Available online at www.sciencedirect.com Molecular Immunology 45 (2008) 1146–1152 The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model Ming-Wei Lu a , Yung-Mei Chao a , Tz-Chun Guo b , Nina Santi b , Øystein Evensen b , Siti Khadijah Kasani a , Jiann-Ruey Hong c , Jen-Leih Wu a,∗ a Laboratory of Marine Molecular Biology and Biotechnology, Institute of Cellular & Organismic Biology, Academia Sinica, Nankang, Taipei 115, Taiwan b Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo, Norway c Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan Received 29 June 2007; received in revised form 12 July 2007; accepted 18 July 2007 Available online 28 August 2007 Abstract Betanodavirus, a small positive-sense bipartite RNA virus notoriously affecting marine aquaculture worldwide has been extensively studied in vitro. However, impending studies in elucidating virus–host interactions have been limiting due to the lack of appropriate animal disease models. Therefore, in this study, we have attempted to successfully establish NNV infection in zebrafish (Danio rerio) showing typical NNV symptoms and which could potentially serve as an in vivo model for studying virus pathogenesis. Zebrafish being already a powerful research tool in developmental biology and having its genome completely sequenced by the end of 2007 would expedite NNV research. We have observed viral titers peaked at 3 days post-infection and histological study showing lesions in brain tissues similar to natural host infection. Further, we used this infection model to study the acute and persistence infection during NNV infection. Interestingly, RT-PCR and immunoblotting assays revealed that the acute infection in larvae and juveniles is largely due to inactive interferon response as opposed to activated innate immune response during persistent infection in adult stage. This study is the first to demonstrate NNV infection of zebrafish, which could serve as a potential animal model to study virus pathogenesis and neuron degeneration research. © 2007 Elsevier Ltd. All rights reserved. Keywords: Betanodavirus; Zebrafish; Animal model; Interferon; Persistent infection 1. Introduction Nervous necrosis virus (NNV) is an important fish pathogen belonging to the virus family Nodaviridae that targets nervous tissues primarily brain and retina. The manifestation of NNV for example, Piscine nodaviruses (betanodaviruses), the causative agents of viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) (Munday et al., 2002) in a wide range of host fish species, has resulted in major economic losses for the marine aquaculture industry. Nodaviruses are small, nonenveloped and isometric particles containing a bipartite genome of two positive-sense RNA molecules; RNA1 encoding the RNA replicase and RNA2 encoding the capsid protein precursor, which are capped but not polyadenylated (Delsert et al., 1997; Lin et al., 2001). ∗ Corresponding author. Tel.: +886 2 27899500; fax: +886 2 27858059. E-mail address: [email protected] (J.-L. Wu). 0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.07.018 Extensive studies have revealed abnormal swimming behavior and sight defect of NNV infected fish. In addition, histological examination of tissues from the central nervous system and the retina of infected fish often reveals areas of conspicuous tissue vacuolation and necrosis (Barke et al., 2002; Dannevig et al., 2000; Johansen et al., 2004). However, the pathogenesis of NNV infection in which acute infection in larvae and juvenile stages caused mass mortality while persistently infecting adult remains poorly understood. To this end, we sought to use zebrafish as a potential NNV disease model to provide insights into NNV pathogenesis and its host immune response. At present, zebrafish is rapidly becoming a valuable molecular genetics model in understanding vertebrate organogenesis and disease development (Glass and Dahm, 2004; Yee and Pack, 2005). To date, several viruses are known to infect zebrafish, such as spring viraemia of carp virus (SVCV), a member of the Rhabdoviridae, that causes significant mortality in common carp (Cyprinus carpio) (Sanders et al., 2003) and snakehead M.