# Avian Influenza Virus Strain Specificity in the Volatile Metabolome
## 4 Discussion  
This study identified four volatiles that varied significantly over time in response to experimental AIV infection in ducks. However, these changes were AIV strain-specific, with no single volatile consistently altered across both viral subtypes (Figure 1A-D). Among these, benzylethyl ether, 2-phenoxyethanol, and 2-hydroxybenzaldehyde are compounds commonly linked to dietary sources or environmental exposure. These volatiles are well-characterized as plant-derived metabolites and are widely used in the flavoring and fragrance industries [14]. Their limited documentation as products of microbial biosynthesis suggests an exogenous origin, likely influenced by host diet or environment. Conversely, 6-methyl-5-hepten-2-one is a well-established microbial metabolite produced by diverse taxa, including _Staphylococcus_, _Streptomyces_, and _Lentilacobacillus_, as well as various fungal genera, such as _Candida_, _Apergillus_, and _Penicillium_[15]. The temporal variation observed in this compound, especially during H5N2 infection, may reflect changes in the host-associated microbiota triggered by viral infection. The link between fecal volatiles and the microbiome triggered by vaccination has been demonstrated in skunks [16]. However, a critical caveat is that the H2N2 and H5N2 infection trials were conducted approximately two months apart, under potentially variable dietary, microbial, or environmental conditions. While birds were housed under identical indoor conditions, the potential for seasonal or environmental variation affecting the microbiota or host physiology cannot be ruled out. No environmental data (e.g., temperature or humidity) were recorded during the study, which represents potential limitations in controlling for back  
Figure 1: (**A–D**) Normalized headspace responses across infection periods for four volatiles with a demonstrated interaction effect between strain and period. Diagonal bars indicate a significant increase (\(p<0.05\)) versus the headspace response observed in the pre-infection period for the given virus strain. Crosshatched bars indicate a significant decrease with respect to the pretreatment value. ground variation in volatile emissions. These temporal differences introduce potential seasonal or environmental confounders that could influence volatile profiles independently of infection status. We acknowledge this as a critical limitation, underscoring the challenges of longitudinal surveillance and the need to control for non-viral sources of variability in metabolomics studies. Cloacal swab analysis, which is primarily used for PCR-based viral detection, may offer additional insights into infection-induced changes in the host's microbiota. The production of 6-methyl-5-hepten-2-one by multiple microbial taxa suggests that this volatile could serve as an indirect indicator of microbial community restructuring. Microbially derived volatiles have been proposed as non-invasive biomarkers for infection and colonization [17]. However, it is important to note that not all microbial volatiles are pathogenic in origin; even commensal species contribute to the volatile metabolome, particularly under shifting ecological conditions. According to ecological competition theory, infection-induced changes in nutrient availability may favor the proliferation of certain microbial taxa, leading to detectable alterations in both community composition and metabolite production. Previously, we hypothesized that the microbial production of acetoin--a fermentation product--could serve as a diagnostic marker of AIV infection, mediated through host metabolic alterations. Specifically, AIV was proposed to increase pyruvate availability for acetoin-producing microbes such as _Bacillus_ spp. [7]. However, in the present study, acetoin was not detected in any cloacal swab samples, even in samples from ducks that were experimentally infected with the same previously used H5N2 strain. This discrepancy may be attributed to differences in sample type and collection context.