39 INFLAMMATION AND INNATE IMMUNE RESPONSE AGAINST VIRAL INFECTIONS

39 INFLAMMATION AND INNATE IMMUNE RESPONSE AGAINST VIRAL INFECTIONS
ABOUT MENINGITIS WHAT IS MENINGITIS? MENINGITIS IS AN INFLAMMATION
ADIPOSE TISSUE INSULIN RESISTANCE AND INFLAMMATION BUT NOT REDUCED

BACTERIAL MENINGITIS WHAT IS MENINGITIS? MENINGITIS IS AN INFLAMMATION
BALANOPOSTHITE BALANITE INFLAMMATION DU GLAND POSTHITE INFLAMMATION
CHRONIC INFLAMMATION TYPES 1 SECONDARY (PROLONGED ACUTE) 2 PRIMARY

Inflammation and innate immune response against viral infections in marine fish

Inflammation and innate immune response against viral infections in marine fish.

Novoa B. ¹, Mackenzie S.²*, Figueras A.¹*

Instituto de Investigaciones Marinas, CSIC. Eduardo Cabello 6, 36208 Vigo, Spain.
Institut de Biotecnologia i de Biomedicina. Universitat Autonoma de Barcelona, Barcelona, Spain

*: Corresponding authors

Dr. Antonio Figueras

Instituto de Investigaciones Marinas, CSIC.

Eduardo Cabello 6, 36208 Vigo, Spain.

Tel: 34 986 21 44 63

Fax: 34 986 29 27 62

E-mail: [email protected]

Dr. Simon MacKenzie

Unitat de Fisiologia Animal

Dept.de Biologia Cellular, Fisiologia i Immunologia

Edifici C, Campus de Bellaterra

Universitat Autonoma de Barcelona

08158 Cerdanyola del Valles

Barcelona, Spain.

Tel: 34-93-5814127

Fax: 34-93-5812390

E-mail: [email protected]

Abstract: Viral infections in fish are common in both natural and cultured fish populations and the spread of infectious disease is a serious threat to both natural ecosystems and commercial exploitations. A significant body of studies have addressed the host response to viral infection including the efficacy of DNA vaccines however we still have a fragmented vision of both pathologies associated with viral infection and the immune response to those across fish species. Many studies have concentrated upon freshwater fish including the zebrafish (Danio rerio) and the Rainbow trout (Oncorhynchus mykiss) whereas the majority of marine fish studies address the Atlantic salmon (Salmo salar). Here we provide a comprehensive review concentrating upon the salient pathological features of the most common viral infections including examples of the Betanodaviruses, Birnaviruses, Rhabdoviruses and the Isavirus in cultured fish with emphasis where possible upon non-salmonid cold water adapted marine species. In parallel we review the current state of the art mainly in reference to gene expression studies describing the host innate immune response concentrating upon the inflammatory response and its relationship toward anti-viral immunity in fish. Due to the complexity of the observed responses and the limitations of candidate gene expression studies to describe global biological processes, recent efforts in the use of microarray analysis for the study of the anti-viral response have been highlighted including members of the Pleuronectiform and the Perciform families. Finally we review the potential of the zebrafish to become a significant biological model in the elucidation of the molecular mechanisms underlying the piscine immune response to viral infection.

Introduction

Teleost fish are the largest group of vertebrates with a complete immune system since they present innate and specific immune mechanisms as mammals. Non-specific or innate immune responses are immediately active and not antigen-specific. Innate immunity maintains the host integrity and is based upon physiological and inflammatory responses. However, sometimes, the damage caused by pathogens in the host may result not only from direct effects produced by their replication or by the release of toxic molecules, but also from indirect effects mediated by an excessive or inadequate immune response.

Innate immunity focuses on highly conserved and essential components of microbes (cell wall structures, nucleic acids) called “Pathogen-associated molecular patterns” (PAMPs). Pathogen recognition involves the interaction of PAMPs with cellular receptors called “pattern recognition receptors” or PRRs such as Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I) receptors. The activation of many of these receptors induces the production of pro-inflammatory cytokines and interferons (IFNs), and also activation of cells involved in inflammation and the induction of adaptive immunity

Innate defence mechanisms provide protection to fish and, as Ellis [1] in his seminal review pointed out, their importance is three-fold: i) non-specific protection does not depend upon pathogen recognition; ii) they are relatively quick to respond, and iii), they are relatively temperature independent.

