Sevenband Grouper Research Articles

Viral reprogramming by nervous necrosis virus alters key metabolites and its pathways in sevenband grouper (Hyporthodus septemfasciatus) gills

Nervous necrosis virus (NNV) which mainly infects sevenband grouper (Hyporthodus Septemfasciatus) is considered a potential threat to the grouper aquaculture industry. The gills being one of the portal of entry and an active site of replication of fish viruses emphasises its role as a key region to study the metabolomic changes caused by viral reprograming and hijacking of metabolic pathways associated with immunity of the host. In the present study, liquid chromatography mass spectrometry (LC-MS) was used to detect changes of endogenous compounds of the grouper after NNV infection. A total of 75 metabolites of ten different pathways were identified. The metabolites were mainly associated with fatty acids, lipids, amino acids and nucleotides. The virus reprogramming lead to the downregulation of majority of the metabolites in their pathways. Arachidonic acid (AA), tryptophan, kynurenine and methandriol were selected as representative metabolites and challenge studies with NNV confirmed the fact that, metabolites controlled the replication of virus in a dose dependent manner. Immune gene expression studies also confirmed the effect of metabolites by upregulated expression of interleukins, cytokines and TLRs which are part of cellular immune response. This study shows the viral reprogramming of NNV in grouper gill cells resulting in alterations in basic metabolic pathways associated with normal functioning of the organism.

Establishment and validation of a 2D primary gill cell culture of the sevenband grouper (Hyporthodus septemfasciatus)

A 2D primary gill cell culture system of the sevenband grouper (Hyporthodus septemfasciatus) was established to validate the pathogenesis of nervous necrosis virus (NNV) as observed in previous studies. This system, developed using the double-seeded insert (DSI) technique, yielded confluent cell layers. Upon challenge with NNV in a setup containing both autoclaved salt water and L15 media in the apical compartment, viral replication akin to that anticipated based on previous studies was observed. Consequently, we advocate for the utilization of primary gill cell culture as a viable alternative to conventional methodologies for investigating host pathogen interactions.

Regulatory effects of potassium channel blockers on potassium channel genes upon nervous necrosis virus infection in sevenband grouper Hyporthodus septumfasciatus

Ion channels in fishes regulate the flow of important ions that play an active role in the excitation and transmission of impulses through neuronal cells. Specific housekeeping genes translates into proteins and selectively permeabilize and facilitate ion crossover transmissions. Potassium (K+) channels play a crucial role in a wide range of functions such as cell volume regulation, hormone secretion, synaptic transmission and muscle contraction. The dysfunction of ion channels result in channelopathies, which hinder critical cellular activities. Recent studies have indicated that viral pathogens tend to regulate cellular ion channels for entry into host cells. Hence, the present study aimed to elucidate the role of K+ channels during nervous necrosis virus (NNV) infections in the sevenband grouper (Hyporthodus septumfasciatus). Real-time PCR with the standardized potassium genes revealed the downregulation of potassium two pore domain channel subfamily member - KCNK10, KCNK9, KCNK2, and KCNK1 genes post infection at both 17 °C and 25 °C whereas an upregulation was noted in the case of gill tissues. SMART analysis revealed a transmembrane region in all genes. Multiple sequence alignment using MultAlin and phylogenetic analysis revealed true homology of potassium genes with other higher vertebrates. In vitro and in vivo challenge study of NNV using Tetra ethyl ammonium (TEA) as potential drug showed inverse relation to that of viral replication and a corresponding downregulation of K+ channel gene expression was observed which was further confirmed by an immunofluorescence assay. These findings indicate that K+ channels play a crucial role during viral infection. Moreover, the observed downregulation can be related to rapid endocytosis resulting from recycling endosomes during a viral infection. Hence, further studies are warranted to better understand the role of K+ channel genes during NNV infection.

