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Infected Common Carp Michal Reichert1*, Anna Lukasik2, Piotr Zielenkiewicz2,3, Marek Matras1, Joanna Maj-Paluch1, Magdalena Stachnik1 and Ewa Borzym1

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Infected Common Carp Michal Reichert1*, Anna Lukasik2, Piotr Zielenkiewicz2,3, Marek Matras1, Joanna Maj-Paluch1, Magdalena Stachnik1 and Ewa Borzym1 Reichert et al. BMC Genomics (2019) 20:46 https://doi.org/10.1186/s12864-018-5266-9 RESEARCH ARTICLE Open Access Host microRNA analysis in cyprinid Herpesvirus-3 (CyHV-3) infected common carp Michal Reichert1*, Anna Lukasik2, Piotr Zielenkiewicz2,3, Marek Matras1, Joanna Maj-Paluch1, Magdalena Stachnik1 and Ewa Borzym1 Abstract Background: The mechanism of latency and the ability of the cyprinid herpesvirus 3 (CyHV-3) to establish life-long infections in carp remains poorly understood. To explain the role of miRNAs in this process we applied a range of molecular tools including high-throughput sequencing of RNA libraries constructed from the blood samples of infected fish followed by bioinformatic and functional analyses which show that CyHV-3 profoundly influences the expression of host miRNAs in vivo. Results: We demonstrated the changed expression of 27 miRNAs in the clinical phase and 5 in the latent phase of infection. We also identified 23 novel, not previously reported sequences, from which 8 showed altered expressions in control phase, 10 in clinical phase and 5 in latent phase of infection. Conclusions: The results of our analysis expand the knowledge of common carp microRNAs engaged during CyHV-3 infection and provide a useful basis for the further study of the mechanism of CyHV-3 induced pathology. Keywords: Cellular miRNA expression, KHV, Herpesvirus, Common carp, Infection Background RNAs (~ 22 nt) transcribed from the genomes of all Koi herpesvirus (KHV), also known as cyprinid herpes- multicellular eukaryotes and some viruses [4, 5]. Studies virus 3 (CyHV-3) belongs to the genus Cyprinivirus of concerning the role of miRNA expression in virus infec- the family Alloherpesviridae comprising of herpesviruses tion has exploded in recent years. The common picture infecting only fish and amphibians [1]. CyHV-3 has that has emerged from virus-host interaction is that virus caused huge economic losses in common and koi carp encoded miRNAs are usually involved in this process and culture industries worldwide since its emergence in the promote viral persistence through multiple mechanisms; late 1990s [2, 3]. All known members of the Herpesviridae evading the immune system, the inhibition of cell apop- family demonstrate the ability to establish life-long infec- tosis and/or viral lytic cycle and the promotion of viral tions in immunocompetent hosts. There are multiple latency [5, 6]. In recent years an increasing body of known mechanisms of this immune evasion but we are evidence suggests that viruses exploit host miRNAs to still far from having a complete understanding of the control the process of infection. Some Herpesviruses are underlying viral strategy. Among them, miRNAs driven not exceptional in this context and are indeed able to gene expression regulation seems to be an important exploit host miRNAs engaged in cellular pathways crucial element of virus-host interplay enabling the creation of a for viral latency [7, 8]. It is interesting to note that only beneficial environment for persistent virus infection. very limited data concerning the role of miRNAs during MicroRNAs (miRNAs) are a class of small non-coding CyHV-3 infection of carp exists to date. We found only two reports [9, 10] about this subject, however, they focus mainly on the role of virus encoded miRNAs while leaving * Correspondence: [email protected] the role of the host components largely uninvestigated. 1Department of Fish Diseases, National Veterinary Research Institute, 57 Partyzantow Avenue, 24-100 Pulawy, Poland This is not surprising as both studies were performed Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Reichert et al. BMC Genomics (2019) 20:46 Page 2 of 16 2 − 1 using an in vitro model of infection namely CCB (com- had an infectivity of 3 × 10 TCID50 ml . Group II was mon carp brain) and KCF − 1 (caudal fin of koi) cell lines. mock infected with the cell culture medium and used as Although analyses from one of the reports also indicate a control. After that, the fish were kept until 8 weeks that cellular miRNAs are involved in the course of after exposure. The progress of the infection was moni- CyHV-3 infection, their value is greatly reduced due to tored using DNA samples prepared from fin sections of the lack of a host immune component, which is a the fish and real-time PCR according to