Turbot Reddish Body Iridovirus Research Articles

The Inactivated ISKNV-I Vaccine Confers Highly Effective Cross-Protection against Epidemic RSIV-I and RSIV-II from Cultured Spotted Sea Bass Lateolabrax maculatus.

The genus Megalocytivirus of the family Iridoviridae is composed of two distinct species, namely, infectious spleen and kidney necrosis virus (ISKNV) and scale drop disease virus (SDDV), and both are important causative agents in a variety of bony fish worldwide. Of them, the ISKNV species is subdivided into three genotypes, namely, red seabream iridovirus (RSIV), ISKNV, and turbot reddish body iridovirus (TRBIV), and a further six subgenotypes, RSIV-I, RSIV-II, ISKNV-I, ISKNV-II, TRBIV-I, and TRBIV-II. Commercial vaccines derived from RSIV-I , RSIV-II and ISKNV-I have been available to several fish species. However, studies regarding the cross-protection effect among different genotype or subgenotype isolates have not been fully elucidated. In this study, RSIV-I and RSIV-II were demonstrated as the causative agents in cultured spotted seabass, Lateolabrax maculatus, through serial robust evidence, including cell culture-based viral isolation, whole-genome determination and phylogeny analysis, artificial challenge, histopathology, immunohistochemistry, and immunofluorescence as well as transmission electron microscope observation. Thereafter, a formalin-killed cell (FKC) vaccine generated from an ISKNV-I isolate was prepared to evaluate the protective effects against two spotted seabass original RSIV-I and RSIV-II. The result showed that the ISKNV-I-based FKC vaccine conferred almost complete cross-protection against RSIV-I and RSIV-II as well as ISKNV-I itself. No serotype difference was observed among RSIV-I, RSIV-II, and ISKNV-I. Additionally, the mandarin fish Siniperca chuatsi is proposed as an ideal infection and vaccination fish species for the study of various megalocytiviral isolates. IMPORTANCE Red seabream iridovirus (RSIV) infects a wide mariculture bony fish and has resulted in significant annual economic loss worldwide. Previous studies showed that the phenotypic diversity of infectious RSIV isolates would lead to different virulence characteristics, viral antigenicity, and vaccine efficacy as well as host range. Importantly, it is still doubted whether a universal vaccine could confer the same highly protective effect against various genotypic isolates. Our study here presented enough experimental evidence that a water in oil (w/o) formation of inactivated ISKNV-I vaccine could confer almost complete protection against RSIV-I and RSIV-II as well as ISKNV-I itself. Our study provides valuable data for better understanding the differential infection and immunity among different genotypes of ISKNV and RSIV isolates in the genus Megalocytivirus.

Genotypic Characterization of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Southeast Asian Aquaculture

Infectious spleen and kidney necrosis virus (ISKNV) is a species within the genus Megalocytivirus (family Iridoviridae), which causes high mortality disease in many freshwater and marine fish species. ISKNV was first reported in Asia and is an emerging threat to aquaculture with increasing global distribution, in part due to its presence in ornamental fish with clinical and subclinical infections. The species ISKNV includes three genotypes: red seabream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and ISKNV. There is an increasing overlap in the recognized range of susceptible fish hosts and the geographic distribution of these distinct genotypes. To better understand the disease caused by ISKNV, a nucleic acid hybridization capture enrichment was used prior to sequencing to characterize whole genomes from archived clinical specimens of aquaculture and ornamental fish from Southeast Asia (n = 16). The method was suitable for tissue samples containing 2.50 × 104–4.58 × 109 ISKNV genome copies mg−1. Genome sequences determined using the hybridization capture method were identical to those obtained directly from tissues when there was sufficient viral DNA to sequence without enrichment (n = 2). ISKNV genomes from diverse locations, environments, and hosts had very high similarity and matched established genotype classifications (14 ISKNV genotype Clade 1 genomes with >98.81% nucleotide similarity). Conversely, two different genotypes were obtained at the same time and location (RSIV and ISKNV from grouper, Indonesia with 92.44% nucleotide similarity). Gene-by-gene analysis with representative ISKNV genomes identified 59 core genes within the species (>95% amino acid identity). The 14 Clade 1 ISKNV genomes in this study had 100% aa identity for 92–105 of 122 predicted genes. Despite high overall sequence similarity, phylogenetic analyses using single nucleotide polymorphisms differentiated isolates from different host species, country of origin, and time of collection. Whole genome studies of ISKNV and other megalocytiviruses enable genomic epidemiology and will provide information to enhance disease control in aquaculture.

