Red Seabream Iridovirus Infection Research Articles

Evaluation of the diagnostic assays detecting red sea bream iridovirus infection at different severity levels

Red sea bream iridovirus (RSIV) is a highly contagious viral infection that affects various fish species and poses a significant threat to the global aquaculture industry. Thus, accurate and timely diagnosis is paramount for sustainable management of fish health. This study rigorously evaluated the diagnostic efficacy of various polymerase chain reaction (PCR) assays, focusing on those recommended by the World Organization for Animal Health (WOAH) and the assays newly proposed by WOAH's Aquatic Animals Health Standards Commission. Specifically, this study assessed conventional PCR, nested PCR, modified 1-F/1-R, and real-time PCR assays using a 95% limit of detection (LoD95%), as well as diagnostic sensitivity (DSe) and specificity (DSp) tests across different RSIV severity grades (G0−G4). In previous studies, the LoD95% for the 1-F/1-R and 4-F/4-R conventional assays were 225.81 and 328.7 copies/reaction, respectively. The modified 1-F/1-R exhibited a lower LoD95% of 51.32 copies/reaction. Notably, the nested PCR had an LoD95% of 11.23 copies/reaction, and the real-time PCR assay had an LoD95% of 12.02 copies/reaction. The DSe varied across RSIV severity grades, especially in the lower G0−G2 grades. The nested PCR and modified 1-F/1-R assays displayed the highest DSe, making them particularly useful for early-stage screening and detection of asymptomatic carriers. In addition, the PCR assays did not cross-react with any other aquatic pathogens except RSIV. Our findings significantly advanced the diagnostic capabilities of RSIVD by suggesting that nested PCR and modified 1-F/1-R assays are particularly promising for early detection. We propose their inclusion in future WOAH guidelines for a more comprehensive diagnostic framework.

Development of a Propidium Monoazide-Based Viability Quantitative PCR Assay for Red Sea Bream Iridovirus Detection

Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 μM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater.

Molecular characterization and expression of CD96 in red seabream (Pagrus major)

CD96 is a membrane-bound receptor discovered in humans in 1992 that is mainly present in natural killer cells and T cells derived from haematopoietic cells and performs immune functions. Based on the sequence of CD96 obtained from red seabream (Pagrus major), phylogenetic analysis with other species, infections of normal fish, Streptococcus iniae and red sea bream iridovirus (RSIV), and expression analysis was conducted using real-time polymerase chain reaction. Phylogenetic analysis showed the highest homology with Sparus aurata, and multiple sequence analysis confirmed the conservation of major domains between different fish species. Normal fish high expression results were confirmed in the head kidney, and spleen, which are the haematopoietic organs of the fish. High expression levels were confirmed in the gills, liver, spleen, and kidney on day three after RSIV infection. After S. iniae infection, high expression was confirmed in the gills and liver on day one, and high expression was confirmed in the spleen from 12 hours. These results show that PmCD96 functions as an immune gene in P. major and is considered a basic research case for CD96 in fish’s hematopoietic organ immune system.

Analysis of the transcriptomic profiles of Mandarin fish (Siniperca chuatsi) infected with red sea bream iridovirus (RSIV).

Red sea bream iridovirus (RSIV) belongs to the family Iridoviridae, genus Megalocytivirus, which could widely infect marine fish, causing diseases and huge economic losses. Now it has been reported that RSIV was also detected in diseased mandarin fish. Transmission electron microscopy and immunohistochemistry showed that spleen was the main target organ in mandarin fish infected with RSIV. To investigate the immune response mechanism of mandarin fish to RSIV infection, transcriptomics of RSIV-infected mandarin fish was analyzed. A total of 53,040 unigenes were obtained, and there were 21,576 and 17,904 unigenes had significant hit the Nr and SwissProt databases, respectively. In RSIV-infected and non-infected spleen tissues, there were 309 differentially expressed genes (DEGs), including 100 up-regulated genes and 209 down-regulated genes. Gene Ontology database (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis were performed to reveal the function information and give a better understanding of the signal transduction pathways of DEGs. Further analysis of the cytokine-cytokine receptor interactions pathway exhibited that the expression of cytokines was widely activated after viral infection. In addition, ten DEGs were randomly selected and verified by quantitative real-time PCR, which revealed a similar expression tendency as the high-throughput sequencing data. These findings present valuable information that will benefit for better understanding of RSIV infection in mandarin fish.