Gilad et al. [14]. common shortcoming of in vitro models [10]. This short- Blood was collected 3 times from the fish of group II; coming is especially significant in the herpesvirus infection one day before exposure, on the 7th day post infection as herpesviruses are masters in evading host immune when clinical signs become evident (lethargy, skin spots response with miRNAs participating significantly in the and skin haemorrhages, sunken eyes) and finally 8 weeks underlying mechanisms. In order to gain knowledge after exposure from the remaining 18 convalescent fish. concerning the role of host miRNAs in the KHV induced Before blood collection the fish were anesthetized by − latency we focused our study on the analysis of host immersion in 50 μgml 1 of tricaine methane sulfonate miRNA expression in CyHV-3 infected common carp. (MS-222). At the end of the experiment fish were eutha- Several studies on the pathogenesis of CyHV-3 infection nised by immersion into a 0.5 g L − 1 tricaine solution indicate that latency may be a distinctive feature of this (Sigma-Aldrich). Blood samples were used for micro representative of the family Alloherpesviridae which is RNA isolation immediately after collection and then the similar to many members of the Herpesviridae family in RNA was kept at -80 °C until required. For library this respect. Although numerous reports suggest that construction and NGS sequencing (Next Generation CyHV-3 may have existed in wild carp long before its Sequencing), the best quality samples of blood RNA recognition in the late 1990s, the detailed mechanism of were chosen from the same three fish (s1, s2 and s3) in its latency and the ability of the virus to establish life-long each phase of the infection (P1-control, P2-clinical, infections is far from being resolved [11–13]. To ex- P3-latent). To this end, the concentration of RNA and plore this intriguing biological phenomenon and bear- the RIN (RNA integrity number) were determined using ing in mind the huge economic losses caused an Agilent 2100 bioanalyzer in conjunction with a RNA world-wide by KHV, we decided to carry out an ex- 6000 Pico LabChips kit. Description of the planned periment that would reproduce the natural infection experiments was submitted to and approved by the of carp. To explain the role of miRNAs in this process we Local Ethics Committee for Animal Experiments at the applied high-throughput sequencing of RNA libraries University of Life Sciences in Lublin. Decision no. 19/2013 constructed from the blood samples of infected fish issued on 12 March 2013. followed by bioinformatic and functional analyses which show that CyHV-3 profoundly influences the expression Small RNA library construction and high-throughput of host miRNAs in vivo. sequencing MicroRNA was extracted from the carp whole blood Methods using a dedicated miRNeasy Mini Kit (Qiagen) and Experimental fish purified according to the manufacturer’s instructions, Forty-five healthy juvenile carp weighing 150 – 300 g, eluated in 40 μl RNAse free water and stored at -80 °C obtained from one of the Polish fish farms in kozienicki until required. district of the mazowieckie voivodship with no history of Next generation sequencing (NGS) using an Illumina KHV, were adapted to experimental conditions for one HiSeq 4000 sequencer was performed by Genomed SA month. The fish were kept in a 400 L tank supplied with (Poland) in partnership with BGI Tech Solutions (Hong − a constant flow of 90 L h 1 of deep well water. Water Kong) according to the manufacturer’s protocol. Briefly, temperature was maintained at 12 ± 1 °C, and a photo- 1 μg of total RNA from each sample was used for library period 24:0 h light/dark regime was followed. Fish were preparation with the NEBNext Multiplex Small RNA Li- fed to satiation twice a day. After adaptation fish were brary Prep Set for Illumina. RNA adapters were ligated randomly divided into groups I and II (30 and 15 fish re- to the 3′ and 5′ end of the RNA molecule and the spectively). Then the fish from group I were exposed to adapter-ligated RNA was reverse transcribed into the supernatant of a KHV-infected CCB cell culture har- single-stranded cDNA. The cDNA was then PCR ampli- vested when the 90–95% cytopathic effect was evident. fied using a common primer and a primer containing KHV of known genotype (U/I) used to infect group I one of 9 index sequences. The introduction of the was isolated from an outbreak of the disease at a com- six-base indices at the PCR step allowed for the multi- mon carp culturing farm and stored at −80o C. Fish were plexed sequencing of different samples in a single lane exposed to infection through bath immersion for 45 min of the flowcell.
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