Segarra et al. Veterinary Research 2014, 45:103 http://www.veterinaryresearch.org/content/45/1/103 VETERINARY RESEARCH RESEARCH Open Access Ostreid herpesvirus type 1 replication and host response in adult Pacific oysters, Crassostrea gigas Amélie Segarra1, Laury Baillon1, Delphine Tourbiez1, Abdellah Benabdelmouna1, Nicole Faury1, Nathalie Bourgougnon2 and Tristan Renault1* Abstract Since 2008, massive mortality outbreaks associated with OsHV-1 detection have been reported in Crassostrea gigas spat and juveniles in several countries. Nevertheless, adult oysters do not demonstrate mortality in the field related to OsHV-1 detection and were thus assumed to be more resistant to viral infection. Determining how virus and adult oyster interact is a major goal in understanding why mortality events are not reported among adult Pacific oysters. Dual transcriptomics of virus-host interactions were explored by real-time PCR in adult oysters after a virus injection. Thirty-nine viral genes and five host genes including MyD88, IFI44, IkB2, IAP and Gly were measured at 0.5, 10, 26, 72 and 144 hours post infection (hpi). No viral RNA among the 39 genes was detected at 144 hpi suggesting the adult oysters are able to inhibit viral replication. Moreover, the IAP gene (oyster gene) shows significant up-regulation in infected adults compared to control adults. This result suggests that over-expression of IAP could be a reaction to OsHV-1 infection, which may induce the apoptotic process. Apoptosis could be a main mechanism involved in disease resistance in adults. Antiviral activity of haemolymph againstherpessimplexvirus(HSV-1)wasnotsignificantly different between infected adults versus control. Introduction infection of C. gigas spat with viral suspension [19,20]. In 1972, virus particles morphologically similar to herpesvi- These abnormal mortalities have not occurred among ruses were first reported in an invertebrate, the Eastern oys- C. gigas adults in France. Nevertheless, virus DNA and ter, Crassostrea virginica, from the USA [1]. Since then, the proteins can be detected in adult oysters in the ab- new family Malacoherpesviridae in the order Herpesvirales sence of mortality [21,22]. Similar observations were has been created in order to include a virus named ostreid reported in vertebrate herpesviruses such as channel herpesvirus type 1 (OsHV-1) infecting different bivalve spe- catfish virus (CCV) in channel catfish adults [23], or cies [2-4]. OsHV-1 is reported in mass mortality outbreaks Oncorhynchus masou virus in salmonid adults [24]. In observed among Pacific oysters less than one year old [5-8]. vertebrates, the strategies that herpesviruses use to infect Considering the economic importance of shellfish culture, cells during lytic or latent infection have been studied tools have been previously developed for rapid virus detec- intensively [25]. However, Information on these aspects of tion [5,9-12] and the OsHV-1 genome has been fully se- infection by OsHV-1 is rudimentary. quenced from purified virus particles [3]. Oysters have an efficient defense mechanism based on Since 2008, mass mortality outbreaks (with rates of up innate immunity. Studies have demonstrated that the to 80%) were reported in C. gigas spat and juveniles in immune system of C. gigas is capable of recognizing Europe, New Zealand and Australia [13-15] and were as- virus infection with resulting variations in expression of sociated with virus variants [16-18]. The role of OsHV-1 genes involved in virus recognition, signaling and im- in these mortalities has been underlined by experimental munity, that is Myeloid Differentiation factor 88, IkB2, Interferon-induced protein 44, Glypican and apoptosis * Correspondence: [email protected] inhibitor [26-29]. Due to the lack of molluscan cell 1Ifremer (Institut Français de Recherche pour l’Exploitation de la Mer), Unité lines for culturing OsHV-1, antiviral activities of mol- Santé Génétique et Microbiologie des Mollusques (SG2M), Laboratoire de lusks are often tested against another virus, using a plaque Génétique et Pathologie des Mollusques Marins (LGPMM), Avenue de Mus de Loup, 17390 La Tremblade, France assay [28,30-32]. Crassostrea gigas heamolymph contains Full list of author information is available at the end of the article © 2014 Segarra et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Segarra et al. Veterinary Research 2014, 45:103 Page 2 of 10 http://www.veterinaryresearch.org/content/45/1/103 a compound(s) that inhibits herpes simplex virus type 1 Mortality was monitored during a period of 144 h and (HSV-1) replication in Vero cells [28,30,31]. To date, in- cumulative mortality was defined daily for each condi- vestigations into in vitro antiviral responses of C. gigas tion (oysters infected with OsHV-1 or