-W. Lu et al. / Molecular Immunology 45 (2008) 1146–1152 rhabdovirus (SHRV) which was shown to cause mortalities exceeding 40% in zebrafish (Phelan et al., 2005). In view of the differential effect of viral infection, we looked into the zebrafish interferon response, in which the zebrafish interferon gene (zIFN) has been recently identified as to having anti-virus function (Altmann et al., 2003) and may contribute to both induction and regulation of the innate and adaptive immunity. Downstream interferon activated Mx gene has also been identified in zebrafish, grouper, salmon, trout, and halibut upon infection with aquatic viruses (Chen et al., 2006; Kibenge et al., 2005; Lin et al., 2005; McBeath et al., 2006) suggesting the importance of the interferon regulatory pathway including RNA-activated protein kinase (PKR) and the 2-5A proteins during viral infection. In our present study, we have observed an elevated interferon expression in infected adult zebrafish relative to infected larvae resulting in higher rate of mortality in the latter. This may indicate that the susceptibility to NNV infection is dependent on the enhancement of IFN system. However, the mechanism in which interferon response is activated upon NNV viral infection needs to be further elucidated. 2. Materials and methods 2.1. Fish AB (−) inbred strain zebrafish were obtained at 2 months stage from Institute of Cellular & Organismic Biology, Academia Sinica, Taiwan. The zebrafish were handled according to Institutional Animal Care and Use Committee guidelines. 2.2. Cell lines and virus The SSN-1 derivate cell line E-11 (Iwamoto et al., 2000) was used to isolate and titrate NNV. Cells were propagated and maintained at 28 ◦ C in L-15 medium (GIBCO) supplemented with 10% fetal bovine serum (Sigma), 100 I.U./ml penicillin, and 0.1 mg/ml streptomycin. The nodavirus used in this study was isolated from malabaricus grouper (Epinephelus malabaricus) juveniles during an outbreak of VER at a fish farm in southern Taiwan. Sequence analysis of partial viral genome (data not shown) revealed that it shares more than 95% homology with other betanodaviruses isolated from fish in Taiwan (Chi et al., 2003). E-11 cells were inoculated with a fourth passage virus supernatant and incubated at 28 ◦ C. When a cytopathic effect (CPE) was observed on 3 days post-infection (p.i.), the culture supernatant was harvested, clarified by centrifugation (3000 × g for 5 min) and stored at 4 ◦ C until challenge. The virus titer in the supernatant was determined by using an infectivity assay and was calculated to be 1 × 108 TCID50 ml−1 . 2.3. Fish challenge Two groups of 60 adult zebrafish, one infected and one mockinfected, were used in the challenge experiment. The fish were held in 5 liter tanks at a water temperature of 28 ◦ C. Challenge was performed by intraperitoneal (i.p.) injection of 1 × 105 1147 TCID50 ml−1 of NNV in 20 l. The mock-infected group was injected with PBS. After challenge, the fish were monitored daily over a 14-day period for signs of disease and mortality. Six fish in each group were sampled each day and tissue samples from different organs were isolated for virus detection by RT-PCR and infectivity assay (TCID50 ). In a separate experiment, fish were maintained and challenged similarly, but three fish from each group were sampled daily for histology and immunohistochemistry. Some fish in parallel were also collected for confirmation of infection by RT-PCR and virus re-isolation. 2.4. Histology and immunohistochemistry (IHC) The heads of fish used for histology were fixed for at least 24 h in neutral phosphate-buffered 10% formalin and embedded in paraffin wax. Five micrometers brain sections were stained with haematoxylin and eosin (H&E) whereas the other parallel sections were processed for immunohistochemical detection of nodavirus protein using anti-NNV antiserum and a streptavidin-biotin-alkaline-phosphatase complex antibody detection technique (Chemicon IHC Select Kit). Mock-infected fish served as negative controls for the experiment. 