Developmental and Comparative Immunology 37 (2012) 429–437

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Developmental and Comparative Immunology

journal homepage: www.elsevier .com/locate /dci

Viral nervous necrosis virus persistently replicates in the central nervous systemof asymptomatic gilthead seabream and promotes a transient inflammatoryresponse followed by the infiltration of IgM+ B lymphocytes

Azucena López-Muñoz a,b, María P. Sepulcre a,b, Diana García-Moreno a,b, Inmaculada Fuentes a,b,Julia Béjar c, Manuel Manchado d, M.Carmen Álvarez c, José Meseguer a,b, Victoriano Mulero a,b,⇑a Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, Murcia, Spainb Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spainc Department of Genetics, Faculty of Sciences, University of Málaga, Málaga, Spaind IFAPA Centro El Toruño, El Puerto de Santa María, Cádiz, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 January 2012Revised 3 February 2012Accepted 7 February 2012Available online 5 March 2012

Keywords:VNNVBlood–brain barrierViral infectionFish

0145-305X/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.dci.2012.02.007

⇑ Corresponding author at: Department of Cell BiolBiology, University of Murcia, 30100 Murcia, Spain. T868883963.

E-mail address: vmulero@um.es (V. Mulero).

The viral nervous necrosis virus (VNNV) is the causal agent of viral encephalopathy and retinopathy(VER), a worldwide fish disease that is responsible for high mortality in both marine and freshwater spe-cies. Infected fish suffer from encephalitis, which leads to abnormal swimming behavior and extensivecellular vacuolation and neuronal degeneration in the central nervous system (CNS) and retina. The mar-ine fish gilthead seabream (Sparus aurata) does not develop VER but it is an asymptomatic carrier ofVNNV. In this study, we report that VNNV was able to replicate and persist for up to 3 months in theCNS of the gilthead seabream without causing any neural damage. In addition, we found an early inflam-matory response in the CNS that was characterized by the induction of genes encoding pro-inflammatorycytokines, a delayed but persistent induction of anti-inflammatory cytokines, and the infiltration of IgM+

B lymphocytes, suggesting that local adaptive immunity played a major role in the control of VNNV in theCNS of this species.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The central nervous system (CNS) has been considered an im-mune-privileged organ due to the existence of a physical barrierbetween the blood and the brain, namely the blood–brain barrier(BBB) (Bechmann et al., 2007). However, the extension of its mean-ing to the context of inhibiting leukocyte recruitment into thebrain is imprecise and some authors have re-defined the BBB as acapillary barrier for solutes, whereas leukocytes are able to crossthe BBB through a multisequential process (Bechmann et al.,2007; Greenwood et al., 2011). On the other hand, following CNSinfection these protective barriers can be compromised, sometimesresulting in severe neurological complications triggered by animbalance or blockage of neural chemistry, which can be fatal(Kang and McGavern, 2010).

The viral nervous necrosis virus (VNNV) is the causal agent ofviral encephalopathy and retinopathy (VER), a worldwide fish

ll rights reserved.

ogy and Histology, Faculty ofel.: +34 868887581; fax: +34

disease that is responsible for high mortality rates in both marineand freshwater species (Munday et al., 2002; Munday and Nakai,1997). Infected fish suffer from encephalitis, which leads to abnor-mal swimming behavior and extensive cellular vacuolation andneuronal degeneration in the CNS and retina (Bovo et al., 1999).The marine fish gilthead seabream (Sparus aurata), which is consid-ered the most important species in southern European aquacul-ture, does not develop VER, but still behaves as an asymptomaticcarrier of VNNV (Castric et al., 2001). Interestingly, it has beenfound that experimental infection with VNNV causes a strong Mxupregulation in the brain of gilthead seabream, which is two ordersof magnitude higher than observed in European seabass Dicentrar-chus labrax (Poisa-Beiro et al., 2008). It has been speculated thatthis strong Mx induction might be responsible for the effectivenessof gilthead seabream to clear the infection and become an asymp-tomatic carrier of the disease (Fernandez-Trujillo et al., 2011;Poisa-Beiro et al., 2008). However, it is not known whether the gilt-head seabream immune system is able to control VNNV infection,avoiding damage to the CNS or, alternatively, whether the VNNV isunable to replicate in the CNS of this species but persists in a latentstage for long period of time. The aim of this study was, therefore,to analyze viral load at early and late stages of VNNV infection, as

430 A. López-Muñoz et al. / Developmental and Comparative Immunology 37 (2012) 429–437

well as the expression profiles on innate and adaptive immunegenes in the head-kidney, a major immune organ of teleost fish,and the brain, the target organ for VNNV.