Although numbers of studies on fish immune responses against viral infections have considerably increased in the last years, we still have a fragmented vision on how fish deal with most viral infections. Most of the publications have been on species adapted to warmer climates (e.g. zebrafish and Japanese pufferfish) or salmonids, while cold-water adapted marine species have received considerably less attention. Moreover, little is known about the mechanisms involved in the carrier state in fish associated in many occasions with viral infections.

In this review we have focused on the innate immune responses, mainly those related with gene expression, elicited by the infection of the most important viruses affecting cultured fish species. They are notifiable diseases (OIE), which means that they are required by law to be reported to government authorities. In addition we have also included nodavirosis due to its increasing importance in marine fish worldwide.

Pathogenesis and inflammatory responses in fish viral diseases

Nodavirus

Viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis (VNN) is a disease caused by several Betanodaviruses (non-enveloped, positive stranded RNA viruses), inducing high mortalities in larval and juvenile stages of infected marine fish. The disease caused by these viruses is characterised by lethargy, abnormal spiral swimming, loss of equilibrium and neurological lesions, with cellular vacuolisation and neuronal degeneration mainly in brain, retina, spinal cord and ganglia of the affected fish [2-10]. Since its first description in larvae and juveniles of sea bass (Dicentrarchus labrax) reared in Martinique [2], the disease has spread to many other marine species worldwide [3- 8], and recently to freshwater fish [9- 10].

Despite the many species affected by this disease, pathogenesis and immune response against nodavirus is not well understood. Nodavirus replication in immune cells appears to be limited, however, blood leukocytes of sea bass are responsive to in vivo nodavirus infection, since a detectable increment of T and B lymphocyte number was observed during nodavirus infection. Moreover, leucocytes from blood, head kidney, and gills showed a higher viability after “in vitro” addition of inactivated viral particles [11].

In vivo studies indicate that nodavirus can be detected early after infection in the blood and kidney where there is an upregulation of proinflammatory cytokines (probably a generalised response against the infection) in sea bream (Sparus aurata) and sea bass. However, after 3 days, the highest viral titer was mainly detected in brain, the target organ for viral replication where a strong inflammatory response was observed [12]. Thus suggesting that this response may be responsible for the observed neurodegeneration and encephalomyelitis associated to nodavirus disease. In fact, this neuroinflammatory reaction (rapid secretion of IFN-γ and proinflammatory cytokines including IL-1β, TNF-α) has been reported in higher vertebrates after viral encephalitis produced by a virus like Herpes simplex virus type-1 [13- 14]. Interestingly, although the TNFα and IL β over-expression in sea bream (non susceptible species) was similar to that observed in the brain of infected sea bass (highly susceptible species), the mRNA expression values for TNFα were much higher in sea bass (>30 times) than in sea bream [12].

Naïve sea bass juveniles intramuscularly infected with a sublethal dose of nodavirus followed after 43 days by a similar boosting showed an upregulation of Cox-2 until boosting, an upregulation of TGF-β and IL-10 after boosting and also the modulation of IL-1, TNF-α which suggests, as Scapigliati et al [11] pointed out, a complex pattern of inflammatory responses during in vivo viral infection in fish species. Increased expression of proinflammatory cytokines may be responsible for the vacuolisation and the neuroinflammatory processes associated with this disease. This has been described for the brain damage associated to the pathogenesis of some neurodegenerative diseases and also during microbial infections of the nervous system including viral encephalitis [15-21].

Proinflammatory and cytokine genes have also been described in characterised EST libraries from nodavirus-infected fish including sea bream [22], Atlantic halibut [23], sea bass [24] and turbot (Scophthalmus maximus) [25]. Nodavirus induced the transitory expression of TNF-α, IRF-1 and Mx in turbot brain. Moreover, the daily administration of corticosteroids (with known anti-inflammatory and immunosuppressive properties) reduced the expression of these genes and it seemed to accelerate the mortality induced by nodavirus. However, if this treatment was delayed 7 days post-infection, the mortality was similar to that of the untreated group. This suggests the importance of an early inflammatory response in nodavirus infection [26].

Another study that analysed the implication of inflammation in nodavirus disease was recently reported by Poisa-Beiro et al. [27]. Using the suppression subtractive hybridisation (SSH) approach, the effect of nodavirus infection on the sea bass head kidney transcriptome was analysed. Lectins, important molecules in innate immunity and regulation of adaptive responses, were found to be differentially expressed among the immune genes in the SSH library. Functional in vitro assays carried out with the recombinant Sbgalectin-1, one of the lectins with an increased expression, highlighted its potential anti-inflammatory activity. A dose-dependent decrease of respiratory burst was observed in head kidney leukocytes after incubation with Sbgalectin-1. Moreover, a decrease in the expression of proinflammatory cytokines (IL-1β and TNF-α) was observed in the brain of sea bass simultaneously injected with nodavirus and Sbgalectin-1 in respect to those infected with nodavirus alone, which suggests a potential anti-inflammatory role for the recombinant galectin-1, as previously proposed in mammals [28]. At the protein level, a tissue-specific induction of Sbgalectin-1 expression in brain after nodavirus infection was observed using Western Blot assays which was not detected neither in the brain tissue of control fish nor in head kidney samples suggesting again its possible role as the target tissue for the virus.