2.5. RNA isolation and RT-PCR RNA was extracted using TRIZOL reagent (Life Technologies) according to the manufacturer’s instructions. The RT-PCR was performed using a Superscript III one-step RT-PCR system (Invitrogen) and each reaction includes 2 l total RNA (20 ng) and a primer set. The primers were designed to amplify the variable T4 region of the coat protein gene (Nishizawa et al., 1994), Mx (sense 5 -AGTACCGGGGAAGAGAGCTA-3 antisense 5 -AAGGTGGCATGATTGT CTGT-3 ), IFN (sense 5 -ATGAGAACTCAAATGTGGAC-3 antisense 5 -TTACA CTCGAGGATTGAC-3 ), and -actin (sense 5 -ATGGATGAGGAAATCGCTG-3 antisense 5 -ATGCCAACCATCACTCCCTG-3 ) gene. 2.6. Microinjection of virus into zebrafish larvae The NNV titer for microinjection was adjusted to 1 × 103 TCID50 ml−1 . Injections were conducted by PLI-100 airinjection apparatus (Medical System Co.). The amount (approximately 0.1 l) injected into larvae was estimated by visualizing the injection volume. After injection, the larvae were incubated in the Petri-dish at 28 ◦ C. 2.7. Immunoblot Fish samples were collected, homogenized, and solubilized in disruption buffer containing 50 mM Tris–HCl (pH 7.0), 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 2.75% sucrose. Samples were then sonicated, boiled, subjected to electrophoresis on denaturing 12% polyacrylamide gels, transferred to nitrocellulose membranes, blocked with 5% nonfat milk, and reacted with antibodies against phospho- 1148 M.-W. Lu et al. / Molecular Immunology 45 (2008) 1146–1152 rylated eIF2␣ protein (Cell Signaling). The membranes were rinsed in phosphate-buffered saline (PBS) and reacted with antirabbit immunoglobulin conjugated to horseradish peroxidase and developed with an enhanced chemiluminescence Western blot detection system kit (Amersham Pharmacia). 2.8. Quantitative RT-PCR for NNV RNA detection A 100 ng aliquot of total RNA was used to quantify NNV-specific RNA levels using an ABI Prism 7000 sequence detector (Applied Biosystems). Real-time reverse transcription-polymerase chain reaction (RT-PCR) amplifications were performed by the High Capacity cDNA Archive kit (ABI, Applied Biosystems) and primer specific for NNV 5 CGAGTCAACACGGGTGAAGA-3 . RT reactions were incubated for 10 min at 25 ◦ C, 2 h at 37 ◦ C and cooling to 4 ◦ C for 5 min. The quantitative RCR protocol provided by the ABI real-time instruments by using Platinum® SYBR® GreenqPCR SuperMix-UDG, and the primers specific for NNV were 5 NTR: 5 -GCCCCTGATGGAGCAGTCT-3 (sense 10 M); 5 AGCACGGTCAACATCTCCAGTT-3 (antisense, 10 M); 45 cycles of PCR were performed with cycling conditions of 3 s at 95 ◦ C, 30 s at 60 ◦ C. Standard curve was generated using vector (pDA8; Lu et al., 2003) bearing NNV s RNA2 gene of variable known concentration. The real-time PCR signals were analyzed in a multiplex format using SDS software (Version 1.7; Applied Biosystems). 3. Results 3.1. NNV replication in different organs Six zebrafish were sacrificed each day over 14 days and pooled tissues from different organs were tested by RT-PCR and TCID50 titration for NNV. Brain, eye, heart, liver and gut were tested positive for NNV, while muscle was negative for NNV by RT-PCR from 3 days p.i. (Fig. 1a). The yields of RT-PCR product from brain, gut and eye were greater compared to heart and liver. It was observed to being most abundant in the brain which is the major target organ for NNV propagation. Virus titers were detected in brain, eye, and gut at 3.6 × 105 , 3.6 × 102 , and 3.6 × 102 TCID50 g−1 , respectively, but not in the heart, liver or muscle (Fig. 1b). Detection of NNV in gut, heart and liver from RT-PCR was probably due to excessive virus introduced during inoculation but had decreased significantly on Fig. 2. NNV replication in infected zebrafish brains at different times postinfection. (a) Agarose gel showing RT-PCR product specific for the NNV coat protein. (b) Virus titers in brains from NNV infected and mock-infected zebrafish. 2 days p.i. (data not shown). In addition, TCID50 assay indicated no considerable amount of virus was in the heart and liver while the low copy number of NNV could still be within the detection of RT-PCR. NNV titers increased in brain tissue (Fig. 2a) from 1.4 × 103 TCID50 g−1 on 1 day p.i. to peak levels of 3.6 × 105 TCID50 g−1 after 3 days p.i. (Fig. 2b). The finding of NNV in the target organs brain and eye implies that the virus can overcome the blood–brain barrier after i.p. injection. 