Molecular characteristics, expression analysis and antiviral activity against RSIV of red sea bream IRF3 and IRF7

Interferon regulatory factor (IRF) 3 and IRF7 are important innate immune-related genes that induce type I interferon (IFN). In this study, PmIRF3 and PmIRF7 were identified from red sea bream (Pagrus major) infected by red sea bream iridovirus (RSIV), and expression and functional analyses were performed. PmIRF3 and PmIRF7 consisted of 465 and 436 amino acids, respectively, and contained a well-conserved IRF domain, IRF3 domain, and serine-rich domain (SRD). PmIRF3 and PmIRF7 are ubiquitously expressed in the liver and gills in normal fish, and with a high expression being recorded in kidney and spleen during RSIV infection. In P. major fin cells (PMF cells), pAcGFP-PmIRF3 and pAcGFP-PmIRF7 were localized to the cytoplasm, and overexpression of PmIRF3 and PmIRF7 using the pcDNA3.1 vector induced type I IFN. In addition, in the experiment in vitro, PMF cells overexpressed with PmIRF3 or PmIRF7 exhibited antiviral activity against RSIV and significantly reduced viral genome copies and virus titer. Therefore, PmIRF3 and PmIRF7 may have antiviral activity through the induction of type I IFN against RSIV infection of red sea bream.

Generation of monoclonal antibodies against heterologously expressed major capsid protein of infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is one of the detrimental viruses causing economic losses in several fish species including Asian sea bass (Lates calcarifer). In this study, monoclonal antibodies specific to major capsid protein (MCP) of ISKNV were generated. The MCP encoding gene of ISKNV genotype II was cloned into pGEX-6P-1 and transformed into E. coli. After induction, a glutathione-S-transferase (GST)-MCP fusion protein was produced and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Five MAbs specific to MCP were obtained after selection against naturally ISKNV-infected Asian sea bass by dot blotting, Western blotting and immunohistochemistry. All MAbs showed no cross-reaction with fish tissues, fish viruses including tilapia lake virus (TiLV), nervous necrosis virus (NNV), scale drop disease virus (SDDV) and eight bacterial species commonly found in diseased fish. The detection sensitivity of MAbs was inferior to that of one step PCR. However, a combination of two MAbs enhanced the detection limit about two-fold. The intense immunoreactivity staining could be observed in liver, kidney, gill, intestine, stomach, and spleen tissues from ISKNV-infected fish by immunohistochemical analysis. All five MAbs can be used to detect naturally ISKNV-infected fish from two different farms and phylogenetic tree analysis based on MCP gene sequences demonstrated ISKNV genotype I infection in diseased fish. In addition, all MAbs displayed immunoreactivity to red sea bream iridovirus (RSIV) in the GF cell culture fluid by dot blotting. Therefore, the generated MAbs could be used for detection of ISKNV infection by various immunological assays and might be able to detect RSIV infection as well.

Red sea bream interleukin (IL)-1β and IL-8 expression, subcellular localization, and antiviral activity against red sea bream iridovirus (RSIV)

Interleukin-1 beta (IL-1β) is transcribed by monocytes, macrophages, and dendritic cells in response to activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) or cytokine signalling and causes a rapid inflammatory response to infection. IL-8, also known as chemokine C-X-C motif ligand (CXCL)-8, is regulated by IL-1β and affects the chemotaxis of macrophages and neutrophils upon pathogen infection. In healthy red sea bream, rsbIL-1β is most highly distributed in the liver, and rsbIL-8 is most highly distributed in the head kidney. In response to RSIV infection, rsbIL-1β and rsbIL-8 mRNA are significantly upregulated in the kidney and spleen. This may be because the primary infection targets of RSIV are the kidney and spleen. In the gills, both genes were significantly upregulated at 7 days after RSIV infection and may be accompanied by a cytokine storm. In the liver, both genes were significantly downregulated at most observation points, which may be because the immune cells such as macrophages and dendritic cells expressing rsbIL-1β or rsbIL-8 migrated to other tissues because the degree of RSIV infection was relatively low. Using a GFP fusion protein, it was confirmed that rsbIL-1β and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. RsbIL-1β overexpression induced the expression of interferon gamma (IFN-γ), myxovirus-resistance protein (Mx) 1, IL-8, IL-10, TNF-α, and MyD88, while rsbIL-8 overexpression induced the expression of IFN-γ, Mx1, rsbIL-1β and TNF-α. In addition, overexpression of both genes significantly reduced the genome copies of RSIV and significantly reduced the viral titers. Therefore, rsbIL-1β and rsbIL-8 in red sea bream play an antiviral role against RSIV through their normal signalling.

