Fish & Shellfish Immunology 53 (2016) 35e49

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Fish & Shellfish Immunology

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Understanding the interaction between Betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy

* Janina Z. Costa , Kim D. Thompson

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, United Kingdom article info abstract

Article history: Over the last three decades, the causative agent of viral encephalopathy and retinopathy (VER) disease Received 27 January 2016 has become a serious problem of marine finfish aquaculture, and more recently the disease has also been Received in revised form associated with farmed freshwater fish. The virus has been classified as a Betanodavirus within the family 4 March 2016 Nodaviridae, and the fact that Betanodaviruses are known to affect more than 120 different farmed and Accepted 15 March 2016 wild fish and invertebrate species, highlights the risk that Betanodaviruses pose to global aquaculture Available online 17 March 2016 production. Betanodaviruses have been clustered into four genotypes, based on the RNA sequence of the T4 var- Keywords: fi fi Betanodavirus iable region of their capsid protein, and are named after the sh species from which they were rst Viral encephalopathy and retinopathy derived i.e. Striped Jack nervous necrosis virus (SJNNV), Tiger puffer nervous necrosis virus (TPNNV), VER Barfin flounder nervous necrosis virus (BFNNV) and Red-spotted grouper nervous necrosis virus Viral characterisation (RGNNV), while an additional genotype turbot betanodavirus strain (TNV) has also been proposed. Vaccines However, these genotypes tend to be associated with a particular water temperature range rather than Disease control being species-specific. Larvae and juvenile fish are especially susceptible to VER, with up to 100% mortality resulting in these age groups during disease episodes, with vertical transmission of the virus increasing the disease problem in smaller fish. A number of vaccine preparations have been tested in the laboratory and in the field e.g. inactivated virus, recombinant proteins, virus-like particles and DNA based vaccines, and their efficacy, based on relative percentage survival, has ranged from medium to high levels of protection to little or no protection. Ultimately a combination of effective prophylactic measures, including vaccination, is needed to control VER, and should also target larvae and broodstock stages of production to help the industry deal with the problem of vertical transmission. As yet there are no commercial vaccines for VER and the aquaculture industry eagerly awaits such a product. In this review we provide an overview on the current state of knowledge of the disease, the pathogen, and interactions between betanodavirus and its host, to provide a greater understanding of the multiple factors involved in the disease process. Such knowledge is needed to develop effective methods for controlling VER in the field, to protect the various aquaculture species farmed globally from the different Betanodavirus genotypes to which they are susceptible. © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction ...... 36 2. History of the disease ...... 36 3. Pathogen...... 36 3.1. Characterisation of the virus ...... 36

* Corresponding author. E-mail address: [email protected] (J.Z. Costa). http://dx.doi.org/10.1016/j.fsi.2016.03.033 1050-4648/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 36 J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49

3.2. Phylogeny and serology ...... 38 4. Thedisease...... 38 4.1. Clinical signs and tissue distribution ...... 38 4.2. Transmission ...... 39 4.3. Route of infection ...... 39 5. Host-pathogen interactions ...... 40 5.1. The pathogen response ...... 40 5.2. The host immune response ...... 40 6. Control measures ...... 41 6.1. Husbandry measures ...... 41 6.2. Therapeutics ...... 41 6.3. Betanodavirus vaccines ...... 42 7. Conclusion ...... 44 Acknowledgements ...... 44 Supplementary data ...... 44 References ...... 44

1. Introduction 2. History of the disease

Since its first outbreak in 1985, the disease Viral Encephalopathy Early studies described the agent of VER as a “picorna-like virus” and Retinopathy (VER) has been increasing in importance to the [7,8], but based on virion size and genome characteristics of Striped aquaculture industry, and is now recognised as a major problem in Jack (Pseudocaranx dentex) nervous necrosis virus (SJNNV), the Mediterranean and Asia marine aquaculture. Outbreaks of VER are pathogen was classified as a member of the Nodaviridae family [9]. associated with high levels of mortality, with up to 100% of fish A Nodaviridae virus was also identified as the pathogen involved in dying from the disease. In most cases of VER, the infection tends to VER outbreaks in European seabass (Dicentrarchus labrax) and be seen in post-hatch larvae, fingerlings and juvenile fish, and those Asian seabass (Lates calcarifer) [10]. In 2000, viruses within the surviving infection are inclined to perform poorly after recovering Nodaviridae family were re-classified into two genera, the Beta- from the disease [1,2]. nodavirus genus grouping the nodaviruses affecting fish, and the Because VER is caused by an RNA virus, which can be trans- genus Alphanodavirus that includes all the insect nodaviruses [11]. mitted both horizontally and vertically, the most sensible way of Betanodavirus is also referred to as NNV (Nervous Necrosis Virus). controlling this disease is to apply effective biosecurity and to Since 1985, VER has been reported worldwide, except for South vaccinate the fish. Although there are a number of promising America where no outbreaks of the disease have been reported studies in the literature focussing on different types of vaccines [12e16]. VER affects both farmed and wild fish, with more than 120 for VER (e.g. recombinant protein, DNA and inactivated vaccines), species belonging to 30 families from 11 different orders being there are still no commercial vaccines available for this disease. susceptible to infection (see supplementary Table 1). For more than This is possibly because the disease is associated with outbreaks a decade, VER was thought to be a problem solely of marine fish, in larvae and juvenile fish, prior to them becoming fully immu- but since 2000 the virus has also been reported in freshwater nocompetent, or perhaps other vaccine formulations/strategies species such as guppy (Poecilia reticulata), tilapia (Oreochromis are required to make the vaccines more efficacious in an aqua- niloticus), sturgeon (Acipenser gueldestaedi), Chinese catfish (Silurus culture setting. The lack of an effective commercial product asotus Linnaeus, 1758), Australian bass (Macquaria novemaculeata), highlights the need for a greater understanding of the interaction Nile tilapia (Oreochromis niloticus) and an endangered blenny spe- between the virus, the immune response of its host and the cies (Salaria fluviatilis) [17e22]. disease process that results. To be in a position to design a more VER is a neuropathogenic disease, and for this reason it was first effective vaccine against VER, we need a broader knowledge of: named sea bass viral encephalitis (SVE) [23]. The disease has also (a) the pathogen (e.g. its routes of infection, how it infects its been referred to as viral nervous necrosis (VNN) [24], fish viral host, how it manipulates the host's response to its own advantage encephalitis (FVE) [10] and viral encephalitis and retinitis [25]. The and evades the host's immune system, the role of the viral pro- disease is now officially named viral encephalopathy and retinop- _ teins in this process and what makes the virus temperature- and athy (VER) by the Office International des Epizooties (OIE), based on host-specific); (b) the host, (e.g. how it fights the viral infection, the histopathology that accompanies the infection [4]. the mechanisms involved and