3.2. Nodavirus detection by histopathology Histopathological lesions involving vacuolated cells were observed in brain tissue from 4 days p.i. onwards. Using immunohistochemistry, positive NNV-specific signal was observed in brain tissue surrounding these lesions (Fig. 3). These results demonstrated that the nodavirus not only replicates but also cause pathological lesions in brain tissue of infected zebrafish. 3.3. Re-isolation of NNV from zebrafish in E-11 cells Brain tissue from zebrafish that tested positive by RT-PCR was used to re-isolate NNV in E-11 cells. Development of CPE manifest the cell death and detachment was observed in the E11 cells 3–4 days after inoculation of the tissue homogenate (Fig. 4). The presence of NNV in cell cultures displaying CPE was confirmed by using RT-PCR and sequence analysis of PCR product. 3.4. Nodavirus infection in zebrafish larvae Fig. 1. Tissue distribution of virus replication in infected zebrafish. (a) Agarose gel showing RT-PCR products specific for the NNV coat protein. (b) Virus titers in different organs from fish exposed to NNV via i.p. injection. B, brain; G, gut; H, heart; E, eye; L, liver; M, muscle; ND, not-detected. The NNV infection can spread both by horizontal and vertical transmission (Peducasse et al., 1999). Hence, we simulated vertical transmission of NNV in zebrafish by injecting the virus to M.-W. Lu et al. / Molecular Immunology 45 (2008) 1146–1152 1149 Fig. 3. Detection of NNV antigen in the brain of infected zebrafish by immunohistochemistry. (a) Negative control obtained from the brain of a mock-infected zebrafish (scale bar = 100 m); (b) brain of an infected zebrafish on day 4 p.i., showing immuno-positive cells associated with brain tissue vacuolization (arrow) (scale bar = 100 m). zebrafish larvae. NNV was propagated in E-11 cells and injected to zebrafish larvae at 1 × 103 TCID50 ml−1 . The result showed 98% mortality at 24 h p.i. compared to 24% mortality in the mock injected group (Fig. 5a). Quantitative RT-PCR confirmed the presence of NNV in dead larvae from the NNV infected group. Furthermore, we also compared the amounts of viral RNA between larval and adult stage by quantitative RT-PCR. The amount of viral RNA2 in larval stage was 0.06 ng, which was much higher than adult brain sample (1.33 × 10−6 ng) (Fig. 5b). lyzed for innate immune response. The IFN-␣ gene was not detectable in the larvae stage but expressed normally in the adult stage (Fig. 6a). The differential expression pattern of activated Mx gene and phosphorylated eIF2␣ gene, which are downstream genes of interferon pathway, between larval and adult stage at 16 h p.i. (Fig. 6a and b) indicated the importance of interferon response during NNV pathogenesis. We further confirmed the observations by introducing interferon in the early stages of zebrafish larvae followed by NNV infection. The dosage of uni- 3.5. The interferon response in larval and adult stage after NNV infection The mortality of larvae is very distinct from adult fish after NNV infection. In larval stage, the mortality is higher than 95% amongst all of marine species which could be infected by NNV (Barke et al., 2002). In this study, we collected the larval and adult stage samples which have been infected by NNV and ana- Fig. 4. Re-isolation of NNV from infected zebrafish. (a) Normal E-11 cell monolayer was grown in L-15 with 10% fetal bovine serum for 3 days. Cells were maintained at 28 ◦ C. (b) Monolayer of E-11 cells showing CPE upon infection with NNV isolated from zebrafish for 3 days. Fig. 5. Cumulative mortality for the three groups of zebrafish larvae and the differential amount of viral RNA between zebrafish larval and adult stage. (a) Control group was untreated. Mock-injection group was injected with L15 medium. NNV injected group was injected with supernatant from a NNV infected cell culture at a dosage of 1 × 103 TCID50 ml−1 . (b) Quantitative RTPCR of the NNV RNA2 was performed in larval and adult stage after NNV infection. 1150 M.