Development and Validation of a Quantitative Polymerase Chain Reaction Assay for the Detection of Red Sea Bream Iridovirus

The analytical and diagnostic performances of methods for detecting red sea bream iridovirus (RSIV), which infects marine fish, have not been evaluated. As disease management and transmission control depend on early and reliable pathogen detection, rapid virus detection techniques are crucial. Herein, we evaluated the diagnostic performance of a TaqMan-based real-time polymerase chain reaction (PCR) assay that detects RSIV rapidly and accurately. The assay amplified the RSIV, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus genotypes of Megalocytivirus and the detection limit was 10.96 copies/reaction. The assay’s performance remained uncompromised even in the presence of nine potential PCR inhibitors, including compounds commonly used in aquaculture. The variation of the cycle threshold values between assays performed by three technicians was evaluated using a plasmid DNA containing the major capsid protein gene sequence. The variation between replicates was low. The diagnostic sensitivity and specificity of the developed assay were evaluated using fish samples (n = 510) and were found to be 100% and 99.60%, respectively. Two technicians evaluated the reproducibility of the assay using fish samples (n = 90), finding a high correlation of 0.998 (p < 0.0001). Therefore, the newly developed real-time PCR assay detects RSIV both accurately and rapidly.

Evaluation of a novel TaqMan probe-based real-time polymerase chain reaction (PCR) assay for detection and quantitation of red sea bream iridovirus

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD95%) was determined to be 5.3 viral genome copies/μL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

Multiplex PCR method for distinguishing two genotypes of Megalocytivirus isolates in Korea

Megalocytivirus is a genus of piscine viruses that belongs to the Iridoviridae family, and this family includes red seabream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and infectious spleen and kidney necrosis virus. RSIV causes high mortality and economic losses in the rock bream (Oplegnathus fasciatus) Korean aquaculture industry. The World Organization for Animal Health’s Office International des Epizooties has provided a manual for RSIV detection. However, distinguishing it from TRBIV has not been confirmed. In this study, a multiplex PCR method was established to detect two genotypes of Megalocytivirus, RSIV and TRBIV. New primer pairs were optimized for the PCR reaction. A mixture of one universal and two specific primer pairs could amplify three distinct products targeting ATPase, DPO, and LRP genes. The sensitivities of primer pairs were evaluated, showing a detection limit of 2.0 × 105 copies, using a plasmid construct, against the three target genes of Megalocytivirus. Moreover, specificity evaluation indicated that primer pairs were not subjected to interference by other virus strains. This evaluation of multiplex PCR showed that it can distinguish two genotypes of Megalocytivirus from 21 Korean Megalocytivirus isolates, including 20 RSIV and one TRBIV. Finally, we describe a new multiplex PCR method to detect different genotypes of Megalocytivirus simultaneously, which makes the diagnosis of viral diseases occurring in the Korean aquaculture industry more convenient.

Infectious spleen and kidney necrosis virus-associated large-scale mortality in farmed giant gourami, Osphronemus goramy, in India.

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.

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.

Identification of the Epitope Recognized by the Anti-Red Sea Bream Iridovirus (RSIV) Monoclonal Antibody M10 Using a Phage Display RSIV Peptide Library

Red sea bream iridoviral disease (RSIVD) spreads readily throughout the marine environment because of its wide variety of host species. Use of a rapid diagnostic method, such as the immunofluorescence antibody test, helps to control the disease, so the anti-RSIV monoclonal antibody M10 (mAb M10) has been developed in Japan. In the present study, we carried out epitope mapping using the phage display method to identify the antigen of mAb M10. A phage display RSIV peptide library was constructed to cover the entire genome of RSIV, then phage clones recognized by the antibody were selected by biopanning. The selected clones harbored partial fragments of the laminin-type epidermal growth factor-like domain (LEGFD) gene. N-terminal and C-terminal deletion peptides were then prepared from the amino acid sequence deduced from the smallest fragment to precisely determine the epitope. Finally, seven amino acids, EYDCPEY, located in the extracellular domain of the LEGFD protein were determined to be the epitope. Identical residues of the epitope were also identified from the LEGFD protein in other megalocytiviruses including the infectious spleen and kidney necrosis virus and turbot reddish body iridovirus. mAb M10 is considered to be widely available for the diagnostics of megalocytivirus infections.

Megalocytiviruses in ornamental fish: A review.

