Risk assessment of wild fish as environmental sources of red sea bream iridovirus (RSIV) outbreaks in aquaculture.

Red sea bream iridovirus (RSIV) causes substantial economic damage to aquaculture. In the present study, RSIV in wild fish near aquaculture installations was surveyed to evaluate the risk of wild fish being an infection source for RSIV outbreaks in cultured fish. In total, 1102 wild fish, consisting of 44 species, were captured from 2 aquaculture areas in western Japan using fishing, gill nets, and fishing baskets between 2019 and 2022. Eleven fish from 7 species were confirmed to harbor the RSIV genome using a probe-based real-time PCR assay. The mean viral load of the RSIV-positive wild fish was 101.1 ± 0.4 copies mg-1 DNA, which was significantly lower than that of seemingly healthy red sea bream Pagrus major in a net pen during an RSIV outbreak (103.3 ± 1.5 copies mg-1 DNA) that occurred in 2021. Sequencing analysis of a partial region of the major capsid protein gene demonstrated that the RSIV genome detected in the wild fish was identical to that of the diseased fish in a fish farm located in the same area in which the wild fish were captured. Based on the diagnostic records of RSIV in the sampled area, the RSIV-infected wild fish appeared during or after the RSIV outbreak in cultured fish, suggesting that RSIV detected in wild fish was derived from the RSIV outbreak in cultured fish. Therefore, wild fish populations near aquaculture installations may not be a significant risk factor for RSIV outbreaks in cultured fish.

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.

Red sea bream iridovirus infection downregulates inflammation-related genes in the spleen of rock bream (Oplegnathus fasciatus).

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 104 TCID50/fish, the viral genome copy number in the spleen was 104.7 ± 0.2 and 105.9 ± 0.4 copies/µg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus.

A New Cell Line Derived from the Caudal Fin of the Dwarf Gourami (Trichogaster lalius) and Its Susceptibility to Fish Viruses.

The detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n = 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis.

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.

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.

Red Sea Bream Iridovirus (RSIV) Kinetics in Rock Bream (Oplegnathus fasciatus) at Various Fish-Rearing Seawater Temperatures.

Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 103.7-106.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 104 RSIV genome copies/fish, RSIV was not detected at 21-30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3-5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.

Whole-genome analysis of red sea bream iridovirus spread in 2021 in Japan provided epidemiological and viral traits insight.

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

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

Interleukin-1 beta (IL-1ß) 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-1ß and affects the chemotaxis of macrophages and neutrophils upon pathogen infection. In healthy red sea bream, rsbIL-1ß is most highly distributed in the liver, and rsbIL-8 is most highly distributed in the head kidney. In response to RSIV infection, rsbIL-1ß 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-1ß 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-1ß and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. RsbIL-1ß 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-1ß and TNF-α. In addition, overexpression of both genes significantly reduced the genome copies of RSIV and significantly reduced the viral titers. Therefore, rsbIL-1ß 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.

Application of Environmental DNA for Monitoring Red Sea Bream Iridovirus at a Fish Farm.

Red sea bream iridoviral disease (RSIVD) causes high economic damage in mariculture in Asian countries. However, there is little information on the source of infection and viral dynamics in fish farms. In the present study, the dynamics of RSIV in a fish farm that mainly reared juveniles and broodstocks of red sea bream (Pagrus major) were monitored over 3 years (2016 to 2018) by targeting environmental DNA (eDNA) of seawater. Our monitoring demonstrated that red sea bream iridovirus (RSIV) was detected from the eDNA at least 5 days before an RSIVD outbreak in the juveniles. The viral loads of eDNA during the outbreak were highly associated with the numbers for daily mortality, and they reached a peak of 106 copies/liter seawater in late July in 2017, when daily mortality exceeded 20,000 fish. In contrast, neither clinical signs nor mortality was observed in the broodstocks during the monitoring periods, whereas the broodstocks were confirmed to be virus carriers by an inspection in October 2017. Interestingly, the viral load of eDNA in the broodstock net pens (105 copies/liter seawater) was higher than that in the juvenile net pens (104 copies/liter seawater) just before the RSIVD outbreak in late June 2017. After elimination of all RSIV-infected surviving juveniles and 90% of broodstocks, few RSIV copies were detected in the eDNA in the fish farm from April 2018 onward (fewer than 102 copies/liter seawater). These results imply that the virus shed from the asymptomatically RSIV-infected broodstock was transmitted horizontally to the juveniles and caused further RSIVD outbreaks in the fish farm. IMPORTANCE Environmental DNA (eDNA) could be applied in monitoring waterborne viruses of aquatic animals. However, there are few data for practical application of eDNA in fish farms for the control of disease outbreaks. The results of our field research over 3 years targeting eDNA in a red sea bream (Pagrus major) fish farm implied that red sea bream iridoviral disease (RSIVD) outbreaks in juveniles originated from virus shedding from asymptomatically virus-infected broodstocks. Our work identifies an infection source of RSIVD in a fish farm via eDNA monitoring, and it could be applied as a tool for application in aquaculture to control fish diseases.

Development of single nucleotide polymorphism (SNP) application for detection and genotyping of RSIV-type megalocytiviruses.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

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.

Rapid diagnosis of two marine viruses, red sea bream iridovirus and viral hemorrhagic septicemia virus by PCR combined with lateral flow assay.