what makes some species sus- ceptible and others asymptomatic carriers) and (c) the disease (e.g. what are the clinical signs of the disease, how is it trans- 3. Pathogen mitted and how it can be managed though appropriate fish husbandry). Although a number of reviews are available 3.1. Characterisation of the virus providing an overview on VER [3e5], and more recently on the immunity of marine fish to Betanodavirus [6], the present review The first characterisation of Betanodavirus particles by electron presents the latest findings relating to the disease, the pathogen microscopy (EM) in 1991, showed that the virus has a typical itself, interactions between Betanodavirus and its host and an icosahedral shape with a diameter of 23e25 nm, while particles overview of the vaccine studies performed to date, in order purified on CsCl gradients have a diameter of 26 nm from side to inform on the development of future prophylactic approaches for side, 29 nm from point to point, and a buoyant density of 1.30 g per controlling VER. cm3 [7]. The nucleic acid and the structural proteins form a non- enveloped particle, with two single-stranded positive-sense RNA J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49 37

Fig. 1. Structure of grouper nervous necrosis virus-like particle (GNNV-LP). (A) A ribbon presentation of the subunit C of GNNV-LP. The disordered N-ARM (residue 1e33, gray), N- arm (residues 34e51, magenta), the S-domain (residues 52e213, red), the linker region (residues 214e220, blue), the P-domain (residues 221e338, cyan) and Ca2þ ion (yellow sphere) are shown. (B) A topology diagram of GNNV CP with the helices and strands in cylinders and arrows, respectively. The 1D topology of the subunit C is color-coded as in B. Adapted from Ref. [36]. molecules with molecular weights of 1.01 106 Da (RNA1) and 214e220) and (iv) the protrusion domain (P-domain, residues 0.49 106 Da (RNA2), and lacking a poly(A) sequence at the 30 221e238) (Fig. 1). Also, the CP of GGNNV is glycosylated [37].Itis terminus [9]. The two major polypeptides present consist of one of the CP that is responsible for the host-specificity observed among approximately 100 kDa encoded by RNA1, and another of 42 kDa Betanodavirus isolates [38], more specifically this specificity is encoded by RNA2 [9,10,26]. A subgenomic RNA3 is produced during located within the variable region T4 [39]. RNA replication and it is co-terminal with the 30 terminus of RNA1 Protein a is the precursor of the capsid protein and is important [27]. for viral assembly [40].InAlphanodavirus, protein a is auto- The RNA sequence of RNA1 contains a single open reading frame catalytically cleaved to form the mature coat protein subunits b (ORF) that encodes “protein A”, which is the viral component of the and g [41], while in Betanodavirus this doublet formation does not RNA-dependent RNA polymerase (RdRp) with a molecular weight occur as a result of auto-catalysis, but instead results from the (MW) of 110 kDa, and is the only enzyme encoded by this virus formation of an intramolecular disulphide bond between cysteines [28,29]. RdRp is non-encapsidated and amongst its functions par- 187 and 201 [42]. Cysteine 201, in conjugation with cysteine 115, ticipates in the selection of RNA template and initiation sites, RNA have been identified as essential for capsid formation and thermal synthesis and adding 50 caps or 30 polyadenylate to the RNA pro- stability of the virus particle [43]. The aspartic acid residues play a duced [29,30]. The size of RNA1 and its coding protein are host- pivotal role. Aspartic acid D75, common to all Betanodaviruses, is species dependent, varying from 983 aa (amino acids) in SJNNV, believed to represent part of a catalytic site that is involved in 981 aa in Atlantic halibut (Hippoglossus hippoglossus) (AHNNV) and capsid protein cleavage, and motif 130DxxDxD135 is associated with 982 aa in greasy grouper (Epinephelus tauvina) (GGNNV) [27,28,31]. calcium ions and is crucial for temperature stability, trimerisation It is also involved in the regulation of the virus' temperature and CP assembly [36,44,45]. The N-terminus of the coat protein, sensitivity [32,33]. rich in basic amino acids (nine arginine and six lysine), is assumed RNA2 has a single ORF that encodes the capsid or coat protein to participate in binding the RNA genome to the internal capsid (CP) (protein a) [34]. Several studies have indicated that the length wall [46], is responsible for the irregularity observed in the mobility of the RNA2 segment varies according to the virus strain analysed of this protein in SDS-PAGE [47], and acts as a molecular switch (GGNNV with 1433 nucleotides (nt), SJNNV with 1410 nt, Sea bass [36]. Amino acids 23 to 31 have been identified as a nucleolus NNV (SBNNV) with 1406 nt, and guppy Poecilia reticulate NNV with localization signal, but movement from the nucleolus to the cyto- just 1367 nt). Differences in the size of RNA2 do not necessarily plasm may depend on a specific nuclear export signal [40]. reflect differences in the size of the CP expressed, as all genotypes During RNA1 replication, the RdRp directs the production of the have the same size of coat protein (i.e. 338 aa), except the CP of subgenomic RNA3 [48]. Within the Nodaviridae family RNA3 en- SJNNV, which is 2 amino acids longer. However, all fish nodavirus codes one or two small non-structural proteins (B1 and B2), and studies appear to quote the same MW for the CP (i.e. 37 kDa) different Nodaviridae species can have one or both ORFs present, [22,27,34,35]. The T ¼ 3 crystal structure of the orange-spotted encoding these proteins. In some nodaviruses with both ORFs, just grouper NNV (Epinephelus coioides) (OGNNV) CP was obtain for the B2 is present during infection [49]. It is generally accepted that subunits A and B (residues 52e338) and subunit C (residues the B1 ORF corresponds to the C terminus of protein A, and B2 is 34e338), while the remainder of the CP molecule, composed of the encoded in the þ1 ORF (with respect to ORF A) and overlaps with N-terminal, was too disordered to be modelled [36]. Their study the C terminus of protein A [50]. Delsert et al. predicted RNA3 to showed that the CP topology has four distinct regions - (i) the N- have a size of around 400 nt [51], while Tan et al. suggested that the terminal arm (N-arm, residues 34e51); (ii) the shell domain (S RNA3 of GGNNV contained putative ORFs for both proteins B1 (111 domain, residues 52e213); (iii) the linker region (residues aa, nt 2692e3027) and B2 (75 aa, 2753e2980) [27]. More recently 38 J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49 the size of RNA3 has been confirmed to be 371 nt (located at nt only cluster geographically restricted to Japanese waters [67]. In the 2730e3100 within the RNA1 sequence), with just 1 ORF present, same year, the occurrence of reassortment events within Betano- consisting of 227 nt encoding B2 [31,52]. A comparative study of the davirus isolates from European sea bass were described for the first four betanodavirus genotypes revealed that the RNA3 of all four time in the Adriatic Sea (Croatia and Italy), with virus having an genotypes lack the B1 ORF [53]. However, in 2009 a study by Chen RNA1-SJNNV genotype and an RNA2-RGNNV genotype (SJNNV/ et al., using a cloned RGNNV B1 gene, suggested that RGNNV B1 has RGNNV) [78]. The opposite type of reassortment is now wide an anti-necrotic death function [54]. spread in Southern Europe and the Iberian Peninsula infecting In fish nodaviruses, B2 is a non-structural protein localised in European seabass, Gilthead seabream (Sparus aurata) and Sene- the cytoplasm during early infection (24 h post-infection, hpi), and galensis sole (Solea senegalensis), with reassorted Betanodavirus at later stages of the infection (48 hpi) is already localised in the having an RNA1-RGNNV genotype and a RNA2-SJNNV genotype nucleus where it accumulates [55,56]. It has been shown to be (RGNNV/SJNNV) [76,79]. And to complicate the classification