viruses Article Piscine Orthoreovirus 3 Is Not the Causative Pathogen of Proliferative Darkening Syndrome (PDS) of Brown Trout (Salmo trutta fario) Robert Fux 1,* , Daniela Arndt 1, Martin C. Langenmayer 1 , Julia Schwaiger 2, Hermann Ferling 2, Nicole Fischer 3 , Daniela Indenbirken 4, Adam Grundhoff 4, Lars Dölken 5, Mikolaj Adamek 6, Dieter Steinhagen 6 and Gerd Sutter 1 1 Institute for Infectious Diseases and Zoonoses, Department for Veterinary Sciences, LMU Munich, 80539 Munich, Germany; [email protected] (D.A.); [email protected] (M.C.L.); [email protected] (G.S.) 2 Bavarian Environment Agency, Unit Aquatic Toxicology, Pathology, 82407 Wielenbach, Germany; [email protected] (J.S.); [email protected] (H.F.) 3 Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg Eppendorf, 20246 Hamburg, Germany; nfi[email protected] 4 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany; [email protected] (D.I.); [email protected] (A.G.) 5 Institut für Virologie und Immunbiologie, Julius-Maximilians-Universität Würzburg, 97078 Würzburg, Germany; [email protected] 6 Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine, 30559 Hannover, Germany; [email protected] (M.A.); [email protected] (D.S.) * Correspondence: [email protected]; Tel.: +49-89-21802536 Received: 10 January 2019; Accepted: 25 January 2019; Published: 28 January 2019 Abstract: 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. Keywords: proliferative darkening syndrome; black trout syndrome; piscine orthoreovirus; orthoreovirus; brown trout; Salmo trutta fario; next generation sequencing; RT-qPCR 1. Introduction The brown trout (Salmo trutta fario) is a predatory fish of the family Salmonidae. Its range of distribution covers nearly the entirety of Europe, and its natural habitats are fast flowing, cool, Viruses 2019, 11, 112; doi:10.3390/v11020112 www.mdpi.com/journal/viruses Viruses 2019, 11, 112 2 of 10 Viruses 2019, 11, x FOR PEER REVIEW 2 of 9 oxygen-rich waters. Since the 1980s, an annually recurring die-off of brown trout takes place in the rhithralregion of region alpine of Bavarian alpine Bavarian limestone limestone rivers. rivers.In the Inaffected the affected river riversections sections,, the mortality the mortality rates rates reach reachalmost almost 100%. 100%. Typically, Typically, diseased diseased brown brown trout trout develop develop a dark a dark or almost or almost black black color color before before they they die. die.Therefore, Therefore, the thedisease disease was was named named “p “proliferativeroliferative darkening darkening syndrome” syndrome” (PDS) oror “black“black trout trout phenomenon”.phenomenon”. SimilarSimilar diseasedisease eventsevents werewere observedobserved inin Austria Austria and and Switzerland. Switzerland. However, However, black black pigmentationpigmentation in in fish fish is is a a rather rather unspecific unspecific symptom, symptom reflecting, reflecting a a response response to to various various factors, factors, and and is is not not pathognomonicpathognomonic forfor aa particularparticular etiologyetiology[ 1[1,2].,2].Therefore, Therefore, it it remains remains unclear unclear whether whether the the term term “PDS” “PDS” describesdescribes thethe samesame typetype ofof diseasedisease inin differentdifferentpre-alpine pre‐alpine regions. regions. TheThe PDSPDS observedobserved inin thethe Bavarian Bavarian rivers rivers shows shows some some interesting interesting characteristics. characteristics. The The clinical clinical diseasedisease andand subsequentsubsequent deathdeath ofof thethe brown brown trout trout is is exclusively exclusively observed observed in in late late summer summer (August (August to to September).September). However,However, thethe syndromesyndrome only only appears appears if if the the respective respective fish fish were were exposed exposed to to water water of of the the affectedaffected riverriver sectionssections duringduring MayMay andand June. June. InIn the the final final stage stage of of PDS, PDS typical, typical clinical clinical signs signs