2. Materials and methods

2.1. Virus and cell culture conditions

NNV previously isolated from sea bass showing encephalopathysymptoms, was propagated on the SSN-1 cell line cultured at 20 �Cin 2% FBS enriched Leibovitz (L-15) medium (Life Technologies).Prior to virus inoculation, SSN-1 cells were maintained at 25 �C inL-15 supplemented with 2% L-glutamine (Sigma–Aldrich), 1% peni-cillin–streptomycin (Life Technologies) and 10% fetal bovine serum(FBS) (Cambrex). Virus inoculation was performed at 60% semi-con-fluence of cell cultures. Virus yield was determined by titration on96-well plates by the TCID50 method (Cunningham, 1973).

2.2. Seabream infection

Experimental infection was performed as described previously(Fernandez-Trujillo et al., 2011) using a total of 800 seabream indi-viduals (30–40 g) kept in the IFAPA Center El Toruño, Cádiz (Spain).Two weeks prior to infection, fish were transferred to the quaran-tine facilities for adaptation and distributed into three (4000 l)tanks. Fish (n = 200) in the control tank were injected intramuscu-larly (i.m.) with PBS, whereas all fish from the two other tanks(n = 300 + 300) were injected i.m. with NNV (104 TCID50/ml). Thisvirus dose was previously tested and seen to induce low mortality(lower than 20%) in European seabass. A similar booster dose wasadministered 47 d post-infection (dpi). Time course sampling was

Fig. 1. VNNV replicates in the CNS but does not cause neural damage. (A) The mRNA levehead-kidney and brain of VNNV-challenged fish at the indicated post-infection times. Thetriplicate samples from five fish. Different letters denote the statistically significant differnot show statistically significant differences from fish collected at 0 dpi. The arrow indicaand VNNV-infected (right panel) specimens at 30 dpi stained with H&E. Note that VNNV

performed as indicated below. No symptomatic fish or mortalitywere observed during the trial.

All experiments complied with the Guidelines of the EuropeanUnion Council (86/609/EU) and were supervised by the BioethicalCommittee of the University of Murcia for the use of laboratoryanimals.

2.3. Analysis of gene expression

For each group/time point, five fish were used. From each spec-imen, brain and head-kidney were removed after infection andboosting at 0 h, 6 h, 24 h, 3 d, 10 d, 12 d, and 30 d post-infection.Samples were immersed in 1 ml of TRIzol Reagent, frozen in liquidnitrogen and stored at �80 �C. Total RNA was extracted with TRIzolReagent (Life Technologies) and treated with DNase I, amplificationgrade (1 unit/lg RNA, Life Technologies). The SuperScript III RNaseH� Reverse Transcriptase (Life Technologies) was used to synthe-size the first strand of cDNA with an oligo-dT18 (immune genes)or NNVCP-R1 primer (specific for gene encoding the VNNV coatprotein) from 1 lg of total RNA at 50 �C for 50 min.

Real-time PCR was performed with an ABI PRISM 7500 instru-ment (Applied Biosystems) using SYBR Green PCR Core Reagents(Applied Biosystems). Reaction mixtures were incubated for10 min at 95 �C, followed by 40 cycles of 15 s at 95 �C, 1 min at60 �C, and finally 15 s at 95 �C, 1 min 60 �C and 15 s at 95 �C. Foreach mRNA, the gene expression was normalized to the ribosomalprotein S18 (rps18) content in each sample using the Pfaffl method(Pfaffl, 2001). In all cases, each PCR was performed with triplicatesamples and repeated with at least two independent samples.The sequence of all primers used is shown in SupplementaryTable 1.

ls of the genes coding for the VNNV CP were determined by real-time RT-PCR in thegene expression is normalized against rps18. Each bar represents the mean ± S.E. of

ences among the groups according to a Tukey’s test. The groups marked with ‘‘a’’ didtes the re-infection. (B) Representative brain sections from PBS-injected (left panel)

infection did not cause neural damage.

