# Avian Influenza Virus Strain Specificity in the Volatile Metabolome
## 3 Results  
Multiple samples collected from duck #7 (H5N2 infection) were identified as highly influential, based on [PERSON]'s T\({}^{2}\) test, so all sampling periods for this subject were removed from the data. Two additional samples from the H5N2 infection (duck #6, day 2, and duck #8, day \(-\)2) were also highly influential and removed as outliers. Repeated measures indicated a significant strain effect (F\({}_{1,62}\) = 0.4816; \(p\) < 0.0001), but no other between-subjects effects were significant (Period: F\({}_{3,62}\) = 0.0169, \(p\) = 0.4682; Strain*Period: F\({}_{3,62}\) = 0.04428, \(p\) = 0.0933). All within-subjects effects were significant: Volatile (F\({}_{121,7502}\) = 33.73, \(p\) < 0.0001), Volatile*Strain (F\({}_{121,7502}\) = 10.70, \(p\) < 0.0001), Volatile*Period (F\({}_{363,7502}\) = 0.6973, \(p\) = 0.0074), and Volatile*Strain*Period (F\({}_{363,7502}\) = 1.115, \(p\) < 0.0001), indicating that some volatiles varied among periods, but this effect depended on the virus strain. An examination of residual plots indicated that they were normally distributed, with constant variance among collection periods. Univariate analyses revealed that four volatiles varied between strains and among sample periods (Table 2). No volatiles were only affected by the period main effect, indicating that the two virus strains did not share the same alteration of any single volatile. Importantly, acetoin was not observed in any samples.  
As compared to the pre-infection period, both increasing and decreasing concentrations of certain volatiles were observed in the headspace of swabs following experimental infection (Figure 1A-D). Among the four volatiles exhibiting a significant interaction  
\begin{table}
\begin{tabular}{c c c c} \hline
**Volatile Name** & **Effect Type** & \(p\)**-Value** & **FDR-Adjusted Alpha** \\ \hline Ethlybenzyl ether & Period*Day & \(p\) < 0.00006 & 0.00041 \\ \hline
2-Phenoxyethanol & Period*Day & \(p\) = 0.00017 & 0.00082 \\ \hline
2-Hydroxybenzaldehyde & Period*Day & \(p\) = 0.00022 & 0.0012 \\ \hline
6-Methyl-5-hepten-2-one & Period*Day & \(p\) = 0.00034 & 0.0016 \\ \hline \end{tabular}
\end{table}
Table 2: Volatiles subject to experimental effects. Decision criteria (alpha values) were determined according to the method of [PERSON] and [PERSON] [13].  
effect between strain and period, perhaps only the pattern of 6-methyl-5-hepten-2-one following infection with H5N2 (increase in the early and mid-periods and returning to the pre-infection level during the late period) is suggestive of a potentially useful diagnostic biomarker for H5N2 AIV infection (Figure 1A). Conversely, patterns of increasing or decreasing concentrations of the other three volatiles appear less informative and are often contradictory. For example, the concentrations of 2-phenoxyethanol remain unchanged for both AIV strains from infection through the mid-period. However, in the late period, the concentration of this volatile increased for the H5N2 strain, while it decreased for the H2N2 strain (Figure 1B). Concentrations of ethylbenzyl ether are similarly constant for both AIV strains through the mid-period; however, late-period concentrations inversely change compared to 2-phenoxyethanol (i.e., this volatile increased for H2N2 and decreased for H5N5; Figure 1C). The concentrations of 2-hydroxybenzaldehyde decreased soon after infection with the H5N2 strain and remained lower throughout the length of the experiment. Conversely, 2-hydroxybenzaldehyde followed a pattern similar to that of ethylbenzyl ether for H2N2 infection. Specifically, the concentration remained constant through the mid-period and increased in the late period (Figure 1D).  
Figure 1: _Cont._  
Figure 1: _Cont_.