include include besidesbesides thethe darkeningdarkening ofof thethe skin, skin, apathy apathy and and highly highly increased increased respiratory respiratory rates. rates. The The pathological pathological lesionslesions comprisecomprise p pronouncedronounced anemia anemia and and leucopenia, leucopenia, hepatic hepatic hemorrhages hemorrhages,, splenic splenic enlargement enlargement,, and andascites ascites (Figure (Figure 1). Histological1). Histological features features include include necrohemorrhagic necrohemorrhagic hepatitis hepatitis and lymphocytic and lymphocytic depletion depletionof the spleen of the [3] spleen. [3]. FigureFigure 1.1. AA brownbrown trouttroutwith with “proliferative “proliferative darkening darkening syndrome”. syndrome” Note. Note the the hepatic hepatic hemorrhages hemorrhages and and thethe enlargedenlarged congestedcongested spleen.spleen. (Photograph(Photograph providedprovided bybythe the BavarianBavarian Environment Environment Agency). Agency). Recently,Recently, KuehnKuehn andand colleaguescolleagues [ 4[4]] reported reported that that a a piscine piscine orthoreovirus orthoreovirus (PRV) (PRV) is is the the causative causative agentagent ofof the the PDS PDS of of brown brown trout trout using using a high a high throughput throughput RNA RNA sequencing sequencing (RNA-seq) (RNA approach.‐seq) approach In that. In study,that study brown, brown trout trout specimens specimens were were exposed exposed in the in the Iller, Iller, a river a river which which is regularlyis regularly affected affected by by PDS. PDS. Afterwards,Afterwards, samples samples were were collected collected at at various various time time points points until until the the occurrence occurrence of of clinical clinical signs signs of of PDS. PDS. UsingUsing transcriptomictranscriptomic analysisanalysis andand RT-qPCR,RT‐qPCR, the the strong strong induction induction of of immune-associated immune‐associated genes genes was was observedobserved supportingsupporting anan infectiousinfectious naturenature ofof the the disease. disease. Samples Samples from from fish fish with with the the corresponding corresponding expressionexpression profileprofile were were used used for for next-generation next‐generation sequencing sequencing (NGS), (NGS) which, which resulted resulted in in the the detection detection of of PRVPRV RNA.RNA. PRVPRV isis a non-envelopeda non‐enveloped virus virus with with a segmented a segmented (L1-3, M1-3,(L1‐3, S1-4),M1‐3, double S1‐4), strandeddouble RNAstranded genome, RNA whichgenome, belongs which to belongs the genus to Orthoreovirusthe genus Orthoreovirus, subfamily, Spinareovirinaesubfamily Spinareovirinaewithin the familywithin Reoviridaethe family. AtReoviridae least three. At least subtypes three ofsubtypes the virus of exist:the virus PRV-1 exist: is frequentlyPRV‐1 is frequently found in farmedfound in Atlantic farmed salmon Atlantic (salmonSalmo salar (Salmo) and salar is associated) and is withassociated the heart with and the skeletal heart muscleand skeletal inflammation muscle (HSMI) inflammation syndrome (HSMI but ) alsosyndrome can be but detected also can in be apparently detected healthyin apparently fish as healthy well as fish in otheras well species as in other of the species genera ofSalmo the generaand Salmo and Oncorhynchus including sea trout (Salmo trutta) [5–8]. The pathogenesis of HSMI is only partially understood. Interestingly, erythrocytes are important target cells for PRV‐1, but infection does not result in severe anemia [9]. In cohabitant infection trials, the virus was detectable in blood Viruses 2019, 11, 112 3 of 10 Oncorhynchus including sea trout (Salmo trutta)[5–8]. The pathogenesis of HSMI is only partially understood. Interestingly, erythrocytes are important target cells for PRV-1, but infection does not result in severe anemia [9]. In cohabitant infection trials, the virus was detectable in blood samples after 4–6 weeks [9] and clinical symptoms occurred after 8–10 weeks [10]. After disease outbreaks, subclinical persistence