-W. Lu et al. / Molecular Immunology 45 (2008) 1146–1152 Fig. 6. Interferon related gene expression in larval and adult stage. (a) Semiquantitative RT-PCR analysis to detect the temporal expression of interferon and Mx gene in larval and adult stage after NNV infection. (b) Immuno-blot of the eIF2␣ phosphorylation protein was performed in larval and adult stage after NNV infection. versal IFN-␣2a (Roche) that was introduced by microinjection was 1000U. We designed four experimental groups, the first group was the positive control for NNV infection, the second group was co-injected with NNV and interferon, the third group was pretreated with interferon for 48 h before inoculated with NNV, and the last group was mock inoculated with PBS. The group that was co-injected with interferon and NNV displayed delayed mortality rate, however, after 96 h the mortality still reached 100%, similar to positive control at 72 h. In addition, the pretreatment with interferon for 48 h could rescue the survival rate of NNV infected group from 0 to 76% which is similar to mock-infected group (Fig. 7a). The viral RNA was also beyond detectable amounts as shown by RT-PCR (Fig. 7b). 4. Discussion Betanodavirus, one of the most important causal agent of viral nervous necrosis resulting in economical losses to the aquaculture industry worldwide. In recent years, betanodaviruses have been detected in a variety of fish species both in temperate and tropical areas, as well as in fresh water species (Azad et al., 2005; Hegde et al., 2003; Johansen et al., 2003, 2004; Sommer et al., 2004; Starkey et al., 2001; Ucko et al., 2004). Experimental infection models have been established in different host species, such as halibut, sea bass and wolfish (Grove et al., 2003; Peducasse et al., 1999; Sommer et al., 2004) but unsuccessful in the mouse system (Banu and Nakai, 2004). In this study, we demonstrated that zebrafish are also susceptible to NNV infection. This is also the first NNV infection model in Fig. 7. The effects of interferon in zebrafish larval stage after NNV infection. (a) Survival rate of groups with differential interferon treatment. The arrow indicates the injection time. (b) Semi-quantitative of the viral RNA amount of differential interferon treatment. zebrafish in which, its genome project is near completion, thus will accelerate the mechanistic study of NNV pathogenesis. RTPCR showed that viral RNA was most abundant in the brain followed by eye and gut but was less in liver and heart. Infectivity assay also confirmed the presence of virus in the brain, eye and gut. The high virus titer in the gut compared to liver and heart, which do not characteristically support replication of NNV, may imply a role of the gut in the disease pathogenesis and as a possible virus entry route via feed, such as contaminated plankton. In persistently infected juvenile Atlantic halibut, viral RNA can also be detected in abdominal organs by RT-PCR, but it is not clear which organ the virus resides in (Johans et al., 2002). Herpes simplex virus, and rabies virus are spread by axonal transport from the skin or muscle to the corresponding dorsal root ganglion or anterior horn cells and then to populations of neurons throughout the CNS. In contrast, arboviruses (mainly togaviruses, flaviviruses, and bunyaviruses) spread to the brain via the blood. In NNV-infected zebrafish, high titers of virus occurred in the gut and eye as well as brain. These results indicated a possible spread of virus from the central lacteal via blood into the brain and eye. However, this hypothesis needs more detailed investigation. Beginning from 14 days post-NNV-infection, brain tissue was collected daily from zebrafish that was in persistent infection. From 3 days to 14 days p.i., no apparent symptom was observed in the adult zebrafish. Hand in zebrafish larvae, however, in the zebrafish larvae, mortality following injection of a brain tissue homogenate from experimentally infected zebrafish reached M.