of either an inhibitor of short interfering RNA (siRNA) silencing, by Betanodavirus even more, it was shown that the same fish can be binding and protecting the viral double-stranded RNA (dsRNA) co-infected by both RGNNV and SJNNV genotypes [80]. Until now [52,57,58], or a necrotic death factor [59]. The B2 protein contains a inter-genotypes reassortants between RGNNV and SJNNV have just mitochondrial target motif (41RTFVISAHAA50), where residues been isolated from the Southern Europe region [81]. The presence Val44, Ile45, Arg52 and Arg53 are fundamental for this targeting [55]. of these re-assortments shows the need for both genome segments The levels of B2 expression are different between acute and to be analysed to correctly classify the virus. chronically infected fish, with chronically infected fish lacking the The occurrence of reassortment is exclusive to RNA segmented presence of the B2 protein [60]. viruses, and provides the virus with a mechanism for faster evo- lution, allowing it to infect new hosts and avoid the host's immune 3.2. Phylogeny and serology response [81,82]. The RNA2 genome of reassortant RGNNV/SJNNV shows changes to that of SJNNV's RNA2. SJNNV is unable to cause Early phylogenetic analysis of fish nodaviruses showed that disease in European sea bass, while RGNNV/SJNNV can induce there are four distinct clusters of isolates: TPNNV (Tiger Puffer clinical signs and mortality in this species [83]. RGNNV, SJNNV and Nervous Necrosis Virus) (Takifugu rubripes), SJNNV (Striped Jack the reassortant RGNNV/SJNNV all cause high levels of mortality in Nervous Necrosis Virus), BFNNV (Barfin Flounder Nervous Necrosis Senegalensis sole, but the reassortant is so virulent that it kills all Virus) (Verasper moseri) and RGNNV (Red-Spotted Grouper Nervous challenged fish within a couple of days of infection with severer Necrosis Virus (Epinephelus akaara) [61]. Two other genetic groups symptoms resulting [83]. The comparison between RNA2 of RGNNV of Betanodavirus have been proposed, one for turbot (Scophtalmus and RGNNV/SJNNV reassortants suggests that amino acids 247 and maximus) (TNV) and another for Atlantic Cod (Gadus morhua) 270 might be involved in the infectivity of the reassorted Betano- (ACNNV) that includes isolates from Atlantic cod, haddock (Mela- davirus [79]. Infectivity trials with RGNNV/SJNNV mutants, with nogrammus aeglefinus) and winter flounder (Pseudopleuronectes amino acid-specific mutations on RNA2 at aa positions 247 and 270, americanus) [13,62]. However, only TNV has generally been resulted in a significant decrease in Senagalensis sole mortality accepted as the fifth cluster, while ACNNV has been included as a compared to fish infected with the reassorted wild type virus [84]. clade within the BFNNV cluster [63e65]. In 2004 another nomen- The wild type RGNNV/SJNNV isolate has six amino acids changes in clature scheme was proposed that was not associated with the fish the temperature-sensitive region (aa 1e445) of RNA1 and requires host, but simply refers to Betanodavirus genotype as I, II, III and IV a distinct temperature range of 18e22 C to be able to infect Sen- (corresponding to RGNNV, BFNNV, TPNNV and SJNNV, respectively) egalensis sole [85,86]. and allows subgroups within the designated genotypes [66]. Probably almost all RGNNV/SJNNV chimeras are the result of a Within this classification, genotype II (BFNNV) has been subdivided single reassortment event that occurred in the Southern Europe into 3 subgroups (a, b and c), reflecting the genomic differences during the early 1980s [81]. Intra-genotype reassortments have between the Canadian Atlantic cod (IIa), the Barfin flounder (IIb) also been observed. In Australia, an isolate with RNA1 and RNA2 and the Atlantic halibut/French European sea bass isolate (IIc). genes from two distinct RGNNV subtypes was found, while in According to this system TNV has been designated as genotype V Malaysia a Betanodavirus composed of an older RNA2 gene and [63,67]. younger RNA1 gene was detected [81]. It has been suggested that genetic diversity among Betanoda- Different studies have shown that SJNNV-polyclonal antibodies virus strains reflects significant phenotypic differences, which may can cross-react with other genotypes, indicating that the virus represent adaptation enabling infection of different host species isolates have epitopes or antigenic regions in common [87e89], but and/or replication at different temperatures [68]. The TPNNV there is no cross-neutralisation among the different genotypes cluster is the only cluster that contains a single fish host species, when using SJNNV-polyclonal or RGNNV-polyclonal antibodies Tiger puffer. Betanodaviruses isolated from other fish hosts in the [75]. A full serological study revealed that the four genotypes fall Genus Takifugu do not group within this cluster, but within the into three distinct serotype groupings, with group A neutralising RGNNV cluster [69]. The correlation between the various genotypes SJNNV, group B neutralising TPNNV and group C neutralising and their specific optimal growth temperature has been demon- RGNNV and BFNNV [90]. Similar results were obtained with RGNNV strated in vitro (BFNNV 15e20 C; TPNNV 20 C; SJNNV 20e25 C monoclonal and polyclonal antibodies that cross-reacted and and RGNNV 25e30 C) [70]. The host-temperature dependence neutralised BFNNV isolates, but not SJNNV isolates [91]. reflects the geographic distribution of the distinct clusters. The BFNNV group are all cold-water Betanodaviruses (Atlantic halibut, 4. The disease Pacific cod (Gadus microcephalus), Atlantic cod, European sea bass, Dover sole (Solea solea) and Barfin flounder) isolated in North 4.1. Clinical signs and tissue distribution America Atlantic waters, Norway, Scotland, France Atlantic coast or Japan [44,71,72]. The RGNNV is the cluster with widest geographic The clinical signs observed differ according to the fish species distribution, and is found all over the Mediterranean basin, USA, infected, but generally include abnormal colouration (pale or dark), French Polynesia, Asia and Australia [12,63,73e78]. Until 2007, anorexia, lethargy, floating upside down at the water surface, when isolated in the Iberian Peninsula, the SJNNV genotype was the resting on the bottom, sluggish behaviour and altered swimming J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49 39 such as rotating, spinning, horizontal looping, abrupt whirling, fertilisation has also been demonstrated [121,122]. It is believed sinking to the bottom, darting or swimming in a corkscrew fashion that the virus is carried in the gonads of broodstock and in several [3,4,8,24,92e98]. other organs (liver, kidney, stomach and intestine), with virus Histological diagnosis of VER is based on the extensive necrosis replication occurring during the stress of spawning, and coloniza- of the central nervous system (CNS), with numerous virus particles tion and replication has been clearly been seen in the testis of both present in the cytoplasm of affected nerve cells, extensive vacuo- European seabass and seabream [119]. During spawning the virus is lation and neuronal degeneration of the mid and hind brain and then shed from the gonads and the digestive tract of the brood- vacuolation of the retina [8,99]. Using EM, it was possible to stock, and subsequently infects eggs, sperm or larvae [119]. observe degeneration of brain and retina cells, and virions present Horizontal transmission of the virus through the surrounding within the cytoplasm of neurones that showed margination of water body has been confirmed from cohabitation trials and sur- nuclear chromatin [8]. In the retina, virions are either membrane vival of purified nodavirus particles in the water [2,122,123]. Such bound to endoplasmic reticulum (ER) or free in the cytoplasm, and transmission is a major problem within aquaculture systems, and is separation of the nuclear membrane and disintegration of the inner possible due to inadequate biosecurity [124] or the presence of cristae of mitochondria can be observed, with only vestiges of the asymptomatic carriers. For example, VER-susceptible European sea plasma membrane remaining [8]. Two degenerative processes, bass were farmed on the same site where the first VER outbreak pyknosis and cell lysis can be observed. Cell lysis occurs much more occurred in sturgeon [17], Gilthead sea bream, a known reservoir frequently and is closely associated with vacuolation, while for the virus, is farmed within the same systems or sites as Euro- pyknotic cells show virus particles densely packed in their cyto- pean sea bass [125], and Atlantic halibut surviving natural infection plasm, they contain a few degenerating mitochondria and cells can act as carrier for the virus one year after the infection, with appear to be highly necrotic [24]. Enlarged, round, basophilic cells, survivors still carrying the virus [1]. have been described in infected Asian seabass larvae, together with Invertebrates also act as natural reservoirs and possible carriers inclusion bodies and cytoplasmic vacuolation in cells of the optic of Betanodavirus. The presence of Betanodavirus has been detected tectum, cerebellum, tegmentum, vagal lobes, medulla oblongata in apparently healthy wild and farmed invertebrates and in in- and spinal cord [100]. The vacuoles observed are often very vertebrates held in public aquaria, such as the bivalves (mussel extensive and the resulting loss of neural substance gives a spon- Mytilus galloprovincialis and clam Ruditapes philippinarum), crus- giform appearance to the tissues, including the retina [100]. There tacean (southern humpback shrimp Pandalus hypsinotus, charybdid also seems to be histological differences according to the age of the crab Charybdis bimaculata, spiny lobster Pamulirus versicolor or fish, and although there are no age related differences in tissue- gastropod Opistobranchia [76,126,127]. Brine shrimp (Artemia sal- distribution of the virus, larvae exhibit heavy necrotization in ina) and rotifer (Brachionus plicatilis), essential for feeding marine their brain, whereas the brains of juveniles and adults show lower fish larvae, can also act as a carrier for the virus [128]. levels of tissue necrotization, even in the presence of higher virus concentrations [101]. A certain level of virus particles needs to be present in the tissue 4.3. Route of infection to cause pathology [102]. It is clear from the nervous tissues affected that Betanodavirus is a neuro-tropic agent, and does not There does not appear to be a single route of infection for cause pathology in non-neuronal tissues [24,93]. However, virus- Betanodavirus. The cerebellum, the optic tectum and the retina are like particles have been observed in gill pillar cells and tissue le- primary sites of lesions in infected Asian seabass, while the spinal sions during natural outbreaks of VER in Atlantic halibut, [96].Itis cord and spinal ganglia are the primary sites of lesions in Japanese possible to detect the virus (RNA1 and RNA2) in non-neuronal parrotfish (Oplegnathus fasciatus) [24,100]. In striped jack, necrosis tissue, including liver, kidney, digestive tract, heart, spleen, intes- and vacuolation of nerve cells are first observed in the spinal cord, fl fi tine, gonadal uid, ns and gills using molecular techniques, such particularly in the area surrounding the swim bladder, suggesting as reverse transcription polymerase chain reaction (RT-PCR), in situ that this area is the initial site of virus replication [129]. These hybridization (ISH), real-time nucleic acid sequence based ampli- authors suggest that the virus then spreads in an anterior direction fi cation (NASBA), quantitative PCR (qPCR or real time PCR) and to the base of the spinal cord and forward to the brain, terminating fi loop-mediated isothermal ampli cation (LAMP) or real-time LAMP in the retina, inferred from the brain and retina lesions that occur e [103 118]. These techniques only detect viral RNA however, and do later in the infection. In a study performed with Atlantic halibut, the fi not con rm whether the virus is infective or not. The titres of initial focus of lesions was observed in the caudal part of the brain betanodavirus particles in these tissues are very low however, with stem, together with the stratified epithelium of the anterior in- less than a 10 fold increase detected during the infection period, testine [130]. The subsequent spread of the infection to the CNS suggesting that although the virus spreads to non-nervous tissues, may have been through axonal transport to the brain stem via no viral replication occurs there [103]. Although, it has recently cranial nerves, including the vagus nerve. been shown that NNV replication takes place in the testis of Eu- It has been suggested that infection of the host may occur ropean seabass and seabream [119]. Molecular studies have through the nasal cavity [25,101]. The authors believe that the virus revealed that the dynamics of betanodavirus infection is fast, and it penetrates the nasal epithelium, disseminating through the olfac- is possible to detect and quantify virus in brain, kidney, spleen, gill, tory nerve and olfactory bulb, to the aboral brain tissue, medulla heart and muscle tissue within 4 h post-intraperitoneal (i.p.) in- oblongata, spinal cord and finally to the retina. jection [120]. The biological pathway of viral entry and infection into the host, and the cell receptors involved are still unclear, although entry of 4.2. Transmission the virus into host cells is believed to occur through endocytosis, and binding to sialic acid on the surface of host cells [131]. More Outbreaks of VER in fish larvae suggest that the disease is recently, HSCP70 (heat shock cognate protein 70) has been iden- vertically transmitted, and the virus has been found in ovarian tified a potential receptor or co-receptor, and has been shown to tissues, sperm, fertilised eggs and hatched larvae. The presence of interact with grouper NNV capsid and plays a crucial rule in the the virus in sperm, with subsequent infection of eggs during early stages of infection [132]. 40 J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49

5. Host-pathogen interactions cell death by affecting mitochondrial morphology and causing MMP loss [141]. The B2 protein is thought to trigger oxidative stress 5.1. The pathogen response by targeting the mitochondria and inducing H2O2 production; the H2O2 then activates the dynamin-related protein (Drp1), which Acute NNV infections in grouper can ultimately lead to persis- associates with the mitochondria resulting in fission and cell death tent infections due to immune evasion strategies by the virus [6]. [142]. The virus has developed mechanisms, which allows it to avoid both innate and cellular immune responses of its host. One of the main 5.2. The host immune response immune evasion mechanisms known for Betanodavirus is the ability of the B2 protein to antagonize the host's RNA interference Viral infections can induce both innate and cellular host im- [56]. By binding to long viral dsRNA, B2 protects the RNA from Dicer mune responses. The importance of innate immunity is well rec- cleavage and subsequent production of siRNA that would otherwise ognised in fish [143]. Cytokines are intercellular signalling induce RNA-silencing by the host's immune response against the molecules that regulate these responses [144] and are involved in Betanodavirus [57,58]. This, in turn, facilitates the accumulation of the induction of innate immunity, generation of cytotoxic T cells viral proteins in infected tissues. and antibody production [145]. They include a wide range of mol- Apoptosis is one of the main host immune defence mechanisms, ecules such as the interferon (IFN), interleukins (IL-1) or tumour but virus can also induce host cells to undergo apoptosis as an necrosis factor (TNF) that can function either as activators or sup- immune evasion mechanism [133]. The induction