NKHs27, a sevenband grouper NK-Lysin peptide that possesses immunoregulatory and antimicrobial activity

As an effective and broad-spectrum antimicrobial peptide, NK-Lysin is attracted more and more attention at present. However, the functions and action mechanism of NK-Lysin peptides are still not comprehensive enough at present. In this study, a sevenband grouper (Hyporthodus septemfasciatus) NK-Lysin peptide, NKHs27, was identified and synthesized, and its biological functions were studied. The results indicated that NKHs27 shares 44.44%∼88.89% overall sequence identities with other teleost NK-Lysin peptides. The following antibacterial activity assay exhibited that NKHs27 was active against both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Listonella anguillarum, Vibrio parahaemolyticus and Vibrio vulnificus. Additionally, NKHs27 showed a synergistic effect when it was combined with rifampicin or erythromycin. In the process of interaction with the L. anguillarum cells, NKHs27 changed the cell membrane permeability and retained its morphological integrity, then penetrated into the cytoplasm to act on genomic DNA or total RNA. Then, in vitro studies showed that NKHs27 could enhance the respiratory burst ability of macrophages and upregulate immune-related genes expression in it. Moreover, NKHs27 incubation improved the proliferation of peripheral blood leukocytes significantly. Finally, in vivo studies showed that administration of NKHs27 prior to bacterial infection significantly reduced pathogen dissemination and replication in tissues. In summary, these results provide new insights into the function of NK-Lysin peptides in teleost and support that NKHs27, as a novel broad-spectrum antibacterial peptide, has potential applications in aquaculture against pathogenic infections.

Temperature dependent cellular, and epigenetic regulatory mechanisms underlying the antiviral immunity in sevenband grouper to nervous necrosis virus infection

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFβ. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

Beta glucan induced immune priming protects against nervous necrosis virus infection in sevenband grouper

In the present study, we studied the effect of β-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of β-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1β production were reduced in β-glucan–primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the β-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with β-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that β-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.

Altered expression of immune factors in sevenband grouper, Hyporthodus septemfasciatus following nervous necrosis virus challenge at optimal and suboptimal temperatures

The nervous necrosis virus (NNV) infection is generally observed in aquafarms when the seawater temperature is higher than 24 °C and the fishes seem to be refractory to disease at suboptimal temperatures below 20 °C suggesting a role of thermoregulation in NNV pathogenesis. The present study profiled the temperature-dependent regulation of cytokines (TNF-α, IL-1β and IFN-γ), innate antiviral factors (IFN-1, Mx, ISG-15), adaptive immune factors (CD-4, CD-8, IgM), signaling regulators (SOCS-1, SOCS-3), transcription factors (STAT-1, STAT-3) and microglial and NCC/NK specific cell markers (TMEM-119 and NCCRP-1) during NNV challenge in seven-band grouper, Hyporthodus septemfasciatus. The co-habitation challenge at 17 °C with showed a sustained expression of proinflammatory cytokines and following rechallenge with a dose of 104 TCID50/100μL/fish at optimal temperature, the survivors also exhibited a stable expression of immune factors. The 100% survival following the challenge at sub-optimal (17 °C) and rechallenge at optimal (25 °C) was due to the stable and sustained activation of the immune response. However, at 25 °C, the rechallenge displayed a priming effect with hyperactivation of the immune system evident from the immune gene expression profile. The mortality pattern observed is co-related with the cytokine storm as is evident from the gene expression profile. Whereas, neither of the adaptive immune markers was suggestive of humoral immune response in the 17 °C groups. Also, the data suggest a possible role of NK cell and microglia in mediating antiviral immune response following infection in the brain at different temperatures, where, former is beneficial in restricting viral infection with higher host tolerance.

Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish.