In nodavirus infections, there is also a strong interferon pathway response: Rise et al. [29] have reported this effect in brain from Atlantic cod (Gadus morhua) with an asymptomatic high nodavirus carrier state. In sea bass, Scapiggliati et al. [11] found a robust amplification in the expression of the antiviral proteins IFN and Mx after both infection and boosting. In sea bream, there was a strong up-regulation of Mx protein in the brain with respect to the one observed in sea bass which could be related to the effectiveness in resolving the infection and could explain why sea bream is an asymptomatic carrier of the disease [12]. An increase of the interferon-induced protein with helicase C domain 1 (mda-5) that regulates type I IFN production was also reported [22]. These results support the fact that fish brain, as in humans, even without being an immune organ, is able to trigger a strong inflammatory response characterised by the expression of inflammatory cytokines and antiviral molecules.

Birnavirus

Infectious Pancreatic Necrosis virus (IPNV) is a bi-segmented double-stranded RNA virus of the family Birnaviridae. It produces a serious viral disease in salmonids, especially at the fry stage [30] but also induces an asymptomatic carrier state in many farmed fish. In Atlantic salmon post-smolts, the disease occurs several weeks after transfer to sea water [31] and the clinical features are similar to those found in rainbow trout [32-33]: severe necrosis of the pancreatic acinar cells and intestinal mucosa, the intestine of moribund fish, usually empty of food, with a whitish yellow exudate and the liver can also show areas of severe focal or generalised necrosis [34]. Viruses with serological relatedness to the IPNV have been reported to cause diseases in some farmed marine fish species, such as turbot (Scophthalmus maximus) [35- 36], halibut (Hippoglossus hippoglossus) [30], cod (Gadus morhua) [37], etc.

Although Wechsler et al. [38] reported that striped bass (Morone saxatilis) infected with IPNV are stimulated to produce circulating neutralising antibodies (which can be depressed by exogenous corticosteroids) several publications have described the implication of the virus upon the suppression of lymphocyte responses. In this sense, the mitogenic response and non-specific cytotoxicity of trout head kidney leukocytes significantly decreased by the inoculation of the virus [39] and also there is a significant reduction of LPS-induced B cell proliferation in infected trout [40]. These results suggest that the suppression of immune responses can be involved in the establishment of the typical carrier state in fish after infection with IPNV.

There are, however, controversial results on the early responses against the infection, mainly on the activation of interferon and inflammatory pathways.

Concerning the inflammatory reaction, interleukin IL-1β is one of the best characterised pro-inflammatory cytokines often used as marker of an activated inflammatory response. IL-1β mRNA expression was assayed in vitro in response to IPNV in adherent cod head kidney cells using quantitative real time PCR and was the only gene related with inflammation responding to IPNV infection showing highest expression at 24 and 48 h [41].

In vivo, however, IL-1β was not induced by the IPNV infection in Atlantic salmon smolts [42] or it was only weakly upregulated (although in this case the first sampling was probably too late to detect it) [43]. In agreement with these results, in cod, the i.p. injection of IPNV induced the expression of gene markers for the innate antiviral defence (ISG15 and LGP2), while expression of interleukin IL-1β was not significantly increased [44]. This could indicate that IL-1β is not involved in the immune response against IPNV. Furthermore TNFα mRNA was not found to be induced after infection [42].

IL-10 is regarded as an anti-inflammatory cytokine and plays a crucial role in the regulation of inflammation. Since it is a Th2 cytokine and inhibits interferon-γ in the mouse, the upregulation of IL-10 could be a mechanism to control or limit the expression of IFN-γ directing the immune system from a Th1 response towards a Th2 response. However, in fish this is not completely understood. In fact, it has been suggested that it may function as an inflammatory cytokine due to a very rapid upregulation after stimulation with LPS similar to IL-1β [45]. In Atlantic salmon smolts challenged intraperitoneally and by cohabitation with IPNV, interleukin-10 was highly induced in head-kidney and spleen [43]. However, in cod, both an in vitro infection of adherent head kidney cells [41] or an intraperitoneal in vivo infection did not significantly induce IL-10 mRNA expression [44].