of apoptosis oc- pressors of immune activity. curs through three pathways - the death receptor or extrinsic Cells of the host immune response use PPRs (pattern recognition pathway (activated by caspase 8), the mitochondrial or intrinsic receptors) to recognise pathogen-associated molecular patterns pathway (activated by caspase 9), and the ER (endoplasmic retic- (PAMPs) on the virus [146]. It has been shown that three classes of ulum) stress mediated pathway (activated by caspase 8) [134]. PRR, the RLRs (retinoic acid-inducible gene I (RIG-1)-like re- Earlier studies demonstrated that protein a induces caspase- ceptors), the TLRs (Toll-like receptors) and the NLRs (nucleotide dependent apoptosis (caspase-3-like and caspase-8-like) [135].It oligomerization domain-like receptors) are involved in recognition has been shown that Betanodavirus can induce post-apoptotic to these virus-specific domains, which in turn, stimulate produc- necrosis cell death in host cells by phosphatidylserine (PS) exter- tion of interferon and other cytokines by the immune cells [147].An nalization (early apoptotic stage), fragmentation of the DNA, and in vitro study using a zebrafish cell line revealed that when infected interfering with the mitochondrial permeability transition pore with Betanodavirus two sets of PPRs were up-regulated, the RLRs (MPTP) that results in the loss of the mitochondrial membrane and the MyD88-dependent TLRs [148]. In the same study, it was potential (MMP) and release of cytochrome c (mid-apoptotic stage) shown that knockdown of RIG-I suppressed group II type I IFN [135e137]. Post-apoptotic necrosis is triggered by caspase 3 and 8, production and suppressed the migration of monocytes, suggesting activated by protein a [137]. The loss of MMP can be blocked by that during a Betanodavirus infection RIG-I plays an important role anti-apoptotic protein zfBcl-xL (a Bcl-2 family member) and by pro- in the inflammatory response and in the up-regulation of group II apoptotic BKA protein, another member of the Bcl-2 family type I IFN. The RLRs act as cytoplasmic viral RNA sensors and have a [136,137]. These studies demonstrate the importance of the Bcl-2 mitochondrial adaptor, the MAVS (mitochondrial antiviral signal- family in Betanodavirus infection, and the fact that a balance be- ling) [147].Anin vivo study using sea perch (Lateolabrax japonius) tween pro and anti-apoptotic proteins ordains how the cells will showed that the expression of MAVs increased in the brain, spleen react to the apoptotic stimulus [138]. Apart from the released of and kidney after infection with sea perch-RGNNV [149]. cytochrome c, this apoptosis process requires the synthesis of the TLR7, which recognizes viral ssRNA [150] has been shown to B2 protein [59]. Further studies have revealed that B2 expression have higher levels of expression in Atlantic halibut infected with does not up-regulate the cytochrome c released, but up-regulates Betanodavirus [151]. Bax (pro-apoptotic molecule of the Bcl-2 family), which triggers Interferons are the main cytokines to induce an antiviral im- mitochondrial-mediated cell death [139]. mune response [152], with IFN type I (IFN a/b or I-IFN) triggering The importance of the interaction between Betanodavirus pro- antiviral innate immune responses and type II (IFN g or II-IFN) is teins and the mitochondria of the host, resulting in host cell death, necessary for triggering adaptive immune responses in fish is now recognised. The B2 protein also induces necrotic cell death [152,153]. IFN type I has been categorized into two groups in fish, by targeting and binding to the mitochondria by forming small group I (4 cysteine residues, 4C I-IFN) with 2 sub-groups (IFNb and complexes with the mitochondrial matrix; it inhibits the activity of IFNc) and group II (2 cysteine residues, 2C I-IFN), with 2 sub-groups complex II and deprives the mitochondria of ATP [55]. As well as the (IFNa and IFNd) showing distinct expression patterns in different activity of protein a and the B2 protein, it has been suggested that cells and tissues [154e156]. Both types I and II IFN have been B1 protein is expressed during the early stage of the infection and detected in fish infected with Betanodavirus. High levels of IFN g interacts with the mitochondria, reducing early apoptotic PS expression were detected in Betanodavirus-infected Atlantic externalization and the MMP loss, which leads to an increase in the halibut [151], while up-regulation of the IFN g induced molecule amount of host cells available for viral replication [54]. IRF1 (interferon regulatory factor 1) has been found in infected The interaction between Betanodavirus and the ER stress- turbot [140]. In vitro studies with RGNNV in grouper brain cell line mediated pathway has been demonstrated by the up-regulation (GB) have shown over expression of IRF3 (interferon regulatory of the chaperone protein GRP78 (glucose-regulated protein) dur- factor 3), an increase in the expression of IFN-related genes and a ing the mid-stage of viral replication, and which interacts with reduction in virus replication in infected cells [157]. It has also been Betanodavirus RdRp in the mitochondria and helps enhance viral shown that I-INF is strongly up-regulated in European sea bass replication [134]. The same study also showed activation of both during Betanodavirus infection, and increased expression of 2C I- ER-associated caspase-12 and PERK phosphorylation. IFN is observed in infected grouper [158,159]. Oxidative stress induced by Betanodavirus has been observed in Mx, a group I type I IFN inducible protein [153,160], is one of the infected turbot brain by the presence of reactive oxygen species anti-viral immune molecules most studied in fish [161,162]. Studies (ROS) [140], and the presence of ROS in GF-1 cells was observed at with grouper and barramundi cell lines have shown that Mx plays an early stage of infection (24 h post infection, h.p.i.) and induced an important role in combating Betanodavirus infection by J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49 41 reducing viral proliferation [162e164], through an interaction with although expression of AMP genes (i.e. complement factor 3, RdRp and re-distribution of the RdRp protein to the perinuclear lysozyme, hepcidin, dicentracin, piscidin or b-defensin) was area for degradation [165]. More recently, it has been suggested detected in the gonad of both species, they were more activated in that endogenous Mx sequesters RdRp for degradation through the susceptible European sea bass, which the authors suggest re- autophagy and lysosomes [166]. Up-regulation of Mx in response to flects differences in susceptibility of the two species to the virus. Betanodavirus infection has also been observed in vivo in European The same research group examined the expression profiles of genes sea bass, Gilthead sea-bream, turbot and grouper [140,158,163,167]. involved in IFN production in the gonads and brain of the same Different Betanodavirus genotypes show differing abilities to species of fish after NNV infection and concluded that the innate induce a Mx response; SJNNV is able to induce much higher levels immune response in the brain of European sea bass was unable to of Mx transcription than RGNNV in European seabass, and this clear the virus and highlighted the importance of gonad immunity might explain the difference that is seen in the susceptibility of this for controlling the spread of the virus to offspring [187]. fish species to these two genotypes, with them being more sus- ceptible to RGNNV [168]. Mx follows the same pattern of up- 6. Control measures regulation in the brain of European sea bass (susceptible species) to that of Gilthead seabream (asymptomatic carrier); however the 6.1. Husbandry measures levels of expression are much higher in Gilthead seabream, which might explain the asymptomatic carrier status in this species [167]. Once Betanodavirus has infected the farm site it can be very The group II