Recently, three types of betanodavirus including red spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), and Korean shellfish nervous necrosis virus (KSNNV) (proposed as a new fifth type) have been detected in shellfish in the marine environment around Korea. To investigate the presence of reassortment between betanodavirus types, the type based on the RNA2 segment of betanodaviruses carried in 420 domestic shellfish (n = 306) and finfish (n = 35), as well as imported shellfish (n = 79), was compared with the type identified by reverse-transcriptase polymerase chain reaction (RT-PCR) for RNA1 segment. Only five samples carrying reassortant betanodaviruses were found, appearing as RG/KSNNV (n = 2), KS/RGNNV (n = 1), and SJ/RGNNV (n = 2) types. From these samples, we successfully isolated two reassortant strains from Korean and Chinese shellfish in E-11 cells and called them KG1-reKS/RG and CM1-reRG/KS, respectively. In the full genome sequences, each RNA segment of the reassortant strains exhibited the same gene length and high sequence homology (≥98%) with the reference strains corresponding to the type of each segment. Both these reassortant strains induced high mortality to sevenband grouper (Epinephelus septemfasciatus) larvae with high viral concentrations in the body (109 viral particles/mg) and severe vacuolation in the retina and brain. These are the first results showing the involvement of the KSNNV type in the reassortment of RNA segments in the reported types of betanodavirus, which could represent a new potential risk in fish.

Mass Production of Virus-Like Particles Using Chloroplast Genetic Engineering for Highly Immunogenic Oral Vaccine Against Fish Disease.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

Spreading of farmed sevenband grouper Hyporthodus septemfasciatus naturally convalesced from prior mild nervous necrosis virus (NNV) infection

As a consequence of nervous necrosis virus (NNV) infection in the East Asian aquaculture industry, sevenband grouper Hyporthodus septemfasciatus in particular has been severely impacted, and is a species that incurs large economic losses. Recently, it has been observed that sevenband grouper in some aquaculture farms do not die even after challenge with NNV. To investigate further, sevenband grouper obtained from an aquaculture farm were intramuscularly inoculated with NNV at 104.3 and 106.3 TCID50/fish (doses that are 100-fold and 10,000-fold the LD50) at eight day intervals, in order to analyze the in vivo multiplication states of NNV, the induction of NNV-specific antibodies and induction of NNV-neutralizing activity. No mortality was observed in the fish challenged once and/or twice with NNV. Multiplication of NNV was observed in 57.1% and 21.4% of fish after the first and second injections, respectively. Although NNV-specific antibodies were reliably detected from one out of eight fish before NNV injection, antibody levels were almost universally elevated following each NNV injection. After the second injection, 85.7% fish were positive for NNV-specific antibodies. Interestingly, almost no NNV-neutralizing activity was detected in any fish sera. These responses are consistent with those of fish that have convalesced from NNV infection. Therefore, we conclude that the fish used in these experiments had experienced mild NNV infections without mass mortality in the past, and naturally convalesced in an aquaculture farm setting.

Proteasome subunit beta type-8 from sevenband grouper negatively regulates cytokine responses by interfering NF-κB signaling upon nervous necrosis viral infection

During viral infection, proper regulation of immune signaling is essential to ensure successful clearance of virus. Immunoproteasome is constitutively expressed and gets induced during viral infection by interferon signaling and contributes to regulate proinflammatory cytokine production and activation of the NF-κB pathway. In this study, we identified Hs-PSMB8, a member of the proteasome β-subunits (PSMB) family, as a negative regulator of NF-κB responses during NNV infection. The transient expression of Hs-PSMB8 delayed the appearance of cytopathic effect (CPE) and showed a higher viral load. The Hs-PSMB8 interacted with NNV which was confirmed using immunocolocalization and co-IP. Overexpression of Hs-PSMB8 diminished virus induced activation of the NF-κB promoters and downregulated the activation of IL-1β, TNFα, IL6, IL8, IFNγ expression upon NNV infection. Collectively, our results demonstrate that PSMB8 is an important regulator of NF-κB signaling during NNV infection in sevenband grouper.