Concerning interferon signalling, as McBeathet al. [42] indicated, the induction of the IFN system by IPNV seems to involve complex virus/host interactions and may play a role in determining states of resistance/susceptibility. Moreover, IFN signalling after IPNV infection may be dependent on the type of cell infected.

In vivo, IPNV has been reported to induce IFN-like activity [46] and expression of interferon and interferon-induced molecules (Mx, ISG15, etc): in Atlantic halibut tissues [47-48], in Atlantic salmon following infection [42, 49], in Atlantic cod [50], etc.

However, there is some controversy as to whether IPNV induces IFN responses in fish cells. In a rainbow trout cell line, IPNV suppresses the early activation of Mx gene expression but this does not happen in salmon macrophages [49]. Jorgensen et al. [51] established a transgenic cell line containing a reporter construct expressing firefly luciferase under the control of the rainbow trout promoter for the IFN-induced Mx1 gene (CHSE-Mx10). These authors reported that IPNV did not activate the Mx promoter in vitro and that the addition of rIFN-α/β to viral infected cells reduced luciferase activity when compared to mock-infected controls, which indicates that the viruses interfere with IFN signalling. This suppression has also been reported after an in vivo infection in rainbow trout when IFN mRNA expression was analysed in the ovary [52].

Intra-peritoneal injection of IPNV also caused a significant induction of type II IFN. IFN-γ has a range of immunomodulatory properties including growth, maturation and differentiation of many cell types, increment of NK cell activity and regulation B cell functions. Moreover, it induces monocyte-like cells to produce CXC chemokines that recruit immune cells to the site of infection [53]. However, it is not clear if the IFN- upregulation after viral infection is related to the activation of antigen-specific cytotoxic CD8⁺ T-cells, macrophages or NK cells [42].

IPNV is known to be sensitive to the antiviral action of IFNs and interferon related genes (Mx, IPS-1) [54-55]. Interestingly, asymptomatic carriers of IPNV, in contrast to post-smolts, did not express Mx transcripts. However, they still had the ability to respond to injection of poly (I:C) [56]. It is clear that IPNV has evolved mechanisms to overcome the IFN responses. Viral proteins VP4 and VP5 seem the most probable candidates responsible for interfering with the IFN-signalling pathway in salmon [57].

Recent studies made in vitro and in vivo have shown that the upregulation of genes encoding proteins involved in viral protein degradation (such as proteasome activating subunit 3, PSME3) and translation inhibition (such as X-linked alpha-thalassemia/mental retardation syndrome, ATRX) could contribute to keep the number of virus particles low during viral persistence [58].

Rhabdovirus

Rhadoviruses are a group of viruses that gather several fish disease causing agents including the highly virulent Infectious Hematopoietic Necrosis virus (IHNV), the Viral Haemorrhagic Septicaemia virus (VHSV) and the Spring Viremia of Carp virus (SVC). Their genome consists of a single-stranded negative-sense RNA which codes for five structural proteins: a nucleoprotein (N), a polymerase-associated protein (P), a matrix protein (M), a RNA-dependent RNA polymerase (L) and a surface glycoprotein (G) responsible for immunogenicity. An additional gene, only present in some fish rhabdoviruses, codes for a non-structural protein Nv, with a possible role in viral growth and pathogenicity [59]. They are important fish viral pathogens, responsible for significant mortalities in farmed salmonids with losses, especially among juveniles, that can reach up to 90%.

Many studies have shown in the last years that rhabdoviruses induce a strong innate immune response characterised by the upregulation of inflammatory and interferon related genes. Using subtractive suppressive hybridisation in trout leukocytes, O’Farrel et al. [60] reported the induction of genes homologous to mammalian interferon responsive genes, three similar to chemo-attractant molecules (CXC chemokine, galectin), and two with nucleic acid binding domains.

In turbot, VHSV induced high TNFα mRNA expression [61] and in rainbow trout there was an increased transcription of IL-1β, IL-8, TGF-β and iNOS mRNAs at early times post-infection, which indicates that an inflammatory response is triggered by the virus or by induced proinflammatory cytokines [62]. IL-1β could be involved in the host protective mechanisms since Peddie et al. [63] reported that trout injected with IL-1β-derived peptides show some resistance to VHSV infection. Other genes such as interleukin-8, the cytotoxic T-cell marker CD-8 and complement factor C3 were also reported to be modulated after an IHNV infection [64].