Virulence difference between red sea bream iridovirus mixed subtype I/II and subtype II and the expression of viral and apoptosis-related genes in infected rock bream (Oplegnathus fasciatus)

In this study, the virulence of the red sea bream iridovirus (RSIV) subtype II (17RbGs isolate) and a novel RSIV mixed subtype I/II (17SbTy isolate), which was genetically characterized in a previous study, were compared. The infectivity to rock bream (Oplegnathus fasciatus) determined by infectious dose (ID50) revealed that 17RbGs isolate was significantly more infective than 17SbTy isolate using both intraperitoneal injection and bath immersion. In a cohabitation challenge test that mimicked natural conditions, the cumulative mortality of the donor (RSIV-injected rock bream) and the recipient (cohabited naïve rock bream) was significantly higher in the 17RbGs group than in the 17SbTy group, regardless of RSIV injected doses, supporting the correlation between genetic mutation and pathogenicity. In addition, the maximum viral shedding ratio identified from RSIV-infected rock bream suggested that viral transmission through infection with the 17SbTy isolate could have a lower relative risk than that of infection with the 17RbGs isolate. In particular, the odds ratio based on the spleen index after 17RbGs infection was 55.00, which was inconsistent with that of 17SbTy infection (19.38), hence supporting the virulence difference between RSIVs. Furthermore, the expression of viral genes, including DNA membrane and myristoylated protein genes with insertion and deletion mutations, and that of caspase-8, which is related to caspase-dependent apoptosis induced by RSIV infection, were significantly upregulated at 11 days post 17RbGs-infection compared to that following 17SbTy infection. Notably, although viral genes were highly expressed in the early infection stage and caspase-8 was upregulated, the low caspase-3 expression may have inhibited apoptosis, reflecting the difference in virulence between different RSIV isolates. Several virulence factors, including pathogenicity, viral shedding ratio, odds ratio, and gene expression, support that RSIV mixed subtype I/II may be a less pathogenic RSIV isolate compared with general RSIV subtype II in a natural environment.

Red sea bream iridovirus infection in cage farmed Asian sea bass (Lates calcarifer): Insights into the pathology, epizootiology, and genetic diversity

Red sea bream iridovirus (RSIV) is an emerging viral pathogen having significant socio-economic and environmental impacts on marine and brackish aquaculture systems. The study documents certain interesting findings of an RSIV disease outbreak that caused 50% cumulative mortality among cage farmed Asian sea bass in India. The confirmatory diagnosis was made based on internationally approved protocols. The affected fish showed splenomegaly, anaemia, and abnormally enlarged cells within the spleen, liver, kidney, and brain. The consistent presence of two fish pathogens, viz., Vibrio alginolyticus and Vibrio fluvialis, in the liver, along with characteristic vibrio-induced pathologies were noted, indicating the possible role of these bacterial pathogens in RSIV pathogenesis during the outbreak. More strikingly, the surviving fish were found to carry the virus even after two months post-outbreak, warranting future investigations on the carrier status of RSIV survivors. Transmission electron microscopy in the survivors demonstrated atypical virus particles within the degenerated cells of the vital organs, suggesting the role of host immune response in preventing the maturation of RSIV particles in the survivors. Phylogram based on MCP and ATPase genes demonstrated the spread of the same RSIV clone across different countries, regardless of the fish species. Results also revealed the presence of two RSIV subtypes. Both subtypes were mainly distributed in marine demersal fishes of Asian countries. Presently, subtype 2 is the most frequently reported one. Subtype 1 revealed higher genetic diversity with a wider geographical spread and host range. Molecular analyses revealed that MCP and ATPase genes could be targeted in RSIV subtyping, with MCP as a better candidate. A purifying selection was observed on the MCP gene that can be applied for diagnostic protocols. In short, the study generates certain thought-provoking insights on RSIV through the analyses of a recent outbreak along with the genetic and epizootiological data of global strains. Further, the paper also forms the first report on the presence of RSIV in surviving fish post-disease outbreak in the absence of clinical signs. Altogether, the results highlight the necessity of strengthening quarantine and RSIV diagnostic measures to have sustainable aquaculture practices.