type I IFN (2C I-IFN) is poorly studied, but a recent difficult to eliminate, since it is one of the most stable viruses to study using 30 days post-hatch groupers showed a correlation affect fish [188]. Implementation of effective disinfection pro- between the number of Betanodavirus copies and 2C-IFN copies, cedures can help to control VER in fish-farms. Studies performed with maximum IFN response observed at 3 d.p.i., followed by a with SJNNV show that virus particles are completely inactivated at peak Mx response at 6e7 d.p.i [159]. pH 12 and with sodium hypochlorite, calcium hypochlorite, ben- Infections studies in vivo have shown that pro-inflammatory zalkonium chloride and iodine [189]. Heat treatment, ultra-violet cytokines, such as IL-1b, IL-6 and TNFa are up-regulated in light (UV) and ozone also efficiently inactivate SBNNV, while Atlantic halibut, European sea bass and turbot in response to NNV formalin, ethanol, methanol, ether and chloroform are not as infection [151,158,167,169]. During Betanodavirus infections in effective at inactivating the virus [190]. The same authors reported grouper the expression of these pro-inflammatory cytokines is that peroxygen and UV irradiation reduce the infectivity of SBNNV, mediated by STAT3 (signal transducer and activator of transcription however, the effect of chlorine, iodine and peroxygen was notice- 3) [170]. The promoter activity of STAT3 increases during infection ably reduced in the presence of organic matter. and this increase is associated with a lower severity in the vacuo- Due to vertical transmission and the high susceptibility of fish to lation and autophagy induced by RGNNV [170]. It has been sug- Betanodavirus at a very early age, it is important to implement gested that the substantial up-regulation of TNFa detected in the measures for reducing Betanodavirus mortality in larval fish and brain of European sea bass compared to that of Gilthead sea bream these include (a) screening broodstock for the detection of Beta- may to be responsible for the vacuolisation and neuro- nodavirus in ovarian biopsies, eggs and sperm by RT-PCR and inflammation seen in the brain of European Sea bass [167]. screening the blood of broodstock for antibodies against the virus Teleosts elicit an antibody response during viral infections [171], by ELISA (with only negative spawners selected for egg produc- and it has been shown that the activation of antibodies during tion); (b) disinfecting fertilised eggs with ozone or electrolyser- Betanodavirus infections is an import host immune response to treated seawater; and (c) screening hatched larvae by RT-PCR to VER, because the antibodies produced can neutralise the virus, prove that they are Betanodavirus-free [108,191e199]. Since Beta- preventing it from causing damage [6]. It is possible to observe up- nodavirus frequently occurs in larvae derived from eggs collected regulation of the IgM gene in fish with both high and lower levels of late in the spawning season, limiting the number of spawnings infection [158,172], and the presence of specific antibodies against reduces the incidence of vertical transmission [200]. Vaccination of the virus have been measured in a variety of fish species e.g. Eu- broodstock is another promising method for stopping the spread of ropean seabass, Asian sea bass, Atlantic halibut, Striped jack and the virus from broodstock to eggs [201]. grouper [105,158,173e178]. The antibodies produced target specific regions of the RGNNV and SJNNV's capsid protein. These regions 6.2. Therapeutics have been identified on the N-terminal, C terminal, and regions comprised by amino acids 91e162, 180e212 and 254e256 The use of chemicals to impair virus replication has been [179e182]. examined, and it has been suggested that Betanodavirus tropism An increase in the expression of T-cell marker genes (TRCb, CD4- involves the monoamine neurotransmitter system [202]. When the 2, CD4, CD8a, CB8b, Lck, NCCRP-1 and ZAP-70) has been observed in potential of using ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole-3- Atlantic halibut, European sea bass and Gilthead sea bream when carboxamide), an anti-RdRp drug, was investigated as a control infected with Betanodavirus [151,158,183]. After oral or bath for Betanodavirus infection, it was found to interact with RGNNV vaccination with inactivated Betanodavirus, gene transcripts for RdRp and inhibit the viral infection in vitro [203]. The viral load was CD8a, MHC-I, MHC-II, IgM and IgT were found to be up-regulated in also reduced using Oligonol (lychee's fruit purified phenolic orange-spotted grouper, while IgT was only up-regulated in the extract) when applied during the early stages of infection, sug- gills of bath-vaccinated fish and in the gut of fish receiving the oral gesting that this drug may interfere with attachment of virus par- vaccine [184]. ticles to the cell [204]. The target tissue of NNV is the CNS, an immunologically- The use of AMPs (antimicrobial peptides) and their ability to privileged site. The gonads are another immunologically- modulate the host's immune response was suggested as a possible privileged site, where the immune response is normally tightly method for treating Betanodavirus infected fish. When two AMPs, regulated to avoid germ cell damage [185], however NNV is known TH1-5 (tilapia hepcidin 1-5) and Epi-1 (epinedicin-1), were to spread by transmission from infected eggs and sperm. In a recent administered to fish, it was shown that TH1-5 helped to reduce the study, the role of antimicrobial peptides (AMPs) was examined in viral load during infection, while treatment with Epi-1 cleared the the gonads of European sea bass and Gilthead sea bream [186], and virus during and after the infection [205]. 42 J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49

Alpha-crystallins are ubiquitous proteins found in all verte- target virus. A RPS value of 88% was obtained when vaccinated fish 3.4 1 brates and have been shown to have a role in the host's stress were infected with 10 TCID50 fish , but the RPS value decreased 4.4 1 response, and they are members of the small heat-shock protein to 35% when fish were challenged with 10 TCID50 fish , sug- family with a chaperone-like function [206]. Using an in vitro sys- gesting that the performance of this vaccine might be insufficient tem, Chen et al. showed the crystallin of grouper to be a stress- for commercial use [176]. On the other hand, the efficacy of a re- induced protein with chaperone-like activities, preventing the ag- combinant partial capsid protein (rT2) from SJNNV was tested in gregation of misfolded proteins with an ability to increase the cells juvenile turbot, and an RPS value of 82% obtained when the protein tolerance to cellular stress, suggesting it may be useful as an anti- was emulsified in oil and administered by i.p. injection [215]. Some oxidant therapy [207]. degree of protection was achieved in juvenile turbot with 10 mg of The fact that IFN responses are non-specific has led to the sug- Escherichia coli-expressed AHNNV capsid protein, also adjuvanted gestion that they can be used as a therapy against viral infections and administered by i.p. injection (i.e. 67% RPS) [216]. More [208]. Significant protection against grouper Betanodavirus was recently, an adjuvanted recombinant capsid protein derived from observed when grouper IFN and salmon IFN when administered i.p. Asian sea bass betanodavirus and administered at a dose of 50 mg or orally to Malabar grouper larvae [209]. fish 1 provided a RPS value of 76% in Asian sea bass juveniles [217]. Virus-like particles (VLPs) are a promising vaccine alternative 6.3. Betanodavirus vaccines for Betanodavirus. When over-expressed the viral structure pro- teins spontaneously self-assemble into virus-like particles that are Vaccination has been used by the aquaculture industry since the antigenically identical to the native virus [218]. VLPs seem as early 1990s and now plays a major role in disease