Reactive oxygen and nitrogen radicals have been also recognised as potential proinflammatory mechanisms during viral infections [65]. In turbot, it was demonstrated that VHSV induces nitric oxide (NO) in head kidney macrophages and that NO has antiviral activity against VHSV [66]. Although no significant changes in ROS production were observed after infection with VHSV [67- 69], in a recent study this response was identified against an avirulent recombinant virus obtained with reverse genetics (Romero et al., unpublished results). The activation of this cellular innate immune system could be related to the induced protection conferred the recombinant virus.

IHNV infection leads to an induction of the MHC class I pathway which results in increased antigen presentation to CD8⁺ cells in trout [70]. Natural Killer and cytotoxic T cells responses are activated after VHSV infection: leukocytes from infected fish showed a higher transcriptional level of the CD8α gene (typical marker for mammalian cytotoxic T cells) and of the natural killer cell enhancement factor (NKEF)-like gene. This indicates that both innate and adaptive cell-mediated immune responses are triggered after VHSV infection [71].

Surface glycoprotein G of fish rhabdovirus has been identified as a potent elicitor of type I interferon (IFN)-mediated antiviral responses [72- 74] and it has been used as the basis for efficient DNA vaccines against rhabdoviral infections [75- 78]. Lorenzen et al [79] suggested that DNA vaccination can be a good tool for studying protective immune responses against these infections. Furthermore the efficacy of DNA vaccines from serologically unrelated rhabdoviruses in O. mykiss suggests that the rhabdoviral G proteins elicit a non-specific anti-viral immune [80]. However, the mechanisms through which resistance is conferred by these vaccines are unknown since sometimes neutralising antibodies do not correlate with protection. Possibly, innate immune components, such as complement, interferon, NK-cells and phagocytic cells, play an important role for activation of a subsequent specific response [79-81]. Inflammatory responses have been also described in DNA vaccinations. Lorenzen et al. [82] described that the injection site of vaccinated fish showed an inflammatory response which was affected by lower temperatures. TNF-α and IL6 transcript production was up-regulated in secondary lymphoid organs (head kidney and spleen) of trout immunised with a plasmid containing the G glycoprotein of VHSV [83].

Sánchez et al. [84] reported that the expression of CC chemokines in trout injected with a plasmid coding for the G glycoprotein gene of VHSV were induced. Cuesta and Tafalla, [85] compared the effects of VHSV on vaccinated or non-vaccinated trout showing that IL-1β, MHC Iα, MHC IIα IFN and Mx mRNAs were significantly up-regulated early after infection. The G glycoprotein has also shown to be a potent trigger of cytotoxic cells [86].

In non susceptible species such as seabream, VHSV was detected in several tissues but did not replicate and although the virus provoked a poor effect on the influx of leukocytes to the peritoneal cavity and phagocytosis activity, other innate functions such as the production of reactive oxygen intermediates (ROI) were increased suggesting that these early innate immune response could be involved in the clearance of the virus [87].

In a recent study, Purcell et al. [88] demonstrated that trout families with different susceptibility to IHNV were able to mount a rapid IFN response which correlated with viral load. The most resistant families had lower viral replication but did not show differences in innate immune gene expression compared to susceptible families. As the authors stated, other barriers to rapid viral replication appear to be involved as immune mechanisms against the infection.

Isavirus

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus and belongs to the genus Isavirus and represents an important threat for Atlantic salmon aquaculture. The ISA virus has a segmented genome composed of eight negative-sense single-stranded RNA (ssRNA) segments [89]. Common clinical signs of the disease usually include inflammation of the liver and spleen, haemorrhaging and anaemia, often leading to death [90].

ISAV infected fish showed increased Mx expression after infection reaching a maximum expression level 6 dpi [42]. In vitro studies also showed that ISAV is an early and powerful inducer of interferon and interferon induced genes (Mx and ISG15) [91- 92]. Mx expression in ISAV infected fish suggests that it may be involved in the pathogenesis of this viral infection. In fact, interferon-signalling antagonist viral proteins have been described [93- 94]. These proteins could be used by the virus as a strategy to evade the IFN system as has been described for mammalian viruses [95]. These results appear to indicate that induction of type I IFN and IFN-dependent genes in ISAV infected fish and cells may not provide protection against the virus.

An increase in IL-1β expression after six days was described in the ISAV infected fish [42]. Although the authors indicate that this can be due to the presence of an introduced bacterial infection, the control tanks containing media-injected fish produced no such increase. This result suggests that IL-1β can have a role during ISAV infection as it has described for other orthomyxoviruses [96]. In vitro, several immediate-induced genes in a macrophage-like cell line were indirectly implicated in pro-inflammatory responses via IL-1 signalling [97], however, this has not be confirmed in in vivo infections. Furthermore, changes in TNF-α mRNA, a key inflammatory regulator, have not been observed following infection with ISAV. Therefore if inflammation has a role in the survival of fish against this infection it remains unknown and requires further study.

