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Inflammation and innate immune response against viral infections in

marine fish.

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

1. Instituto de Investigaciones Marinas, CSIC. Eduardo Cabello 6, 36208 Vigo, Spain.

2. Institut de Biotecnologia i de Biomedicina. Universitat Autonoma de Barcelona, Barcelona, Spain

*: Corresponding authors

Dr. Antonio FiguerasInstituto de Investigaciones Marinas, CSIC. Eduardo Cabello 6, 36208 Vigo, Spain.Tel: 34 986 21 44 63Fax: 34 986 29 27 62E-mail: antoniofigueras@iim.csic.es

Dr. Simon MacKenzieUnitat de Fisiologia AnimalDept.de Biologia Cellular, Fisiologia i ImmunologiaEdifici C, Campus de BellaterraUniversitat Autonoma de Barcelona08158 Cerdanyola del VallesBarcelona, Spain.Tel: 34-93-5814127 Fax: 34-93-5812390 E-mail: Simon.MacKenzie@uab.es

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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.

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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

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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

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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-

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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

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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].

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Concerning interferon signalling, as McBeath et 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].

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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].

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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.

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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

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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

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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

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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-

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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

16

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.

17

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.

18

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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35

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.

36

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]

37

Figure 1.