A. López-Muñoz et al. / Developmental and Comparative Immunology 37 (2012) 429–437 431

2.4. Light microscopy and immunohistochemistry (IHC)

Tissue fragments were fixed overnight in 4% paraformaldehydesolution, embedded in Paraplast Plus (Sherwood Medical) and sec-tioned at a thickness of 5 lm. After dewaxing and rehydrating,some sections were stained with hematoxylin and eosin (H&E),while others were subjected to an IHC method (Mulero et al.,2008) using the G7 monoclonal antibody (mAb), which is specificto seabream acidophilic granulocytes (Sepulcre et al., 2002), com-mercial anti-IgM mAb (Aquatic Diagnostic Ltd.) (Sepulcre et al.,2011) or rabbit polyclonal antibodies to VNNV (Bovo et al.,1999). The sections were incubated for 40 min in peroxidase-quenching solution (H2O2 in methanol, 1:9) to eliminate theendogenous peroxidase activity and then rinsed in PBS and inPBS containing 0.01% bovine serum albumin (BSA) and 0.2% TritonX-100 (PBT). After 30 min incubation with PBS containing 0.5% BSAto block the non-specific reaction, they were rinsed in PBT, incu-bated for 2 h at room temperature with a 1:100 dilution of theG7 or anti-IgM mAbs, washed in PBT and exposed to anti-mouseIgG-peroxidase antibodies (Sigma–Aldrich) diluted 1:200 for 1 hat room temperature. The sections immunostained with the anti-VNNV antibodies were washed in PBT, exposed to anti-rabbit IgG(whole molecule)-biotinylated antibody produced in swine (DakoCytomation) diluted 1:100 for 1 h at room temperature, washedin PBT and incubated for 1 h at room temperature with the avi-din–biotinylated peroxidase complex (Dako Cytomation) accord-ing to the manufacturer’s instructions. All samples were finally

Fig. 2. VNNV infection modulates the expression of gene encoding TLRs. The mRNA levelthe head-kidney and brain of PBS-injected and VNNV-challenged fish at the indicated porelative to the mean of PBS-injected fish. Each bar represents the mean ± S.E. of triplicate sbetween the groups according to a Tukey’s test. The groups marked with ‘‘a’’ did not showre-infection.

washed in PBT, in 0.5 M TRIS–HCl buffer (pH 7.6) and the peroxi-dase activity was then revealed by incubation for 5 min at roomtemperature with 0.05% 3,30-diaminobenzidine tetrahydrochloride(DAB) in TRIS–HCl buffer containing 0.05% H2O2. Sections werethen slightly counterstained with haematoxylin, dehydrated,mounted in DPX and examined under an Axiolab microscope(Zeiss). The photographs were obtained with a CoolSNAP (RSPhotometrics) digital camera using a computer with CoolSNAP(RS Photometrics) software. The infiltration of IgM+ cells was calcu-lated as the number of extravascular positive cells in each opticalarea at �40 magnification. Three areas of each section from six dif-ferent brain sections at 30 dpi were analyzed.

2.5. Statistical analysis

Data were analyzed by ANOVA and a Tukey multiple range testto determine the differences among groups.

3. Results

3.1. VNNV replicates in the brain of seabream without causing neuraldamage

We designed a real-time PCR-based protocol to quantify viralload in the brain of infected seabream. As the VNNV is a ssRNApositive-strand virus, we used a specific primer for reverse

s of the genes coding for the indicated TLRs were determined by real-time RT-PCR inst-injection times. The gene expression is normalized against rps18 and is shown asamples from five fish. Different letters denote the statistically significant differencesstatistically significant differences from mock-injected fish. The arrows indicate the

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transcription of the gene encoding the VNNV coat protein (CP)followed by real-time PCR. The results show that the CP genewas detected in the brain of infected animals immediately afterinjection (5–15 min) (Fig. 1A), suggesting that the virus reachedthe target organ very quickly. Notably, the mRNA levels of theVNNV CP drastically increased at 5 dpi, peaked at 30 dpi and re-mained high after re-infection for the rest of the trial (Fig. 1A).No expression of the gene encoding the VNNV CP was detectedin non-infected specimens throughout the trial (data not shown).