Iridoviruses, especially megalocytiviruses, are related to severe disease resulting in high economic losses in the aquaculture industry worldwide. The ornamental fish industry has been affected severely due to Megalocytivirus infections. Megalocytivirus is a DNA virus that has three genera; including red sea bream iridovirus, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus. Megalocytivirus causes non-specific clinical signs in ornamental fish. Cell culture, histology, immunofluorescence test, polymerase chain reaction (PCR) assay, and loop-mediated isothermal amplification assay have been used to diagnose megalocytiviruses. Risk factors such as temperature, transportation (export and import), and life stages of ornamental fish have been reported for the previous cases due to Megalocytivirus infections. In addition, other prevention and control methods also have been practiced in farms to prevent Megalocytivirus outbreaks. This is the first review of megalocytiviruses in ornamental fish since its first detection in 1989. This review discusses the occurrences of Megalocytivirus in ornamental fish, including the history, clinical signs, detection method, risk factors, and prevention measures.

Novel peptide nucleic acid-based real-time PCR assay for detection and genotyping of Megalocytivirus

Megalocytivirus, the causative agent for Red Sea Bream Iridoviral Disease (RSIVD), is classified into three genotypes based on its phylogeny: Red Sea Bream Iridovirus (RSIV), Infectious Spleen and Kidney Necrosis Virus (ISKNV), and Turbot Reddish Body Iridovirus (TRBIV). To overcome drawbacks such as limitation of genotypic variant type discrimination and unexpected PCR reaction from an SYBR green-based genotyping method by high-resolution melting analysis, peptide nucleic acid (PNA)-based real-time PCR assay for detecting and genotyping Megalocytivirus was developed. Compared with DNA hybridization, PNA has more suitable hybridization traits that allowed the difference in melting temperature (Tm) even by a single nucleotide mismatch. Based on the consensus sequences of the viral major capsid protein gene, four PNA probes labeled with respective fluorescence at their 3′ ends were designed as reporter molecules. PNA-based real-time PCR assay allowed quantification of Megalocytivirus copies using a synthetic DNA as standard (detection limit of 103 copies) and discrimination of the genotypes based on Tm in a single-tube reaction. Furthermore, the results of this new method were consistent with those of sequencing analysis of Megalocytivirus-infected field and artificial samples, proving its accuracy and efficiency. The novel PNA-based real-time PCR assay could contribute to the detection as well as discrimination of the genotypes of Megalocytivirus in a single simple assay, and could be used as a highly robust diagnostic tool in the field of aquatic animal diseases.

Genetic diversity of Megalocytivirus from cultured fish in Korea

Since the first outbreak of Megalocytivirus infection in cultured marine fish in the 1990s, the Red sea bream iridovirus (RSIV) subtype II and Turbot reddish body iridovirus (TRBIV) type Megalocytivirus have been prevalent in Korea. Based on the major capsid protein (MCP) genes, the RSIV subtype II, as the predominant genotype, was identified in marine and freshwater fish including thread-sail filefish (Stephanolepis cirrhifer), so-iuy mullet (Planiliza haematocheila), starry flounder (Platichthys stellatus), and golden mandarin fish (Siniperca scherzeri). Recently, a new genetic variant of RSIV type (subtype I and II), SB5-TY originating from sea bass (Lateolabrax japonicas), was identified by their sequence identity with the four ankyrin repeat domains of RSIV (401R, 424R, 534L, and 641L). This result corroborate the evidence that increased genotypic variation in RSIV-type megalocytiviruses has been detected.

Genotype determination of megalocytivirus from Indonesian Marine Fishes

Murwantoko, Sari DWK, Handayani CR, Whittington RJ. 2018. Genotype determination of megalocytivirus from Indonesian Marine Fishes. Biodiversitas 19: 1730-1736. Megalocytivirus is the newest genus within the family of Iridoviridae which can be divided into groups represented by red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV) and turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV). This virus caused serious systemic disease in cultured marine fishes for consumption and ornamental freshwater fishes with significant mortality. The objective of this study was to determine the genotype of megalocytivirus which infected marine fishes from Lampung, Karimun Jawa, Situbondo and Batam based on major capsid protein (MCP), ATPase, DNA polymerase, CY15 and IRB6. The liver, spleen and kidney tissues of humpback grouper (Cromileptes altivelis), tiger grouper (Epinephelus fuscoguttatus), baramundi (Lates calcarifer) were fixed in 10% phosphate-buffered formalin for histological and fixed in 70% ethanol for molecular analysis. Molecular analysis was performed by amplification of MCP, ATPase, DNA polymerase, CY15 and IRB6 genes and followed by sequencing. Genotype was determined by alignment of the sequences with various genotypes of megalocytivirus from Genbank. Histological examination showed that hypertrophy, inclusion body forming bearing cells were found in liver, spleen and kidney tissues. Polymerase chain reaction with MCP primer produced specific DNA bands. Those results confirmed the infection of megalocytivirus on marine cultured fish samples. The analysis from 10 isolates on five genes revealed that two genotypes of megalocytivirus as infectious spleen and kidney necrosis virus (ISKNV) and red sea bream iridovirus (RSIV) genotypes were existed in Indonesia. The ISKNV genotype was confirmed in fish samples from Lampung, Jepara, Bali; while RSIV genotype was found in fishes from Batam, and Situbondo. Interestingly, both ISKNV and RSIV genotypes were confirmed in fish samples from Karimun Jawa. This paper is the first report on the present of ISKNV and RSIV genotypes in Indonesia based on MCP, ATPase, DNA polymerase, CY15 and IRB6 genes.