Functional genomics in viral infection using microarrays

The objectives of transcriptomics to disease control management with reference to viral infection take on three significant forms: 1) the identification and development of biomarkers for prognosis and breeding programmes, 2) the design, development and evaluation of vaccines and 3) the comparative immunology of host-pathogen interactions (Fig. 1). The impact of microarray technologies upon the above over the last decade is steadily increasing and significant advances in sequencing technology aligned to whole genome programmes suggests a bright future [98]. To date, as shown in Table 1, the majority of studies have been conducted in Salmoniformes addressing IHN and ISA infection in in vivo infection studies although in vitro studies have also been carried out. In the Pleuronectiformes all published studies to date address in vivo infection with either VHS or Nodavirus. In the following sections we will describe the salient features of these studies in reference to each viral group.

Nodavirus infection

Park et al [25] used a cDNA turbot microarray to address the transcriptional responses of this fish species to Nodavirus infection at 3, 6, 24 and 72 hours post infection. Of the 1920 genes studied on the microarray, a total of 94 genes were differentially expressed in the kidney of the nodavirus-infected turbot. Mx, interferon inducible protein 35 (IFI35), saxitoxin binding protein 1, serum lectin isoform 4, serum-inducible protein kinase were differentially up-regulated genes. Genes involved in complement pathway and coagulation cascade were also significantly up-regulated (kinnogin I, haptoglobin, thrombin, and proteinase activated receptor 3). Thus suggesting that the Pleuronectiformes display a similar IFN driven response as observed in the Perciformes to Nodavirus with a parallel innate immune response.

Rhabdoviral infection

A good example of the potential of microarray analysis has been the elucidation of innate and adaptive immune responses to IHN, VHS and hirame rhabdovirus (HIRR) infection across 2 distinct phylogenetic groups (S. salar, O. mykiss and P. olivaceus). In the Japanese flounder the responses to DNA vaccines containing the viral G proteins of VHSV and/or HIRRV were analysed in a series of reports using a cDNA microarray enriched with 213 immune-related genes [99- 101]. All DNA vaccines containing the viral G glycoprotein conferred specific protection to fish challenged 1 month after vaccination. In these studies, the majority of differentially up-regulated genes responding to VHSV and HIRRV infection were identified 3 and 7 days d.p.v. The authors suggested that the type 1 Interferon (IFN) system may be of significance due to the number of IFN-related genes consistently up-regulated across vaccinations in their studies including interferon-stimulated gene 15kDa (ISG15), interferon-stimulated gene 56kDa (ISG56) and the Mx protein [101]. In concordance with these observations results from tissue surrounding the intra-muscular site of IHNV-DNA vaccination, profiled using the 16K GRASP cDNA array, in the Rainbow trout highlighted up-regulation of IRF-3, Mx, Vig-1 and Vig-8 [72]. These results from both species suggest that the host-expressed viral glycoprotein (DNA vaccine) induces a systemic non-specific type 1 IFN innate immune response. However the development of adaptive immunity including the functional role of specific T and B lymphocyte populations in the viral response that would shed light upon the mechanisms of action of DNA vaccine-induced protection is yet to be clearly identified.

Evidence for adaptive immunity was initially reported in the rainbow trout head kidney responding to in vivo virulent IHN, attenuated IHN and bacterial lipopolysaccharide challenge [98]. Using the 1.8k SFA2.0 immunochip (enriched for mRNA relevant to the immune system) to analyse acute (1-3 days) changes in response an IHN-dependent shift in the transcriptional programme of the head kidney was observed. This was described by an over-representation of the MHC class II, immunoglobulin and MMP/TBX4 response coupled to an inhibition of TNF-alpha, MHC class I and several macrophage and cell cycle/differentiation markers. Thus suggesting an inhibition of the proinflammatory response in IHN-infected trout head kidney tissue.