Fig. 3. VNNV infection induces a transient inflammatory response. The mRNA levels odetermined by real-time RT-PCR in the head-kidney and brain of PBS-injected and VNnormalized against rps18 and is shown as relative to the mean of PBS-injected fish. Eachdenote the statistically significant differences between the groups according to a Tukey’from mock-injected fish. The arrows indicate the re-infection.

Although none of the infected animals died or showed anysymptom, the persistent replication of VNNV in the brainprompted us to evaluate whether the virus was able to produceneural damage. Surprisingly, the brains of infected animals didnot show any obvious histopathological alteration (Fig. 1B). Inaddition, the VNNV was not detected in the brain of infected sea-breams, as assayed by IHC, while a strong immunoreaction was ob-served in the brain of infected European seabass specimens (datanot shown).

f the genes coding for the indicated pro- and anti-inflammatory molecules wereNV-challenged fish at the indicated post-injection times. The gene expression isbar represents the mean ± S.E. of triplicate samples from five fish. Different letters

s test. The groups marked with ‘‘a’’ did not show statistically significant differences

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3.2. VNNV infection promotes a transient local and systemicinflammatory response

In order to understand the local (brain) and systemic (head-kidney) host innate immune response to VNNV infection, we firstevaluated the expression of genes encoding several toll-like recep-tors (TLRs), including soluble TLR5 (TLR5S), which recognizes fla-gellin and is an excellent inflammation marker for both viral andbacterial infections (Sepulcre et al., 2007; Tsukada et al., 2005);TLR22, which recognizes double stranded RNA that might be gen-erated during replication of nodavirus (Albarino et al., 2001); andTLR9, which senses DNA and might be used as a marker of lympho-cyte infiltration according to its high expression by seabream lym-phocytes (Cuesta et al., 2008). The results show that while themRNA levels of soluble TLR5, and to some extent of TLR22, didnot change in the brain of infected animals, those of TLR9 startedincreasing at 12 dpi, following a plateau at 30 dpi (Fig. 2). However,we observed increased transcript levels of TLR5S and TLR22 soonafter re-infection.

The mRNA levels of genes encoding pro-inflammatorymediators were then examined. Interleukin-1b (IL-1b) transcriptincreased soon after viral inoculation in both brain and head-kidney, while those of tumor necrosis factor a (TNFa) and cycloox-ygenase-2 (COX2, ptgs2 gene) increased at later times afterinfection (Fig. 3A). The expression of the anti-inflammatory cyto-kine transforming growth factor b1 (TGFb1) gene increased at

Fig. 4. VNNV infection hardly affects the expression of the gene encoding IFN. The mRNAhead-kidney and brain of PBS-injected and VNNV-challenged fish at the indicated postrelative to the mean of PBS-injected fish. Each bar represents the mean ± S.E. of triplicate sbetween the groups according to a Tukey’s test. The groups marked with ‘‘a’’ did not showre-infection.

Fig. 5. VNNV infection induces the expression of the gene encoding the macrophage mardetermined by real-time RT-PCR in the head-kidney and brain of PBS-injected and VNnormalized against rps18 and is shown as relative to the mean of PBS-injected fish. Eachdenote the statistically significant differences between the groups according to a Tukey’from mock-injected fish. The arrows indicate the re-infection.

5 dpi in the brain and the head-kidney and then remained stablethe rest of the trial (Fig. 3B). These results suggest that, despitethe persistence of the VNNV in the brain of infected animals, itdid not promote any long lasting inflammatory response thatmight exacerbate neural damage.

When the antiviral response to VNNV was examined, it wasseen that the mRNA levels of the type I interferon (IFN) gene werebarely affected by the infection in the brain or the head-kidney(Fig. 4). This result sharply contrasts with the strong and persistentinduction of the gene encoding Mx in these organs during VNNVinfection (Fernandez-Trujillo et al., 2011).

