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The proliferative darkening syndrome (PDS) is a lethal disease of brown trout (Salmo trutta fario) which occurs in several alpine Bavarian limestone rivers. Because mortality can reach 100%, PDS is a serious threat for affected fish populations. Recently, Kuehn and colleagues reported that a high throughput RNA sequencing approach identified a piscine orthoreovirus (PRV) as a causative agent of PDS. We investigated samples from PDS-affected fish obtained from two exposure experiments performed at the river Iller in 2008 and 2009. Using a RT-qPCR and a well-established next-generation RNA sequencing pipeline for pathogen detection, PRV-specific RNA was not detectable in PDS fish from 2009. In contrast, PRV RNA was readily detectable in several organs from diseased fish in 2008. However, similar virus loads were detectable in the control fish which were not exposed to Iller water and did not show any signs of the disease. Therefore, we conclude that PRV is not the causative agent of PDS of brown trout in the rhithral region of alpine Bavarian limestone rivers. The abovementioned study by Kuehn used only samples from the exposure experiment from 2008 and detected a subclinical PRV bystander infection. Work is ongoing to identify the causative agent of PDS.