathogenic avian influenza HA clade 2.3.4.4 outbreak in North America

Summary phylogenetic tree avian influenza PB2 segment

Time-rooted Maximum clade credibility phylogenetic tree of the polymerase subunit (PB2) segment of highly pathogenic avian influenza viruses isolated from wild birds and poultry during the 2014-2015 outbreak in North America. Nodes dates are the mean posterior estimate and are annotated with phylodynamic estimates of uncertainty if posterior probability was > 0.5. NEXUS fil

Birth death multitype model initialization file, HA segment

xml file created from the BEAUTI template for the birth-death multi type model (https://github.com/denisekuehnert/bdmm) to implement Bayesian estimation of phylodynamic parameters in BEAST v2.4.3 (available from www.BEAST2.org) with 1 MCMC chain of 10 million iterations. Set-up for analysis of 85 aligned whole segment sequences of the hemagglutinin (HA) gene of isolates from the 2014-2015 highly pathogenic avian influenza HA clade 2.3.4.4 outbreak in North America