3.3. VNNV infection promotes the infiltration of lymphocytes to thebrain

Determination of the transcription levels of the gene coding forthe macrophage colony-stimulating factor receptor (MCSFR), a spe-cific marker for the monocyte/macrophage lineage in the seabream(Mulero et al., 2008), showed that they did not significantly changein the brain of infected animals but slightly increased in the head-kidney at 5 and 12 dpi and 6 and 24 h after re-infection (Fig. 5). Inaddition, no infiltration of acidophilic granulocytes was observedin the brain of VNNV-infected animals, as assayed by IHC (datanot shown). By contrast, the transcript levels of the genes encodingthe T lymphocyte markers T-cell receptor (TCRb), CD8a and CD4,increased in the brain of infected animals between 5 and 12 dpi,

levels of the gene coding for type I IFN were determined by real-time RT-PCR in the-injection times. The gene expression is normalized against rps18 and is shown asamples from five fish. Different letters denote the statistically significant differencesstatistically significant differences from mock-injected fish. The arrows indicate the

ker MCSFR in the head-kidney. The mRNA levels of the gene coding for MCSFR wereNV-challenged fish at the indicated post-injection times. The gene expression isbar represents the mean ± S.E. of triplicate samples from five fish. Different letters

s test. The groups marked with ‘‘a’’ did not show statistically significant differences

Fig. 6. VNNV infection induces the expression of the genes encoding the T lymphocyte markers TCRb, CD8a and CD4 in the brain. The mRNA levels of the genes coding for theindicated T lymphocyte markers were determined by real-time RT-PCR in the head-kidney and brain of PBS-injected and VNNV-challenged fish at the indicated post-injectiontimes. The gene expression is normalized against rps18 and is shown as relative to the mean of PBS-injected fish. Each bar represents the mean ± S.E. of triplicate samples fromfive fish. Different letters denote the statistically significant differences between the groups according to a Tukey’s test. The groups marked with ‘‘a’’ did not show statisticallysignificant differences from mock-injected fish. The arrows indicate the re-infection.

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while they barely changed in the head-kidney (Fig. 6). In addition,the transcript levels of TCRb and CD8a, while not of CD4, werehigher in the brain of VNNV-infected animals after re-infection,than in uninfected controls (Fig. 6). The mRNA levels of the geneencoding the heavy chain of seabream immunoglobulin M (IgM)showed a similar tendency; that is, a strong increase between 5and 12 dpi, when a plateau was reached (Fig. 7). Interestingly,the expression levels of the gene coding for the heavy chain ofthe recently discovered mucosal IgT (Zhang et al., 2010), werenot significantly affected in VNNV-infected animals (Fig. 7). Asthe overall results suggested the infiltration of CD8+ T lymphocytesand IgM+ B lymphocytes in the brain of VNNV-infected seabreams,we examined the expression levels of the chemokines, CC-chemo-kine ligand 4 (CCL4) and IL-8, having unexpectedly found that theirtranscript levels did not change in the brain of infected animals(Fig. 8).

The infiltration of IgM+ cells in the brain of VNNV-infected ani-mals was confirmed by IHC (Fig. 9). These results showed the pres-ence of abundant IgM+ cells inside the capillaries as well asabundant IgM+ cells scattered among neuronal cell bodies of thecerebral cortex of VNNV-infected specimens (Fig. 9). The numberof infiltrated IgM+ lymphocytes per field in the cerebral cortex ofcontrol and VNNV-infected animals at 30 dpi was 3.3 ± 1.4 and12.0 ± 2.4, respectively (p < 0.01).

4. Discussion

The VNNV is a neurotropic virus, which is able to infect virtuallyall marine and freshwater fish species (Munday et al., 2002; Mun-day and Nakai, 1997). Infected fish from most species suffer fromencephalitis, leading to abnormal swimming behavior, extensivecellular vacuolation and neuronal degeneration in the central ner-vous system and retina (Bovo et al., 1999). Interestingly, the gilt-head seabream, does not develop VER, while behaves as anasymptomatic carrier of VNNV (Castric et al., 2001). Despite the ef-forts made by different laboratories to unveil the mechanism ofVNNV dissemination and access to the CNS, the host immune re-sponse and the mechanisms involved in VNNV persistence inasymptomatic carriers, all these aspects are still largely unknown.Our study reveals for the first time that the VNNV is able to repli-cate in the gilthead seabream CNS, even though it causes no obvi-ous tissue damage and could not be detected by IHC. Therefore, wethink that although the VNNV may replicate in the brain of sea-bream specimens, the amount of viral particles are below thedetection limit of the IHC assay. It is unlikely that the virus is un-able to appropriately assemble in the seabream brain, since thisspecies has been found to be able to transmit VNNV infection tosusceptible species, such as the European seabass (Castric et al.,2001). This result, together with the absence of clinical signs of