Efficacy of live NNV immersion vaccine immunized at low temperature in sevenband grouper, Epinephelus septemfasciatus

The objective of this study was to investigate safety and efficacy using a low-temperature immunization protocol with NNV in sevenband grouper, Epinephelus septemfasciatus. Further, NNV specific antibody post immunization and intramuscularly challenge was also evaluated. Immunization at low temperature resulted in a low titer virus infection in brain tissues without any clinical symptoms of infection such as sluggish behavior and/or spinning, rotating swimming being observed, and no mortality was observed. Post challenge, NNV titer NNV giving an RPS of 100 %, increased in brain tissues of naïve (non-immunized) sevenband grouper NNV giving an RPS of 100 %, with a cumulative mortality of 100 % at 25 days post-infection. No mortality or disease symptoms NNV giving an RPS of 100 %, as NNV giving and of 100 %, observed in the groups immunized at low temperature with live NNV giving an RPS of 100 %. NNV giving an RPS of 100 %. NNV specific antibody was not detected in live NNV vaccinated sevenband grouper. This is the first study that confirms that field-scale NNV immersion vaccine can protect sevenband grouper against lethal infection with NNV at natural seawater temperature under the gradually increased from 14.3–24.8 °C.

Early viral uptake and host-associated immune response in the tissues of seven-band grouper following a bath challenge with nervous necrosis virus

In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation.

Comparative immunohistological study on using capsaicin, piperine, and okadaic acid for the transepithelial passage of the inactivated viral and bacterial vaccines in fish.

The practical difficulty of parenteral application of fish vaccines against devastating fish diseases diverted the interest toward oral vaccination. Search for effective methods to enhance the oral uptake of viral and bacterial vaccines is continuing. The current research focus on a new role of mucosal fish vaccine adjuvants inducing the antigen uptake by enhancing vascularity or increasing intestinal permeability. Some inflammatory substances cause reversible pathology to the intestinal epithelium, which could be employed for the transepithelial passage of vaccine particles. The natural inflammatory substances used were capsaicin, piperine, and okadaic acid as 1 mg, 2 mg, and 1 μg/fish, respectively. Two inactivated vaccines were used as antigens to test the effect of these inflammatory substances in two different fish hosts. Tested vaccines were inactivated redspotted grouper nervous necrosis virus vaccine in sevenband grouper (Epinephelus septemfasciatus) and inactivated Edwardsiella tarda vaccine in red sea bream (Pagrus major) fish models. The inflammatory substances and each vaccine were anally intubated to fish. Capsaicin proved to be effectively aiding the transepithelial passage of vaccine particles more than piperine, while okadaic acid had no detectable effect.

Development of a lateral flow immuno-chromatic strip assay for the detection of nervous necrosis virus (NNV, RGNNV genotype)

Viral nervous necrosis (VNN), a nodaviral disease, currently represents a global threat to sustainable marine aquaculture. An efficient and rapid on-site detection method is in need for the early diagnosis of VNN. We have developed and validated a lateral flow immuno-chromatographic assay (LFIA) using the previously reported six anti-nervous necrosis virus (NNV) monoclonal antibody (mAb) for the point-of-care detection of NNV (RGNNV genotype). mAb 2B1 was used to bio-conjugate NNV antigen with colloidal gold nanoparticles, and mAb 2B11 was used to capture antigen bound colloidal gold on the test line. A downstream control line was lined with goat anti-mouse IgG to capture unbound gold nanoparticle to validate the performance. The test result can be viewed within 10 min after the application of virus culture supernatants and brain tissue lysates in sample buffer to the sample well in separate tests. The limit of detection of the LFIA test was found to be 105.05 TCID50/100 μL without any cross-reactivity to other fish viruses. Field-level validation of the assay using brain tissue of RGNNV-infected and non-infected sevenband grouper (Hyporthodus septemfasciatus) showed a specificity of 100% and sensitivity of 94.92% with 96.81% diagnostic effectiveness. The developed LFIA strip test will be a useful device for the early detection of NNV in farm-level.

Validation of housekeeping genes as candidate internal references for quantitative expression studies in healthy and nervous necrosis virus-infected seven-band grouper (Hyporthodus septemfasciatus)

BackgroundIn the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus.MethodsExpression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0.ResultsTissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals.ConclusionsThese results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.