-W. Lu et al. / Molecular Immunology 45 (2008) 1146–1152 1151 Fig. 8. The molecular mechanism of NNV infection in acute and persistence infection. The interferon was activated after NNV infection in adult stage, however, no activation observed in larval stage. The downstream antiviral responses of interferon pathway in the larval stage were also not activated leading to acute infection and mass mortality. In contrast with larval stage, the adult stage interferon related antiviral genes were expressed normally and repressed virus replication thus leading to persistent infection. The 2-5A system has not been reported in fish. 98% within 24 h p.i. These results mirror infection in natural hosts of NNV in which mortality primarily occurs in larvae and juveniles. There are some pathogens infecting human also shown to have the same phenomenon, such as alphavirus, JEV, and enterovirus. Therefore, it is vital to elucidate the mechanism that discriminate acute infection in the juvenile from persistence or non-infective stage in the adult. The acute and persistent infection in the in vivo host is highly related to immunity, signal transduction and viral replication. The viral persistence involves several mechanisms especially host innate/adaptive immune response. In HCV, control of acute primary viral replication is associated with expansion of antiviral CD4+ (helper) and CD8+ (cytotoxic) T cells (Bowen and Walker, 2005). The innate immune response also plays a very important role in acute persistent infection. In a recent paper, the in vitro results indicated that Mx gene expression may play an important role in NNV persistence in BB cell (Wu and Chi, 2006). However, the involvement of interferon response in the age-dependent viral disease remains unclear. We tried to use zebrafish as an infection model to understand the relationship between interferon response and NNV infection. According to our results, the virus titer and mortality rate was significantly different between larval and adult stage (Fig. 5). This phenomenon was similar to the acute and persistence infection in NNV pathogenesis in nature. We first examined the interferon related expression profile of NNV infected zebrafish in larval and adult stage. We found that interferon related gene could not be activated by NNV in larval stage. The similar observation was also reported in NNV infected adult sea bass and sea bream recently (Poisa-Beiro et al., 2008). That might be the reason for causing acute infection in larval stage but persistent infection in adult stage. The same phenomena was also observed in the mice system, that is mice lacking functional IFN-I receptors are extremely susceptible to a wide variety of viral infections (Alsharifi et al., 2006). In the gain of function experiment, we observed the inhibition of virus replication after interferon treatment. The reaction time of interferon treatment was also recorded to be 48 h. In conclusion, these results indicated that we could generate the possible mechanism that leads to the difference between acute and persistence infection in NNV pathogenesis (Fig. 8). The zebrafish infection model will provide a convenient system to study NNV pathogenesis and its potential drug-mediated intervention. Other important advantages of zebrafish are the availability of developmental and genomic databases. Additionally, their immune systems are well characterized compared to other fish species. By detecting the IFN-related gene expression in our zebrafish infection model, we first suggest a hypothesis that the antagonistic effect seen in acute and persistent infection in NNV is age dependent. In addition, since the innate immunity is conserved amongst fly, plant and mammals, the finding and discovery in this study could significantly increase our understanding of innate immunity in human thereby lead to medical application. This model can be further used for virus pathogenesis studies, such as apoptosis regulation, signal transduction and immune response. Acknowledgements This work was funded by the Taiwanese Council of Agriculture (95AS-5.1.6-FA-F1), National Science Council of Taiwan (NSC 95-2313-B-001-004) and Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan. References Alsharifi, M., Regner, M., Blanden, R., Lobigs, M., Lee, E., Koskinen, A., Mullbacher, A., 2006. Exhaustion of type I interferon response following an acute viral infection. J. Immunol. 177, 3235–3241. 1152 M.-W. Lu et al. / Molecular Immunology 45 (2008) 1146–1152 Altmann, S.M., Mellon, M.T., Distel, D.L., Kim, C.H., 2003. 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