Red sea bream iridovirus (RSIV) and Viral hemorrhagic septicemia virus (VHSV) are the most important viral marine pathogens in South Korea because RSIV and VHSV infect and cause high mortality rates in major fish species such as Paralichthys olivaceus and Sebastes schlegelii. These viruses can be transmitted both vertically and horizontally, and early diagnosis is imperative. In this research, RSIV and VHSV viral genomes are detected by PCR-lateral flow assay (LFA). PCR-LFA is sensitive, capable of detecting a viral genome at a concentration of 2-200 fg/µL. Development of this detection method is very meaningful because LFA is simple, requiring a minimum of personnel training to perform. Additionally, LFA requires less time than other detection methods and can be an immediate detection tool that is indispensable in preventing rapid viral spread.

PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan.

The Megalocytivirus genus includes three genotypes, red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV), and has caused mass mortalities in various marine and freshwater fish species in East and Southeast Asia. Of the three genotypes, TRBIV-like megalocytivirus is not included in the World Organization for Animal Health (OIE)-reportable virus list because of its geographic restriction and narrow host range. In 2017, 39 cases of suspected iridovirus infection were isolated from fingerlings of giant sea perch (Lates calcarifer) cultured in southern Taiwan during megalocytivirus epizootics. Polymerase chain reaction (PCR) with different specific primer sets was undertaken to identify the causative agent. Our results revealed that 35 out of the 39 giant sea perch iridovirus (GSPIV) isolates were TRBIV-like megalocytiviruses. To further evaluate the genetic variation, the nucleotide sequences of major capsid protein (MCP) gene (1348 bp) from 12 of the 35 TRBIV-like megalocytivirus isolates were compared to those of other known. High nucleotide sequence identity showed that these 12 TRBIV-like GSPIV isolates are the same species. Phylogenetic analysis based on the MCP gene demonstrated that these 12 isolates belong to the clade II of TRBIV megalocytiviruses, and are distinct from RSIV and ISKNV. In conclusion, the GSPIV isolates belonging to TRBIV clade II megalocytiviruses have been introduced into Taiwan and caused a severe impact on the giant sea perch aquaculture industry.

Molecular genetic characterisation and expression profiling of calpain 3 transcripts in red sea bream (Pagrus major).

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Data on molecular characterization and expression of the C-reactive protein (CRP) gene from rock bream, Oplegnathus fasciatus.

C-reactive protein (CRP) is a member of the pentraxin family and is an acute-phase response to plasma protein; its level in blood increases rapidly in response to trauma, inflammation, and infection. In the present study, we analysed the molecular characteristics of the Oplegnathus fasciatus CRP (RbCRP) gene sequence using multiple alignments and phylogenetic analyses of the deduced amino acids. In addition, we also examined RbCRP gene expression in rock bream infected with the pathogens Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) or red sea bream iridovirus (RSIV) and in healthy rock bream individuals. In healthy individuals, RbCRP was ubiquitously expressed in all 14 tested tissues, mainly in the trunk kidney and head kidney. Expression of RbCRP was notably upregulated in the spleen and whole kidney after RSIV infection. This study can provide basic data on the innate immune system of the rock bream to viral and microbial infections.

Data on molecular characterisation and expression analysis of the interferon-related developmental regulator 2 (IFRD2) gene from red sea bream, Pagrus major.

The interferon-related developmental regulator 1 (IFRD1) protein is expected to play a role in the regulation of inflammatory responses in adult mice, since it is known to repress transcription of NF-κB in myoblasts that regenerate skeletal muscle after traumatic injury Micheli et al., 2011. The IFRD2 gene is expressed in many tissues including skeletal muscle, kidney, heart, brain, lung, placenta and liver in adult humans and is highly expressed in adult human skeletal muscle and heart. In mice, interferon-related developmental regulator 2 (IFRD2) may be associated with early haematopoiesis after gastrulation and in the hepatic primordium Buanne et al., 1998. In this study, we analysed the molecular characteristics of the IFRD2 gene identified from Pagrus major (PmIFRD2) and performed multiple alignments and phylogenetic analyses of the protein sequence. In addition, we examined the expression pattern of IFRD2 in healthy red sea bream tissues and the temporal expression pattern after challenging with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV)]. This study characterises the non-specific immune response of the red sea bream after viral and microbial infections.

The impact of pooling samples on surveillance sensitivity for the megalocytivirus Infectious spleen and kidney necrosis virus.

Movements of large volumes and species varieties make the ornamental fish industry a high-risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.

Data on molecular characterization and gene expression analysis of secretory carrier-associated membrane protein 5 (SCAMP5) from the red sea bream (Pagrus major).

Secretory carrier membrane proteins (SCAMPs) are widely distributed integral membrane proteins implicated in membrane trafficking. Secretory carrier membrane protein 5 (SCAMP5) is expected to be involved in regulation of the immune response because it is expressed in a variety of immune tissues and promotes the secretion of cytokines in monocytes and macrophages. In this study, we performed an analysis of the molecular characteristics and phylogenetic of the SCAMP5 gene identified in Pagrus major (PmSCAMP5). In addition, we analysed PmSCAMP5 gene expression levels in the tissues of red sea bream infected with various pathogens [Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and Red sea bream iridovirus (RSIV)], and we analysed PmSCAMP5 gene expression levels in the tissues of healthy red sea bream. This study was carried out to provide basic data on the non-specific immune system of the red sea bream.