Functional characterization of seven-band grouper immunoglobulin like cell adhesion molecule, Nectin4 as a cellular receptor for nervous necrosis virus

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.

Immunoglobulin-like cell adhesion molecules, nectins - Characterization, functional prediction and expression profiling from seven-band grouper, Hyporthodus septemfasciatus

Here, we report the characterization of four calcium-independent cell adhesion molecules, viz- nectin-1, 2, 3 and 4 from seven-band grouper Hyporthodus septemfasciatus a high valued marine finfish susceptible for nervous necrosis virus (NNV) infection. Structural analysis of the nectin molecules revealed an N-terminal V domain, two constant C2 domains, a transmembrane domain, and a cytoplasmic tail. All the nectin molecules except nectin-4 shared a C-terminal interaction motif (E/AxYV). Phylogenetic analysis revealed a close relationship between seven-band grouper nectins and zebrafish nectins. Also, nectins ubiquitously expressed in all the tissues of healthy fish with higher transcript abundance in the brain, gill and anterior kidney. Significant up-regulation of nectin-4 expression was observed in the brain of NNV challenged fish followed by the gill and kidney. Further, docking simulations of nectins with NNV capsid proteins revealed a strong interaction with nectin-1 and nectin-4. Our findings suggest that nectins might have a possible role to play in NNV neuropathogenesis. The present observations provide potential clues for further elucidating the involvement of nectins in NNV pathogenesis and could help in the development of new antiviral therapeutics inhibiting the NNV entry to the cell.

Complete genome sequence and pathogenic analysis of a new betanodavirus isolated from shellfish.

We determined the complete genomic RNA sequence of a new type of betanodavirus Korea shellfish nervous necrosis virus (KSNNV) isolated from shellfish. Compared with other isolates representing four genotypes of betanodaviruses, the identity of the whole nucleotide sequence of the virus was in the range of 76%-83% with the presence of specific genetic motifs and formed a separate new branch in the phylogenetic analysis. In pathogenic analysis by immersion method, KSNNV-KOR1 shows 100% cumulative mortality like SFRG10/2012BGGa1 (RGNNV) in newly hatched sevenband grouper and mandarin fish, which is clearly different from those found in negative control groups. There were no significant differences in increasing rates of mortality and viral intra-tissue concentration of larval fishes infected with KSNNV-KOR1 at both 20 and 25°C water temperature. Histopathological examination of each fish species in the moribund stage revealed the presence of clear vacuoles in both brain and retinal tissues similar to typical histopathology features of RGNNV. In the present study, we first report a new betanodavirus from shellfish as the aetiological agent of viral nervous necrosis disease in fish with complete genomic nucleotide sequence and pathogenic analysis.

Development of double labeling in situ hybridization using RNA probes for genome detection of nervous necrosis virus (NNV)

In situ hybridization (ISH) of genomic segments using RNA-RNA hybrid for nervous necrosis virus (NNV) detection has not been reported yet. The objective of this study was to develop RNA-ISH using RNA probes for the detection of NNV in infects SSN-1 cells or sevenband grouper Hyporthodus septemfasciatus. Two viral RNA segments viz., RNA1 and RNA2 were synthesized by in vitro transcription and labeled with fluorescein UTP and dignoxigenin dUTP, respectively. These labeled RNA probes specifically detected NNV in infected SSN-1 cells. We also applied double labeling RNA-ISH with two-color staining of RNA probes. The results showed that these two viral genomic segments were localized in same regions although RNA1 was also expressed separately. These findings suggest that RNA1 overexpression may be important for sufficient assembly of infectious particles. The RNA-ISH showed that both RNA segments were localized in the tectum opticum, torus semicircualris, cerebellum, thalamus, hypothalamus, and medulla of experimentally infected brain tissues. Especially, RNA segments were highly localized around the ventricle, suggesting that ventricle might play a vital role in the spread of NNV. This technique can be useful for understanding the localization of NNV and the relationship between clinical sign and viral expression.