Development of a method for experimental infection of Pacific bluefin tuna with red seabream iridoviral disease

The market for Pacific bluefin tuna (Thunnus orientalis) is expanding as global consumption increases. Meeting this demand has led to overfishing and consequent depletion of wild populations, resulting in a focus on aquaculture as an alternative to fishing. However, under the crowded conditions typical of tuna aquaculture settings, infectious diseases are a common problem associated with considerable economic losses, especially with regard to Pacific bluefin tuna, which is a high-priced species. Studying diseases in Pacific bluefin tuna requires techniques that enable experimental infection; however, no promising methods are available due to the difficulty of handling these fish, including transportation, retention, and injection. In the present study, therefore, we developed a method for experimental infection of juvenile Pacific bluefin tuna with red seabream iridovirus (RSIV), which causes an acute and highly contagious disease in this species. The route of experimental infection was validated using Pacific bluefin tuna and greater amberjack (Seriola dumerili), which is used as a model species susceptible to the disease. Oral infection with the virus was found to be suitable for reproducing red seabream iridoviral disease (RSIVD). A high copy number of the RSIV genome was detected in Pacific bluefin tuna that died following RSIV experimental infection, indicating that the virus efficiently replicated in the infected fish. In addition to virus detection, a histopathologic abnormality specific to RSIV infection was detected in the infected fish. These results indicate that experimental infection via the oral route is effective for reproducing RSIVD in Pacific bluefin tuna.

Development of a high-dose vaccine formulation for prevention of megalocytivirus infection in rock bream (Oplegnathus fasciatus)

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 1010 copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 1010 copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 109 copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 108 copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 104 copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.

Complete genome analysis of a red seabream iridovirus (RSIV) isolated from Asian seabass (Lates calcarifer) in India

Red sea bream iridovirus (RSIV) is the causative agent of the iridoviral disease with high mortality rates in cultured fish. Our laboratory reported the first case of RSIV infection in India which resulted in mass mortalities of Asian seabass, Lates calcarifer. The RSIV-LC strain isolated from infected fish was subjected to complete genome sequencing and analysis. The complete genome of RSIV-LC was found to be of 111,557 bp in size having a G + C content of 53 %. The complete genome has 114 open reading frames (ORFs) of which 38 ORFs were predicted as functional proteins while the rest were hypothetical proteins. Among the ORFs 26 were found to be core genes reported earlier to be homologous in iridovirus complete genomes. Phylogenetic tree constructed based on the 26 core gene sequences, major capsid protein and ATPase genes revealed RSIV-LC in this study to belong to the genus Megalocytivirus of the RSIV-Genotype II. The present study provides the first report of the complete genome sequence and annotation of the RSIV strain isolated from India.

Efficacy of a bivalent inactivated vaccine against red seabream iridovirus and Streptococcus iniae in red seabream, Pagrus major