injected with ster- after an OsHV-1 infection have not been undertaken. ile artificial seawater). Although experimental protocols have been developed recently to reproduce the viral infection in laboratory Plasma and tissue sample collection conditions [19,20] the dynamic processes of infection For both families (A1 and A2), haemolymph was col- have not been studied in experimental conditions at the lected from six live oysters with a sterile syringe (1 ml, adult stage. Terumo) from the pericardial cavity and pooled for each In this context, adult oysters appear as animals of tank, condition and family. These samples were collected interest to study host and OsHV-1 interactions. Based at 0.5, 10, 26, 72 and 144 hours post-infection (hpi). on previous observations adult oysters are assumed to Haemolymph pools were centrifuged at 300 rpm during be more resistant to viral infection. 10 min at 4 °C then filtered at 0.22 μm in order to study In this study, we explored what happens in adult antiviral activity. All plasma samples (without haemo- oysters after a virus injection through monitoring cytes) were stored at −80 °C. mortality, antiviral activities and viral and oyster gene Two pieces of mantle were sampled from two live in- expression. dividuals per tank (6 individuals per time/condition) from both families. A piece of mantle (50 to 100 mg) Material and methods was disposed in a tube containing 1 mL of TRIZOL® Animals and experimental infection design Reagent™ (Ambion®) and frozen at −80 °C for further Two families of Pacific oysters, Crassostrea gigas, with RNA extraction. The second piece of mantle was dir- different genetic backgrounds were used in this study. ectly frozen at −20 °C for DNA extraction. Two families, A1 and A2, were produced using different genitors (one male and one female for each family) in DNA and RNA extraction and cDNA synthesis the Ifremer facilities located in La Tremblade (Charente DNA extraction was performed using a QiAamp tissue Maritime, France) in 2010. They were characterized by mini kit® (QIAgen) according to the manufacturer’sproto- high survival rates during their first year of life when col. Elution was performed in 100 μL of AE buffer pro- spat were reared in the field [33]. Experimental infec- vided in the kit. tions were carried out using oysters from both families Total RNA was extracted using TRIZOL® Reagent™ (two years old, 6 cm in length for A1 and 19 months (Ambion®) according to the manufacturer’s recommen- old, 4 cm in length for A2). For each family, 240 oysters dation. Total RNA was treated with Turbo™ DNAse were “anaesthetized” during 4 h in a solution containing (Ambion®) to remove genomic DNA. The RNA quality magnesium chloride (MgCl2, 50 g/L) in seawater (1 v)/ and quantity were determined using NanoDrop 2000 distilled water (4 v) [34]. One hundred μL of an OsHV-1 (Thermo Scientific) and Bioanalyser 2100 (Agilent). (μVar [16]) suspension at 1.106 copies of viral DNA/μL First-strand cDNA synthesis was carried out using the were injected into the adductor muscle of 120 oysters SuperScript ® III First-Strand Synthesis System (Invitrogen) per family [19,20]. Similarly, 120 adults from each family using 8000 ng of treated RNA. Reactions lacking RT were were injected with 100 μL of sterile artificial seawater as performed after each DNAse treatment using real-time controls. Pacific oysters were then placed in four tanks PCR in order to control the absence of oyster and/or virus (30 oysters per tank/condition) containing 5 L of filtered genomic DNA. seawater (1 μm) at 22 °C. One tank per condition was dedicated to survival record and the three other were OsHV-1 DNA quantification dedicated to sample collection. The absence of bacterial Viral DNA quantification was carried out by real-time contamination of the viral suspension was tested before PCR in duplicate using a Mx3000 Thermocycler (Agilent) each challenge. The viral suspension was spread onto a according to previously described protocols [11,35]. Amp- Petri dish which contained marine agar and no bacteria lification reactions were performed in a total volume of were observed. 20 μL to study oyster gene or viral gene expression. Each Twenty oyster spat (7 months old, 3 cm in length) well contained 5 μLofDNA,10μL of Brillant III Ultra- were simultaneously injected in order to confirm that Fast SYBR® Green PCR Master Mix (Agilent), 2 μLofeach the virus suspension was infectious and mortality was primer (3 μM) and 1 μL of distilled water. Real time PCR monitored without sample collection. Spat were pro- cycling conditions were as follow: 3 min at 95 °C, followed duced at the Ifremer facilities (La Tremblade, Charente by 40 cycles of amplification at 95 °C for 5 s, 60 °C for Maritime, France) from wild oysters which were col- 20 s.