Inflammation and innate immune response against viral infections

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Inflammation and innate immune response against viral infections Powered By Docstoc
					 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 Figueras
      Instituto de Investigaciones Marinas, CSIC.                                      Formatted: Spanish (Spain,
                                                                                       International Sort)
      Eduardo Cabello 6, 36208 Vigo, Spain.
      Tel: 34 986 21 44 63
      Fax: 34 986 29 27 62
      E-mail: antoniofigueras@iim.csic.es


      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: 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.


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


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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,

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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.



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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

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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.



                                                                                               6
        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           Formatted: English (United
                                                                                                  Kingdom)
was probably too late to detect it) [43]. In agreement with these results, in cod, the i.p.       Formatted: English (United
                                                                                                  Kingdom)
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             Formatted: English (United
                                                                                                  Kingdom)
against IPNV. Furthermore TNFα mRNA was not found to be induced after infection
[42].                                                                                             Formatted: English (United
                                                                                                  Kingdom)
         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 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.



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        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                 Formatted: English (United
                                                                                                    Kingdom)
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),

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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 TNF 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


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in increased antigen presentation to CD8+ cells in trout [70]. Natural Killer and                Formatted: English (United
                                                                                                 Kingdom)
cytotoxic T cells responses are activated after VHSV infection: leukocytes from
infected fish showed a higher transcriptional level of the CD8 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        Formatted: English (United
                                                                                                 Kingdom)
used as the basis for efficient DNA vaccines against rhabdoviral infections [75- 78].            Formatted: English (United
                                                                                                 Kingdom)
Lorenzen et al [79] suggested that DNA vaccination can be a good tool for studying               Formatted: English (United
                                                                                                 Kingdom)
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                Formatted: English (United
                                                                                                 Kingdom)
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].                 Formatted: English (United
                                                                                                 Kingdom)
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].                       Formatted: English (United
                                                                                                 Kingdom)
       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-                      Formatted: English (United
                                                                                                 Kingdom)
vaccinated trout showing that IL-1, MHC I, MHC II 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].

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       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-                Formatted: English (United
                                                                                                   Kingdom)
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              Formatted: English (United
                                                                                                   Kingdom)
strategy to evade the IFN system as has been described for mammalian viruses [95].                 Formatted: English (United
                                                                                                   Kingdom)
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.

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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)


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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

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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

                                                                                           14
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

                                                                                               15
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



                                                                                            16
       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.

                                                                                            17
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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.




                                                                                           34
  Figure 1.


Fish species   Pathogen Stimulus Tissue/Cell Type                   Platform      Reference


                                                                    1.8k SFA2.0
Salmo salar    ISAV     in vitro   ASK cells                                      [97]

                                                                    1.8k SFA3
               ISAV     in vivo    Spleen, gills, heart and liver                 [102]

                                                                    cGRASP
               ISAV     in vivo    Head Kidney                                    [103]

                                                                    cGRASP
               ISAV     in vitro   TO cells                                       [104]


Onchorynchus
                                                                    cGRASP
mykiss       IHNV       in vivo    muscle                                         [72]

                                                                    1.8k SFA2.0
               IHNV     in vivo    Head kidney                                    [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]




                                                                                              35
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