prevention in effective as inactivated betanodavirus and are a safe alternative to aquaculture, with several bacterial and viral vaccines commercially live attenuated vaccines because they do not contain any genetic available for fish. Despite intensive research efforts directed to- material from the virus and thus do not have the potential to be wards the development of a vaccine against Betanodaviruses, no infectious. The humoral immune response of malabar grouper commercial vaccine is yet available for VER apart from an inacti- (Epinephelus malabaricus) and dragon grouper (Epinephelus lan- vated RGNNV vaccine against VER of sevenband grouper in Japan ceolatus) were studied when immunised with a betanodavirus VLP [210]. expressed in Escherichia coli [219]. The authors found high levels of Fish surviving Betanodavirus infection have been shown to antibodies in fish by 4 w.p.v., which were capable of neutralising produce neutralising antibodies, which is thought to explain their native virus, and that booster vaccination elevated these levels, resistance to natural re-infection [176]. This observation suggests although high doses of VLPs actually resulted in a decrease in that vaccination represents a logical and effective means of con- antibody production. They also showed that an adjuvant was un- trolling VER. Various studies over the past two decades have shown necessary to elevate this antibody response and that immunisation that vaccination offers some protection against subsequent beta- with the VLPs could stimulate high antibody titres in vaccinated nodavirus infection. These have included the use of inactivated fish for more than 5 months. The immune response (antibody virus, recombinant viral capsid protein or virus-like particles response and expression profiles for immune-related genes) of expressed in various expression systems. Evaluation of the efficacy orange-spotted grouper, Epinephelus coioides vaccinated with of these has tended to focus on measuring the antibody response betanodavirus VLPs produced in Escherichia coli was also examined after vaccination or determining the relative percent survival (RPS) [220]. The authors found that a single dose of VLPs was able to after subsequent infection. produce high titres of neutralising antibody as early as 1 w.p.v., There have been a number of studies using inactivated beta- while activation of genes associated with cellular and innate im- nodavirus as potential vaccines against Betanodavirus. In one study, munity in the liver, spleen and head kidney were observed as early juvenile sevenband grouper were vaccinated with an i.p. injection as 12 h.p.v. In 2006, Thiery et al. examined the efficacy of RGNNV- of formalin-inactivated RGNNV [211]. Neutralising antibodies were genotype VLPs in European sea bass using coat protein expressed in detected from 10 days post vaccination (d.p.v.) until the end of baculovirus and found both the immune response and the pro- study at 160 d.p.v. and RPS values of 67% or higher were obtained in tective effect against viral challenge were dose dependent with RPS vaccination fish. Protection was still evident by 9 weeks post values as high as 89% obtained 30 days after challenge [221]. The vaccination (w.p.v.) in a field trial in which fish were exposed to a yeast Saccharomyces cerevisiae was also used to produce RGNNV natural viral infection (85% RPS). Neutralising antibodies were VLP particles, which were then administered to convict grouper 7 1 induced in these fish, when an inoculation dose of 10 TCID50 fish Epinephelus septemfasciatus as i.p. and oral vaccines [222]. The was used, and fish appeared protected when they has an antibody production of neutralising antibodies was observed with both titre of 1:200 or greater [178]. In similar studies, brown-marbled vaccination methods, while RPS values differed considerably, with grouper, Epinephelus fuscogutattus [212], and Asian sea bass [177] RPS values of 100% obtained with fish vaccinated by i.p. injection were vaccinated with a single intramuscular (i.m.) injection of and 50% for fish receiving the oral vaccine. formalin-inactivated RGNNV, which was shown to induce a sig- The feasibility of using synthetic peptides as Betanodavirus nificant increase in neutralising antibodies and levels of protection vaccines has also been examined. Knowledge of protective epitopes when vaccinated fish were experimentally infected with a homol- is required to develop such vaccines [223]. Preliminary studies have ogous isolate of the virus. There was also evidence of suppressed indicated their potential use and suggest that it is the N-terminal viral replication in the brains and kidneys of vaccinated fish. peptide of the virus, which is involved in protection [179,224]. There has been much interest in using recombinant protein Despite several advantages over conventional vaccines, peptide expression to develop a vaccine for VER. As early as 1995, Nakai vaccines are typically poorly immunogenic and adjuvants formu- et al. showed that a recombinant capsid protein from SJNNV, lation is an important consideration to enhance the host's response expressed in Escherichia coli, was able to elicit virus-neutralising to them. There are currently no peptide vaccines licensed for antibodies in striped jack, when administered to fish by i.m. in- commercial use, however [225]. jection [213]. As well as successfully inducing high titres of neu- DNA vaccines are expression based vaccines, which instead of tralising antibodies, it was subsequently shown that this type of using antigens to elicit an immune response, use a gene (or genes) vaccine could reduce mortalities in sevenband grouper [176] and that encodes the protective antigen(s) [226]. DNA vaccines offer the humpback grouper (Cromileptes altivelis) [214] using RGNNV as the advantage of conserving the native structure of the protein [227] J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49 43 and as well as having the ability to stimulate antibody production, expressed as early as 7 d.p.v., and induced levels of protection of 64% trigger helper T-cells and cytotoxic T-cells (CTLs) [228]. However, and 70% RPS in two separate vaccination trials. Following on from DNA vaccines have not readily gained public acceptance as they are their work and accounting for the fact that VER occurs in very young ® perceived to be a product of genetic manipulation, although Apex - fry, the authors appreciated that it was important to lessen the IHN, the only plasmid DNA-based vaccine to be commercialised, response time of the larvae to the vaccine and also to increase the has been licensed for use in Canada in 2005 against infectious vaccine's efficacy [233]. The authors chose to use Vibrio anguillarum, hematopoietic necrosis virus (IHNV). Work has been carried out to a strongly immunostimulatory fish pathogen as an alternative examine the feasibility of using DNA vaccines in the control of VER. expression host to express recombinant NNV coat protein. Inacti- It has been shown that rhabdovirus DNA vaccines can induce a non- vated recombinant V. anguillarum were encapsulated into Artemia specific immune response that confers protection against other and when its efficacy was compared to that of the E. coli-expressed viruses. This cross-protection was tested in a challenge study in oral vaccine, they found it give greater and earlier protection than which a DNA-VHSV vaccine protected 2.2 g turbot against beta- their previous E. coli-expressed oral vaccine. It also elicited a higher nodavirus infection [229]. A RPS value of 100% was obtained when Mx expression in the brain and viscera of vaccinated fry together the fish were challenged 8 d.p.v., however the RPS dropped to 63% with higher titres of NNV specific antibodies [233]. when fish were challenged 35 d.p.v. This high level of protection Other studies have used bath vaccination as an alternative early on in the infection is a consequence of short-lived cross- immunisation route. The