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Piscine Orthoreovirus Subtype 3 (PRV-3)

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Piscine Orthoreovirus Subtype 3 (PRV-3) Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss) Niccoló Vendramin, Dhamotharan Kannimuthu, Anne Berit Olsen, Argelia Cuenca, Lena Hammerlund Teige, Øystein Wessel, Tine Moesgaard Iburg, Maria Krudtaa Dahle, Espen Rimstad, Niels Jørgen Olesen To cite this version: Niccoló Vendramin, Dhamotharan Kannimuthu, Anne Berit Olsen, Argelia Cuenca, Lena Hammerlund Teige, et al.. Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss). Veterinary Research, BioMed Central, 2019, 50 (1), pp.14. 10.1186/s13567- 019-0632-4. hal-02488837 HAL Id: hal-02488837 https://hal.archives-ouvertes.fr/hal-02488837 Submitted on 24 Feb 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vendramin et al. Vet Res (2019) 50:14 https://doi.org/10.1186/s13567-019-0632-4 RESEARCH ARTICLE Open Access Piscine orthoreovirus subtype 3 (PRV-3) causes heart infammation in rainbow trout (Oncorhynchus mykiss) Niccoló Vendramin1*† , Dhamotharan Kannimuthu2†, Anne Berit Olsen3, Argelia Cuenca1, Lena Hammerlund Teige2, Øystein Wessel2, Tine Moesgaard Iburg1, Maria Krudtaa Dahle4, Espen Rimstad2 and Niels Jørgen Olesen1 Abstract Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in diferent salmonid species. PRV-1 causes heart and skeletal muscle infammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaun- dice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Onco- rhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purifed PRV-3 particles to assess the causal relationship between the virus and development of heart infammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histo- pathological fndings occurred in the late phase of the trial and was associated with increased expression of CD8 , indicating cytotoxic T cell proliferation. The fndings indicate that, under these experimental conditions, PRV-3 infec+ - tion in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection. Introduction the virus relies on in vivo experimental challenge in sus- Piscine orthoreovirus (PRV) causes or are associated ceptible fsh. with emerging diseases in salmonid aquaculture. PRV Tree subtypes of PRV have been reported and are belongs to the family Reoviridae, sub-family Spinareoviri- denoted as PRV-1, PRV-2, and PRV-3. Subtypes 1 and 2 nae, genus Orthoreovirus. It has a double stranded RNA have recently been identifed as aetiological agents of dis- genome consisting of 10 segments [1]. Te virion has a ease in salmonids through infection with purifed virus. double protein capsid with icosahedral symmetry and no PRV-1 causes heart and skeletal muscle infammation envelope [1]. PRV resists cultivation in cell culture mon- (HSMI) in Atlantic salmon (Salmo salar) [3]. Te disease olayers, however, ex vivo infection of naïve red blood was frst diagnosed in 1999 in Norway [4, 5]; HSMI has cells has been demonstrated [2]. Efcient propagation of also been reported in Scotland and Canada [6]. In Nor- way, the causative relationship between PRV-1 and HSMI was demonstrated [3]. As the disease name implies, the *Correspondence: [email protected] major histopathological fndings are in located in the †Niccoló Vendramin and Dhamotharan Kannimuthu contributors equally heart and red skeletal muscle. Afected fsh show pan- to this work carditis with epicarditis, mononuclear cell infltrations in 1 National Institute of Aquatic Resources, Technical University of Denmark, Kongens Lyngby, Denmark the atrium and spongy and compact layers of the cardiac Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vendramin et al. Vet Res (2019) 50:14 Page 2 of 13 ventricle and necrotic cardiomyocytes. Severely afected In this study, we set out to demonstrate an eventual fsh also have red muscle infammation [7]. causality between a disease recently described in rain- PRV-2 was shown to cause erythrocytic inclusion body bow trout [11], and the associated Piscine orthoreovirus syndrome (EIBS) in coho salmon (Oncorhynchus kisutch) (PRV) [13]. To achieve this goal, PRV-3 was purifed from in 2016 [8] in Japan. Te anaemic condition denoted as experimentally infected rainbow trout blood and used in EIBS was frst described in 1977 in rainbow trout [9] and the challenge model previously developed to compare the in 1987 in chinook salmon from the Pacifc Northwest infection progress and disease development with a con- of North America [10], however, as these occurrences trol group injected with PRV-3 infected blood. predate the discovery of PRV-2, it remains unknown as Te viral RNA kinetics in the heart and spleen and PRV to whether these historical EIBS cases are equivalent to protein expression in erythrocytes were assessed, along the PRV-2 causing EIBS as diagnosed in Japan. To date, with the development of heart pathology. In addition, the PRV-2 has not been reported outside of Japan. immune response was monitored through gene expres- PRV-3 was detected in 2013 following a thorough sion analysis and detection of PRV specifc antibodies. investigation of unexplained mortalities in young rain- bow trout (Oncorhynchus mykiss) farmed in fresh water Materials and methods in Norway [11]. After detection of the virus, infection Virus purifcation trials were conducted to assess its pathogenicity and the Te challenge material, consisting of purifed PRV-3 risk associated with its introduction to salmonid aqua- particles or PRV-3 infected blood, originated from culture in Europe. Tose experimental trials showed that intra peritoneal (i.p.) injected fsh from a PRV-3 chal- PRV-3 replicated in rainbow trout blood and efciently lenge trial described earlier [13]. PRV-3 particles (isolate transmitted to naïve host in a cohabitation trial, while NOR/060214) were purifed from the PRV-3 infected its capacity of replicating in Atlantic salmon is limited blood (500 µL, Ct 19.7) exactly as stated in [13]. Fractions [12]. Te PRV-3 genome was sequenced and compared of 0.5 mL were collected using a syringe with a 23 G nee- with PRV-1 showing that PRV-3 is more closely related to dle. Te density of the fractions was determined by cross PRV-1 than PRV-2 [13]. referencing to the refractive index [18]. Te quantity of PRV-3 antigenic properties were analyzed with anti- PRV-3 in the fractions was determined using RT-qPCR. bodies raised against homologous proteins of PRV-1. Fractions with a density corresponding to that of PRV Western blot analysis of PRV-3 infected blood cells were chosen for dialysis. Te purity of the samples was have demonstrated that polyclonal rabbit antisera raised inspected by transmission electron microscopy (TEM) against the homologous proteins of PRV-1 cross-reacted and analyzed by Next Generation Sequencing (NGS) as with the PRV-3 proteins σ1, σ3, σNS, µ1, µNS, and λ1 described by Dhamotaran et al. [13]. [13]. Te orthoreoviruses are, in general, ubiquitous in Experimental challenge their respective niches. Te mere detection of the virus Rainbow trout were obtained from eyed eggs provided by is therefore not necessarily indicative of disease causal- a Danish commercial fsh farm ofcially registered free of ity. For example, PRV-1 is present in almost every cohort IPNV, IHNV, VHSV and bacterial kidney disease (BKD). of farmed Atlantic salmon in the marine phase [3] and After iodophor disinfection, the eggs were hatched and PRV-3 was detected in non-diseased adult rainbow trout fsh were grown in the wet laboratory facilities of the in Norway [14]. Recently the virus was detected in several European Union Reference Laboratory for fsh disease European countries, including Scotland, Germany, Italy (EURL, Copenhagen, Denmark)
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