Phylogenomic characterization of two novel members of the genus Megalocytivirus from archived ornamental fish samples.

The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

A natural infection by the red sea bream iridovirus-type Megalocytivirus in the golden mandarin fish Siniperca scherzeri.

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)-subgroup I. Additionally, this train clustered with the ehime-1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV-type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18×102 to 7.95×106 copies/mg of tissue individually, suggesting that the infected fish were in the disease-transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV-type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.

Cloning and expression study of an IRF4a gene and its two transcript variants in turbot, Scophthalmus maximus

Interferon regulatory factor 4 (IRF4) is known to be involved in antiviral response as well as regulation of functional and developmental processes in lymphomyeloid cell lineages in mammals. In this study, the gene of IRF4a and its two transcript variants (named IRF4a1 and -2) were cloned from turbot, Scophthalmus maximus, the tissue distributions and in vivo immune responsive expression patterns of the two transcripts were subsequently examined. The Scophthalmus maximus (Sm)IRF4a gene is 8367 nucleotide (nt) in length, consisting of eight exons and seven introns. The SmIRF4a1 transcript is 3185 nt long, containing an open reading frame (ORF) of 1401 nt that encodes a polypeptide of 466 amino acids (aa). The SmIRF4a2 transcript is 2265 nt long and identical with the SmIRF4a1 from position 1 to 1171, containing an ORF of 1164 nt that encodes a truncated protein of 387 aa as a result of a frame shift in exon 6 which introduces a premature stop codon. The deduced aa sequence of SmIRF4a1 posses a DNA-binding domain (DBD), a nuclear localization signal (NLS), a serine-rich domain (SRD) and an IRF association domain (IAD), while SmIRF4a2 lacks the C-terminal 52 residues of the IAD and the downstream C-terminal extension, instead, they are replaced by a 8-aa segment although the three upstream domains are intact. Quantitative real-time PCR analysis revealed a broad tissue expression for both SmIRF4a1 and -2 with the former showing a significantly higher expression in all examined tissues except skin. Expressions of two transcript variants after stimulation with polyinosinic:polycytidylic acid [poly(I:C)] and turbot reddish body iridovirus (TRBIV) were tested in gills, spleen, head kidney and muscle. A two-wave of induced expression pattern was observed for both transcripts with either stimulus treatment during a 7-day time course. SmIRF4a2 responded more promptly to the stimuli and showed a higher level of inducibility in the early phase while SmIRF4a1 was strongly detected in the later phase. These data suggest an important role of SmIRF4a2 in the fast immune response under a background of SmIRF4a1-dominant antiviral response in the IRF4a system of turbot.

Cloning and expression analysis of a Toll-like receptor 21 (TLR21) gene from turbot, Scophthalmus maximus

Toll-like receptor 21 (TLR21) is a non-mammalian TLR recognizing unmethylated CpG DNA and considered as a functional homolog of mammalian TLR9. In the present study, a TLR21 gene was cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (Sm)TLR21 gene is an intronless gene with a length of 3527 bp and encodes a peptide of 984 amino acids. The deduced protein possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 16 LRR motifs, a transmembrane (TM) region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost TLR21s. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SmTLR21 mRNA in all twelve examined tissues with higher levels in the lymphomyeloid-rich tissues like spleen and head kidney. Further, upon stimulation with polyinosinic: polycytidylic acid [poly(I:C)], turbot reddish body iridovirus (TRBIV) and CpG oligodeoxynucleotides (CpG-ODN) 2395, the SmTLR21 mRNA expression was up-regulated in the gills, head kidney, spleen and muscle. The maximum increases of SmTLR21 transcript levels ranged from 1.3 to 8.1-fold and appeared at 3 h to 5 day post-injection depending on different organs and stimuli. These findings suggest that SmTLR21 may play an important role in the immune responses to the infections of a broad range of pathogens that include RNA and DNA viruses and bacteria.