Fig. 7. VNNV infection induces the expression of the gene encoding the IgM heavychain in the brain. The mRNA levels of the genes coding for the indicated Blymphocyte markers were determined by real-time RT-PCR in the brain of PBS-injected and VNNV-challenged fish at the indicated post-injection times. The geneexpression is normalized against rps18 and is shown as relative to the mean of PBS-injected fish. Each bar represents the mean ± S.E. of triplicate samples from five fish.Different letters denote the statistically significant differences between the groupsaccording to a Tukey’s test. The groups marked with ‘‘a’’ did not show statisticallysignificant differences from mock-injected fish. The arrows indicate the re-infection.

Fig. 8. VNNV infection barely affect the expression of the gene encoding thechemokines CCL4 and IL-8 in the brain. The mRNA levels of the genes coding for theindicated chemokines were determined by real-time RT-PCR in the brain of PBS-injected and VNNV-challenged fish at the indicated post-injection times. The geneexpression is normalized against rps18 and is shown as relative to the mean of PBS-injected fish. Each bar represents the mean ± S.E. of triplicate samples from five fish.Different letters denote the statistically significant differences between the groupsaccording to a Tukey’s test. The groups marked with ‘‘a’’ did not show statisticallysignificant differences from mock-injected fish. The arrows indicate the re-infection.

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disease in experimentally infected fish and the lack of reports onnatural VNNV outbreaks in this species (Munday et al., 2002),firmly support the idea that this virus causes a persistent infectionin the gilthead seabream, since latent infections can be controlledby the immune system, though usually re-emerge and cause dis-ease symptoms during periods of immunosuppression.

It has been speculated that strong Mx induction in the brain ofVNNV-infected gilthead seabream is responsible for clearing thevirus (Fernandez-Trujillo et al., 2011; Poisa-Beiro et al., 2008).However, we found a surprisingly weak and transient inductionof the gene encoding type I IFN, which was mostly restricted tothe brain, thus suggesting that either the Mx expression was di-rectly induced by the VNNV or other type I IFN genes existing inthe seabream, as occurs in other teleost species (Zou andSecombes, 2011), would be induced by VNNV. Although we cannotcompletely rule out an IFN-independent induction of Mx, as hasbeen shown for MxC in the zebrafish (Levraud et al., 2007;Lopez-Munoz et al., 2010), this seems unlikely since the threeMx genes known in the seabream were strongly upregulated inthe brain and the head-kidney of VNNV-infected fish (Fernandez-Trujillo et al., 2011), it being extremely unlikely that all of themare IFN-independent. Whatever the case, our results suggest thatthe induction of the three Mx genes in the brain of VNNV-infectedseabreams helps to dampen viral replication, by preventing CNSdamage, although they are unable to clear the virus.

Another interesting observation from this study is the transitoryexpression of IL-1b in both the brain and head-kidney of infectedanimals and the delayed and long lasting, though weak, inductionof TNFa and COX2, which suggest that the high viral load of VNNVin the brain does not promote a lasting inflammatory response thatmight contribute to neural damage. In fact, a stronger expression of

IL-1b and TNFa has been observed in the brain of European seabasscompared with gilthead seabream (Poisa-Beiro et al., 2008). It istempting to speculate, therefore, that the late and long-lastinginduction of TGFb1 in the brain and head-kidney of infected gilt-head seabream specimens might be involved in preventing anexacerbated inflammatory response in the CNS, which would bedetrimental for the host. This is plausible since the induction ofan anti-inflammatory lectin, namely galectin-1, has been observedin the brain of European seabass upon VNNV infection (Poisa-Beiroet al., 2009). However, the induction of an early inflammatory re-sponse might be involved in viral clearance, since the administra-tion of several corticosteroids accelerates VNNV dissemination andmortality onset in turbot (Scophthalmus maximus), with a concom-itant reduced TNFa expression in the brain (Montes et al., 2010).