efficacies of formalin-inactivated Beta- protective anti-viral defence mechanisms from the innate immune nodavirus, with or without encapsulation, when used to immunise response. In parallel to the AHNNV recombinant vaccine discussed orange-spotted grouper larvae (0.2 g) by bath, showed that the above, the same authors produced a DNA vaccine against AHNNV inactivated vaccine was significantly improved by nano- [216]. In protection trials, this vaccine was unable to induce an encapsulation, using a patented technology of Alarvita Biolife Co immune response or protect challenged fish, unlike the recombi- [234]. The authors also showed that un-encapsulated virus inacti- nant protein, which gave an RPS value of 67% by i.p. injection. vated with 0.4 mM binary ethylenimine (BEI) gave higher levels of Although transcription and translation of the DNA construct was survival (79 < RPS<95) than virus inactivated with 0.1e0.2% evident in the muscle tissue in vivo, a humoral immune response formalin (39 < RPS<43). The BEI-inactivated betanodavirus pro- was not induced in these fish. More recently it has been shown that tection peaked at 30 (d.p.v.) and was retained for at least 3 months. DNA vaccination of 10e15 g Asian seabass with an Asian sea bass Due to the fact that there is strong evidence that Betanodavirus Betanodavirus construct induced a significant increase in the serum is vertically transmitted and present in broodstock fish antibody level by 3 w.p.v, and elicited a RPS value of 77% [230]. The [122,173,196,235], an alternative approach to vaccinating larvae and Asian seabass in their study were 10e15 g, while the turbot used in juvenile fish is to vaccinate the broodstock, thereby reducing the the study of Sommerset et al. (2003) were 2.2 g, and this may reflect risk of vertical transmission of the virus in the offspring. Successful the difference in the response to the VER DNA vaccine. The use of an transfer of specific antibody has been detected in offspring of other appropriate adjuvant may be key for developing a successful DNA fish species after having immunized the broodstock prior to vaccine for VER. Immunising orange-spotted grouper larvae with breeding [236e238]. Vaccination of potato grouper (Epinephelus an MGNNV (Malabar grouper NNV)-DNA vaccine, adjuvanted with tukula) broodfish (35e60 kg) with an intramuscular injection of class A CpG ODN (oligodeoxynucleotide) [231] induced moderate/ inactivated betanodavirus in Freund's complete adjuvant reduced high levels of protection in a dose-dependent fashion at 1 w.p.v., the risk of vertical transmission by the virus [201]. After five with RPS values of 43% and 47% obtained when fish were chal- months post-vaccination, the levels of specific antibodies in lenged with a viral dose of 2 106 TCID50 and 2 107 TCID50, offspring from the vaccinated broodstock were still elevated when respectively, and a RPS of 71% was obtained 2 w.p.v. when chal- compared to those from non-vaccinated broodstock, and while the lenged with the higher dose. The vaccine on its own was able to virus was detectable in the eggs of non-vaccinated fish, it could not elicit both innate and adaptive immune responses with associated be detected in the eggs from vaccinated fish. up-regulation of TLR9, Mx, IL-1b, specific antibodies and Th1 and The administration of immunostimulatory compounds has been Th2 markers T-bet and GATA-3, respectively. The use of the adju- proposed as a way of enhancing the immune response of larval fish, vant elicited slightly higher levels of RPS when fish was challenged prior to their adaptive immune system becoming fully developed at the highest viral concentration at 1 w.p.v., which might be e.g. recombinant Reishi immunomodulatory protein [239]. Poly I:C correlated with the higher levels of up-regulation of TLR9, Mx and administered with live NNV has been shown to confer protection in IL-1b observed in vaccinated fish at 7 d.p.v. sevenband grouper, both under laboratory conditions [240] and in Many of the vaccine studies discussed have been performed in the field [241], whereby injection of poly I:C 20 days after a VER non-juvenile fish by i.m. or i.p. injection. Betanodavirus tends to outbreak improved the survival of infected fish, and the duration of affect young fish however, especially larvae and fry, making this protection lasted for more than 10 months. The same group administration of vaccines by injection difficult and often inap- showed that vaccination of fish with a live vaccine at a suboptimal propriate since the fish are not fully immunocompetent. Other temperature of 17 C improved the survival of fish to VER when routes of vaccination have been investigated to overcome the dif- reared at an optimal temperature of 26 C [242]. Tanaka et al. ficulty of mass vaccinating small fish, although there is a worry that previously showed that water temperature can influence the small fish are insufficiently immunocompetent to respond to the development of VER, and that earlier and higher levels of mortality vaccine. occur at higher water temperatures [243]. Encouraging results were obtained using a recombinant NNV The protection elicited by simultaneous vaccination with an capsid protein, expressed in E. coli, and encapsulated into Artemia, as aquabirnavirus (ABV) mixed with inactivated RGNNV was investi- an oral vaccine for orange-spotted grouper larvae [232]. One of the gated in sevenband grouper [244]. Fish were injected with either major problems with oral vaccination is digestion of antigen in the live ABV (i.m.), inactivated RGNNV (i.p.) or with both, and subse- gastrointestinal tract, so in this study the authors claim that using quently challenged with RGNNV. Fish receiving ABV and inacti- Artemia to deliver the antigen has the advantage that they are a vated RGNNV together showed an RPS of over 60% as early as 3 d.p.i. natural starter feed for larval fish, helping to ensure antigen uptake. The ABV seemed to have a potential immunomodulatory activity, The authors showed that the antigen was taken up in the hindgut of offering non-specific protection against RGNNV at a very early stage the grouper and specific antibodies against the antigen were in the infection. 44 J.Z. Costa, K.D. Thompson / Fish & Shellfish Immunology 53 (2016) 35e49

7. Conclusion study of pathological findings in Atlantic halibut, Hippoglossus hippoglossus (L.), throughout one year after an acute outbreak of viral encephalopathy and retinopathy, J. Fish. Dis. 27 (2004) 327e341. Successful control of VER within aquaculture systems is not [2] M. Arimoto, K. Maruyama, I. Furusawa, Epizootiology of viral vervous vec- straightforward and is influenced by a number of factors high- rosis (VNN) in Striped Jack, Fish. Pathol. 29 (1993) 19e24. lighted in this review. Betanodaviruses are very resilient in the [3] B.L. Munday, T. Nakai, Special topic review: nodaviruses as pathogens in larval and juvenile marine finfish, World J. Microb. Biot. 13 (1997) 375e381. aquatic environment and are hard to irradiate from farm sites once [4] B. Munday, J. Kwang, N.J. Moody, Betanodavirus infections of teleost fish: a introduced. Their transmission is an important consideration when review, J. Fish. Dis. 25 (2002) 127e142. devising control strategies, with broodstock and larval fish being [5] M. Shetty, B. Maiti, K. Shivakumar Santhosh, M.N. Venugopal, I. Karunasagar, Betanodavirus of marine and freshwater fish: distribution, genomic organi- sources of replicating virus, and asymptomatic carriers responsible zation, diagnosis and control measures, Indian J. Virol. 23 (2012) 114e123. for horizontal transmission. Husbandry measures are effective in [6] Y.M. Chen, T.Y. Wang, T.Y. Chen, Immunity to betanodavirus infections of reducing VER outbreaks, but are not sufficient to completely control marine fish, Dev. Comp. Immunol. 43 (2014) 174e183. [7] G. Breuil, J.R. Bonami, J.-F. Pepin, Y. Pichot, Viral infection (picorna-like virus) them. 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