The red seabream (Pagrus major) is an economically important marine fish species that is cultured in Japan. Natural outbreaks of red seabream iridovirus (RSIV) and/or Streptococcus iniae can cause massive mortalities and severe economic losses in red seabream production. No effective single-injection vaccine for protecting red seabream against both diseases has yet become commercially available. Here, we evaluated the efficacy of a bivalent formalin-killed vaccine (PISCIVAC™ Irido Si) against RSIV and S. iniae infections in red seabream. In laboratory trials, intraperitoneally vaccinated fish reared at 25 °C were protected against challenges with RSIV and S. iniae as early as 3 days post vaccination (dpv), with a relative percent survival (RPS) of 69% and 90%, respectively. The protection was also observed in fish reared at 15 °C, with a RPS of 88% and 89% after challenging the fish with RSIV and S. iniae at 10 dpv and 14 dpv, respectively. Furthermore, intramuscular vaccination effectively protected fish against RSIV (RPS of 100% and 65–96% at 10 and 20 dpv, respectively) and S. iniae (RPS of 92–100% at 14 dpv). The vaccine conferred protection against RSIV infection for at least 2 months and against S. iniae infection for at least 6 months in the field trial. These results indicate that PISCIVAC™ Irido Si is effective for protecting red seabream against RSIV and S. iniae infections.

Multiple Passages of Grunt Fin Cells Persistently Infected with Red Seabream Iridovirus (RSIV) at 15ºC or 30ºC to Yield Uninfected Cells.

Red seabream iridovirus (RSIV), a member within genus Megalocytivirus (Iridoviridae), causes serious economic losses to marine fish aquaculture industry in East Asia. In this study, we established a Blue Striped Grunt Haemulon sciurus fin (grunt fin; GF) cell line persistently infected with RSIV (PI-GFRSIV) by subculturing GF cells that survived RSIV inoculation. PI-GFRSIV cells were morphologically indistinguishable from naive GF cells. They could stably produce RSIV at approximately 104.9 ± 0.5 genomes per microliter after 24 passages over 18 months. The optimum temperature to produce RSIV in PI-GFRSIV cells was 25°C. These cells also produced RSIV at 15, 20, and 30°C with multiple subcultures. The amount of RSIV yielded from PI-GFRSIV cells decreased gradually by multiple subculturing at 15°C or 30°C. Red seabream iridovirus was no longer detected from PI-GFRSIV cells after subcultures at these temperatures. These PI-GFRSIV cells freed from RSIV infection exhibited a level of RSIV productivity similar to those of naive GF cells after inoculation with RSIV. Therefore, we consider that these PI-GFRSIV cells were no longer infected with RSIV after multiple subculturing at 15°C or 30°C. Received October 15, 2015; accepted June 27, 2016.

Establishment of rock bream Oplegnathus fasciatus embryo (RoBE-4) cells with cytolytic infection of red seabream iridovirus (RSIV)

Red seabream iridovirus (RSIV) is a member of genus Megalocytivirus in the family Iridoviridae. RSIV infection causes significant economic losses of marine-fishes in East Asian countries. Grunt fin (GF) cell line has been commonly used for culturing RSIV. However, it is not suitable for definite evaluation of infectivity titer of RSIV because cells infected with RSIV are not completely cytolysed. Thus, we established a new cell line, RoBE-4, from rock bream (Oplegnathus fasciatus) eyed-egg embryos in this study. Morphologically, RoBE-4 cells were fibroblastic-like. They have been stably grown over two-years with 60 passages using Leibovitz’s L-15 medium containing 10% (v/v) fetal bovine serum. RoBE-4 cells infected with RSIV exhibited cytopathic effects (CPE) with cell rounding. They were cytolysed completely after ≥2 weeks of culture. Numerous RSIV particles with icosahedral morphology of approximately 122nm in diameter were observed in cytoplasmic area of infected RoBE-4 cells. The RSIV-suceptibility and amount of extracellular RSIV released by RoBE-4 cells were 100-fold higher than those by GF cells. RSIV cultured with RoBE-4 cells was highly virulent to rock bream in infection experiments. Therefore, using RoBE-4 cells instead of GF cells will enable accurate and sensitive measurement of RSIV infectivity. In addition, RoBE-4 cells might be used to produce RSIV vaccine in the future with significant reduction in cost.

English

Red seabream iridovirus (RSIV), a member of the Iridoviridae family, is the causative pathogen of some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of RSIV infection. In the present study, a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of RSIV infection in fishes was developed. Using a set of synthesized primers matching a specific region of the RSIV genome (GenBank accession no.: AB666336.1), the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficiently detect RSIV infection in red sea bream. Our results provide a simple and convenient method for the detection of viral infection in aquatic organisms. Key words: Red sea bream, Iridovirus, polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP).