To the best of our knowledge, the infiltration of immune cells inthe CNS of fish during VNNV infection has never been examined.We found no evidence supporting the infiltration of the two pro-fessional phagocytic cell types of the gilthead seabream, namely,macrophages and acidophilic granulocytes. Interestingly, however,we observed the induction of the gene encoding T and B lympho-cyte markers, TCRb/CD8a and IgM, respectively, and the moremoderated induction of CD4, a marker for T helper lymphocytes.Although no antibodies are available for T lymphocytes in the gilt-head seabream, we were able to confirm by IHC the presence ofIgM+ cells in the brain of VNNV-infected fish. This result, togetherwith the induction of the gene encoding TLR9, which was found tobe highly expressed in seabream lymphocytes (Cuesta et al., 2008),strongly suggests that IgM+/TLR9+ B lymphocytes infiltrate thebrain of infected animals. Clarifying the role of T and B lympho-cytes in VNNV-infection would need a functional experimental ap-proach in genetically tractable animal models, such as the

Fig. 9. VNNV infected fish show abundant IgM+ cells in the brain. Representativebrain sections from PBS-injected (upper panel) and VNNV-infected (lower panel)specimens at 30 dpi immunostained with the anti-IgM monoclonal antibody. Thenuclei were slightly counterstained with hematoxilin. Note that although a fewintravascular IgM+ cells (arrows) were observed in control and infected specimens,abundant extravascular IgM+ cells (arrow heads) were only observed in VNNV-infected brain.

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zebrafish, which is susceptible to VNNV (Lu et al., 2008) and whichwould allow the depletion of specific cell types (Pisharath and Par-sons, 2009). Nevertheless, the recent observation of increased lev-els of TCRb, LCK, CD8a, CD4 and IFNc transcripts in the brain inAtlantic halibut (Hippoglossus hippoglossus) experimentally in-fected with VNNV (Overgard et al., 2011) further suggests theinvolvement of T lymphocytes in VNNV clearance.

Crossing the BBB or the blood–cerebral spinal fluid barrier re-quires specialized viral adaptations. One particularly importantstrategy used by viruses is to enter the peripheral nervous systemand travel via axon fibres to the CNS, since these peripheral nervesextend beyond the protective barriers of the CNS (McGavern andKang, 2011). Another common strategy used by neurotropic virusis to cross the vascular endothelium by altering endothelial cellbehavior through promoting increased production of chemokinesand the altered expression of tight junction proteins (Afonsoet al., 2008; Casiraghi et al., 2010; Gralinski et al., 2009; Vermaet al., 2010), the increased expression of vascular cell adhesionmolecule 1 (VCAM1) (Verma et al., 2010) and decreased transendo-thelial electrical resistance (Gralinski et al., 2009). Finally, anotherway that viruses can enter the CNS is through a ‘Trojan horse’mechanism, in which infected monocytes/macrophages (Alexakiand Wigdahl, 2008; Clay et al., 2007) and, probably B lymphocytes(Chapagain and Nerurkar, 2010; Monaco et al., 1996), carry

pathogens from the blood across the BBB. Although further exper-iments are required to elucidate the mechanism used by VNNV toenter the CNS, our results argue against a ‘‘Trojan horse’’ mecha-nism in VNNV infection, since the virus reached the CNS beforeany obvious infiltration of leukocytes.

In conclusion, our study demonstrates that VNNV was able toreplicate and persist for up to 3 months in the brain of the giltheadseabream without causing any neural damage. The immune re-sponse to VNNV in the brain is characterized by an early inflamma-tory response, a persistent antiviral and anti-inflammatoryresponse and the infiltration of IgM+ B lymphocytes, suggesting thatviral replication in the brain is controlled by adaptive immunity.

Acknowledgements

We thank Drs. F. Buonocore, E. Randelli and G. Scapigliati forsharing with us unpublished sequences of seabream CD4 andCD8a genes, Drs. G. Bovo and A. Toffan for the anti-VNNV antibody,Dr. C. Kim (University of Stirling) for the anti-IgM mAb, and Drs. B.Novoa and A. Figueras for the brain sections of VNNV-infected sea-bass. This work was supported by the Sixth Framework Programmeof the European Union (grant FOOD-CT-2005-007103) and the Uni-versity of Murcia (fellowship to A.L.-M.). Sponsors were not in-volved in the study design, in the collection, analysis andinterpretation of data; in the writing of the manuscript; and inthe decision to submit the manuscript for publication.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.dci.2012.02.007.

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