ISA infection

Jørgensen et al [102] reported an extensive tissue analysis (Table 1), using the 1.8k SFA2.0 immunochip, of a highly virulent ISA infection (Glesvaer 2/90) in Atlantic salmon to identify differences between early and late mortalities aiming to characterise molecular determinants of resistance. A progressive increase in IgT mRNA peaking >30 post infection in parallel to a concomitant decrease in IgM expression was recorded. A suite of regulated mRNAs related to B lymphocyte differentiation/maturation and activation of T lymphocyte-mediated immunity including; CD4, TGFβ, CD8a and IFNγ was reported providing further evidence of a co-ordinated regulation of innate and adaptive response to viral infection. Furthermore using linear discriminant analysis based upon QPCR, a minimum set of genes (5-lipoxygenase activating protein, cytochrome P450 2K4, galectin-9 and annexin A1) were selected from an unbiased microarray data set, using only expression profiles and no inference of function, and were shown to predict which class, early or late mortality, an individual fish would belong to. In relation to this a recent publication using the 32K cGRASP cDNA array addressed ISA infection in the salmon head kidney over a more acute time period (1-16 days) using a different serovar of ISA (NA-HPR 4 or HPR21) [103]. Results obtained suggest a low level response due to the low number of differentially expressed mRNAs identified over early stages of infection characterised by innate immunity (TRIM and chemokines). This was followed by a strong inhibition of mRNAs related to oxygen transport and erythrocytes that was proposed to reflect late stage anaemia during ISA infection.

Both ASK (Atlantic Salmon Kidney) [97] and TO (Atlantic Salmon macrophage/dendritic-like) [104] cells lines have also been used to probe the molecular basis of pathogenesis of cytopathic ISAV infection using the 1.8k SFA2.0 immunochip and 16K cGRASP array respectively. Interestingly both studies highlightcell-specific responses related to cellular susceptibility to ISA infection, where ASK cells display a strong response to ISA [97] and an ISAV strain-specific response (NBISA01, RPC/NB-04-085-1, RPC/NB-01-0593-1) where strains with lower pathogenicity caused larger transcriptomic remodelling when measured as transcript diversity [104]. Both studies then applied an extensive panel of QPCR primers (>20) derived from microarray data in order to characterise marker genes for ISA infection.

In summary, the application of microarrays to questions addressing viral infection in fish has generated a significant set of studies and preliminary tools which have been mainly aimed toward the study of disease processes in species of commercial interest. Studies upon rhabdoviruses have been directly linked to DNA vaccine testing whereas ISA studies as a whole aim toward the development of genetic markers for the disease. The complex biology of the immune response including different spatial-temporal expression profiles, multiple cell types and distinct body locations make complete mapping of a response a difficult and expensive activity. However foundations have been laid down and make an important contribution toward development in this field. Of particular interest is the identification of adaptive immune responses at very early stages of viral infection and in some tissues a suppression of inflammatory responses. However the intensity of tissue-specific inflammatory responses and its role in pathological manifestations of viral infection remains to be explored i.e. brain versus haematopoietic tissue response. These and future studies will provide important insights toward diagnostic/biomarker development and the understanding of the biology underlying vaccine-induced protective immunity in fish.

Potential of zebrafish (Danio rerio) as a model for the study of viral diseases

Zebrafish (Danio rerio) has been extensively used to study vertebrate development and recently interest has grown in the fields of human disease, cancer and immunology [105- 111]. The zebrafish with a complete (innate and adaptive) immune system has advantages over other vertebrate infection models, such as mice, because of its small size, relatively rapid life cycle and ease of breeding, transparency of early life stages and rapid growth allowing a high number of genetic screens and real-time visualisation. This has been shown already in a number of studies on bacterial diseases such as Streptococcus iniae, Salmonella typhimurium and Vibrio anguillarum [112- 115] and also zebrafish infection with Mycobacterium marinum has been proposed as a model for tuberculosis [116]. Infections with zebrafish have also been proposed to study fish viral diseases. Vaccine and treatment trials, sometimes highly expensive with commercial species, can be conducted at a reduced cost with this model. In addition, the zebrafish is the only lower vertebrate model where powerful genetic approaches can be conducted in order to ascertain the role played by particular genes in disease resistance. Sullivan and Kim [117] published a comprehensive review of the capabilities and potential of the zebrafish model system with an overview of information on zebrafish infectious disease models. So far this fish has been infected with IHNV, VHSV, IPNV, SVCV, Snakehead rhabdovirus (SHRV) and nodavirus [118- 128].

In many of these studies, similar symptoms to those present in susceptible commercial species were detected in zebrafish after the infection and mortalities can be reproducible. La Patra et al. [118] infected zebrafish hematopoietic precursors with IHNV and IPNV where a transient effect decreasing the number of red cells was detected. The kinetics of hematopoietic defects between IHNV and IPNV infection differed but fish infected with either virus had recovered by 6 days post-infection.