First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus

CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

Quantitative change of red seabream iridovirus (RSIV) in rock bream Oplegnathus fasciatus, following Poly(I:C) administration

Serious economic losses due to red seabream iridovirus (RSIV) infection have occurred in fingerlings and market-sized rock bream Oplegnathus fasciatus in Korea. Previously, it was demonstrated that fishes immunized with a live fish RNA virus following polyinosinic–polycytidylic acid [Poly(I:C), an inducer of interferon (IFN)] administration were protected from the viral infection [Poly(I:C) immunization with live virus]; however, rock bream administered Poly(I:C) were not protected from RSIV infection, although IFN was expressed well in rock bream that received Poly(I:C). In this study, we investigated kinetics of the RSIV in rock bream that did or did not receive Poly(I:C). No significant difference was observed in the mortality pattern due to RSIV infection regardless of Poly(I:C) administration, indicating that rock bream administered Poly(I:C) were not protected from RSIV infection. No significant difference was observed in the pattern of RSIV kinetics between the fishes that did or did not receive Poly(I:C). We would conclude that RSIV is resistant to IFN induction in rock bream.

Molecular cloning and expression analysis of two lipopolysaccharide-induced TNF-α factors (LITAFs) from rock bream, Oplegnathus fasciatus

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α factor (LITAF) plays an important role controlling the expression of TNF-α and the other cytokine genes in the presence of LPS. However, two LITAF homologues have not been characterized in fish. In this study, we cloned two distinct LITAF (RbLITAF1 and RbLITAF2) cDNAs from rock bream (Oplegnathus fasciatus) and characterized their expression profiles after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding regions of RbLITAF1 and RbLITAF2 cDNAs were 492 bp and 417 bp, encoding 153 and 138 amino acid residues, respectively. The genes consisted of a LITAF domain. RbLITAF1 was highly expressed in the spleen and heart of healthy rock bream, whereas RbLITAF2 was highly expressed in the gill, intestine and stomach. In spleen, the gene expression of RbLITAF1 and RbLITAF2 were increased until 5 days post-infection (dpi), and then decreased at 7 dpi. In kidney, E. tarda and RSIV infection led to induction of the RbLITAF1 gene at 1 dpi, RbLITAF2 gene was down-regulated after pathogen infection. These results suggest that RbLITAFs may be involved in the LITAF-mediated immune response and regulate systemic immune responses against pathogen infection.

Potential for a live red seabream iridovirus (RSIV) vaccine in rock bream Oplegnathus fasciatus at a low rearing temperature

Serious economic losses have occurred in fingerlings and market-sized rock bream Oplegnathus fasciatus in Korea due to red seabream iridovirus (RSIV) infection. We demonstrated previously that viral multiplication in fish is downregulated by maintaining fish at far from optimum temperatures at the onset of disease. We applied this concept to develop a live RSIV vaccine in rock bream. Mortalities in rock bream that were inoculated with RSIV and reared at 21–30°C were ≥90%, whereas no mortality was observed in fish that received an RSIV inoculation and were reared at ≤18°C. RSIV kinetics revealed that RSIV multiplied rapidly in fish reared at 24.3±1.3°C, and achieved the critical level for rock bream (approximately 109.0 genomes/mg) within 28 days. In contrast, the RSIV genome was detected on day 10 in fish that received an RSIV inoculation at 15.5°C, and peaked on day 28 at 105.91±0.54 genomes/mg, then decreasing gradually, and were then maintained under the detection level beginning on day 84 after RSIV inoculation. Furthermore, the fish surviving the RSIV infection at low rearing temperature were strongly protected from re-challenge with homologous RSIV; the threshold level of RSIV for rock bream to mount a protective immune response was ≤105.4 genomes/mg. Cohabitation experiments revealed that the spread of RSIV from rock bream vaccinated with a live RSIV could be low if it is limited to fish in the late stage (≥84 days of elapse) after vaccination. Thus, it was concluded that when rock bream are reared at ≤18°C and inoculated with RSIV, the survivors can mount a protective immune response against RSIV, suggesting a positive effect of a live RSIV vaccine for rock bream.