Sanders et al. [122] showed the susceptibility of zebrafish to SVCV. Mortality exceeded 50% in fish exposed to 10⁵ PFU of SVCV/ml at 20ºC. Affected zebrafish were anorectic and listless, with epidermal petechial haemorrhages followed by death. Fish presented lesions such as multifocal brachial necrosis and melanomacrophage proliferation in several tissues. Interestingly, López-Muñoz et al. [129] found that although larvae present a functional antiviral system, they are not able to mount a protective antiviral response against a waterborne SVCV infection. Similar results were found by Phelan et al. [128] in infections with snakehead rhabdovirus (SHRV). Zebrafish from 24 h to 30 days post-fertilisation were susceptible to infection by immersion in 10⁶ TCID₅₀ of SHRV/ml, and adult zebrafish were also susceptible to intraperitoneal infection. Mortalities exceeded 40% in infected fish (both larvae and adults), and clinical presentation of infection included the typical signs of rhabdoviral infections. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection.

Novoa et al [130] proposed zebrafish as a model for the study of vaccination against VHSV. Using an avirulent recombinant vaccine previously used for rainbow trout [131], zebrafish were protected against the VHSV infection.

Lu et al. [121] successfully established a nodavirus (NNV) infection in zebrafish. Infected fish exhibited typical nodavirus symptoms. Viral titers peaked at 3 days post-infection and histopathology showed lesions in the brain tissue similar to natural host infection. These authors suggest that the susceptibility to NNV infection is dependent on the enhancement of IFN system.

Conclusions

Due to the significance of viral infection and related mortalities in fish both in natural (e.g. VHS outbreaks in the Great Lakes of the U.S. 2005-7) and in commercially cultured fish populations there is a strong interest aimed toward understanding viral infection in fish and the development of methods including vaccination to combat such outbreaks. In this review we have covered the majority of significant viral infections where a complex picture is emerging between different viral infection strategies and corresponding immune responses. Studies using microarray platforms have significantly contributed in this area and underpinning molecular mechanisms are emerging however much work remains. In our opinion a central issue that remains to be resolved is the intensity of the host response in a specific tissue targeted by viral infection. Here the fundamental role of the inflammatory response and its involvement in either resolution of viral infection or dysfunctional responses leading to the establishment of asymptomatic carriers or extensive tissue damage leading to a negative outcome is central. Due to the complexity and relatively unknown nature of these responses i.e. the underlying molecular regulation, studies using a candidate gene approach are clearly limited. In view of the ‘toolbox’ available to fish immunologists which has a strong bias toward gene expression studies we propose that functional genomics, microarrays and RNA-Seq, will play an increasingly significant role toward the elucidation of the molecular mechanisms involved in the piscine anti-viral response.

Acknowledgements

We want to thank the funding from the project CSD2007-00002 “Aquagenomics” of the program Consolider-Ingenio 2010 from the Spanish Ministerio de Ciencia e Innovación.

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Figure legend.

Figure 1. Eschematic representation about the interaction on different new biotechnological tools used to understand the fish expression profile agaisnt a pathogen with the aim to obtain genetic markers or putative vaccine adjuvants.

Table 1. Summary of the studies conducted using microarray Technologies.

Fish species	Pathogen	Stimulus	Tissue/Cell Type	Platform	Reference

Salmo salar	ISAV	in vitro	ASK cells	1.8k SFA2.0	[97]
	ISAV	in vivo	Spleen, gills, heart and liver	1.8k SFA3	[102]
	ISAV	in vivo	Head Kidney	cGRASP	[103]
	ISAV	in vitro	TO cells	cGRASP	[104]

Onchorynchus mykiss	IHNV	in vivo	muscle	cGRASP	[72]
	IHNV	in vivo	Head kidney	1.8k SFA2.0	[98]


Paralichthys olivaceus	VHSV	in vivo	Head Kidney	1.2K cDNA	[99]
	VHSV	in vivo	Head Kidney	1.2K cDNA	[100]
	HIRRV	in vivo	Head Kidney	1.2K cDNA	[101]


Psetta maxima	Nodavirus	in vivo	head kidney	1.9K cDNA	[25]

Figure 1.

39 INFLAMMATION AND INNATE IMMUNE RESPONSE AGAINST VIRAL INFECTIONS

DOCUMENTATION FOR DATA ON BIOMARKERS OF INFLAMMATION PAGE 3
IDIOPATHIC (STERILE) CYSTITIS (INFLAMMATION OF THE BLADDER OF UNIDENTIFIABLE
IN VIVO IMAGING OF LUNG INFLAMMATION WITH NEUTROPHILSPECIFIC 68GA

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