Detection of infectious spleen and kidney necrosis virus (ISKNV) and turbot reddish body iridovirus (TRBIV) from archival ornamental fish samples

Although infections caused by megalocytiviruses have been reported from a wide range of finfish species for several decades, molecular characterisation of the viruses involved has been undertaken only on more recent cases. Sequence analysis of the major capsid protein and adenosine triphosphatase genes is reported here from formalin-fixed, paraffin-embedded material from 2 archival ornamental fish cases from 1986 and 1988 in conjunction with data for a range of genes from fresh frozen tissues from 5 cases obtained from 1991 through to 2010. Turbot reddish body iridovirus (TRBIV) genotype megalocytiviruses, previously not documented in ornamental fish, were detected in samples from 1986, 1988 and 1991. In contrast, megalocytiviruses from 1996 onwards, including those characterised from 2002, 2006 and 2010 in this study, were almost indistinguishable from infectious spleen and kidney necrosis virus (ISKNV). Three of the species infected with TRBIV-like megalocytiviruses from 1986 to 1991, viz. dwarf gourami Trichogaster lalius (formerly Colisa lalia), freshwater angelfish Pterophyllum scalare and oscar Astronotus ocellatus, were infected with ISKNV genotype megalocytiviruses from 2002 to 2010. The detection of a TRBIV genotype isolate in ornamental fish from 1986 represents the index case, confirmed by molecular sequence data, for the genus Megalocytivirus.

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron-like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1-TRBIV-MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT-PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT-PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post-vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon-induced antiviral molecules were observed in the vaccinated fish 60 h post-vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.

Development of oral DNA vaccine based on chitosan nanoparticles for the immunization against reddish body iridovirus in turbots (Scophthalmus maximus)

Turbot reddish body iridovirus (TRBIV), a piscine iridovirus, causes viral reddish body syndrome (VRBS) in turbot and is highly lethal to the fish. Infection of the virus hampers the development of turbot aquaculture. Hence, it is important to develop a preventive vaccine. In our previous study, we constructed and evaluated the immunization efficacy of the recombinant plasmid, pEGFP-N2-TRBIV-MCP. In the present study, an oral nanoparticle-based DNA vaccine against TRBIV was developed to evaluate the immunization effectiveness in turbots (Scophthalmus maximus) through oral administration. We prepared chitosan nanoparticles enwrapping plasmid DNA using ionic gelation process, and the characteristics of chitosan nanoparticles were analyzed by transmission electron microscope and Malvin ZS analyzer. The results revealed that the chitosan nanoparticle plasmids encoding DNA (pDNA-CS-NPs) were spherical shape with the particle size and zeta potential of 145.5±1.5nm and 24.3±0.5, respectively. The loading rate and encapsulation efficiency were 92.8±1.38% and 63.7±0.89%, respectively. In vitro transfection, pDNA-CS-NPs was transferred into eukaryotic cells, turbot cell TK line, and transient expression of pDNA-CS-NPs was detected by RT-PCR and laser scanning confocal microscope. After oral administration, the expression of the antigen gene could be detected in different tissues of turbots (S. maximus) at 20 and 40days post-vaccination. The pDNA-CS-NPs clearly induced an innate and specific immune response, and significantly up-regulated the expression of MHCIα, IIα, IFN-1, IFN-g, Mx-1, IL8 and Mx. In addition, strong protection with relative survival rate of 68% was recorded in the orally-vaccinated turbots, which could produce the detectable level of anti-TRBIV antibody during 90days. These data in this study suggested that pDNA-CS-NPs were promising carriers for plasmid DNA vaccine through oral vaccination. This method could be extended to the development of other oral vaccines and applied in other fish species against TRBIV. Statement of relevanceIn this work, an oral nanoparticle-based DNA vaccine against TRBIV was developed to evaluate the immunization effectiveness in turbots (S. maximus) through oral administration. We prepared chitosan nanoparticles enwrapping plasmid DNA using ionic gelation process, and the characteristics of chitosan nanoparticles were analyzed by transmission electron microscope and Malvin ZS analyzer. I hope this paper is suitable for aquaculture.

Initiation of two ovarian cell lines from Fugu rubripes (Temminck et. Schlegel)

The ovary is an excellent system for studying stem cell renewal and differentiation, which is under the control of ovarian somatic cells. In order to understand oogenesis in Fugu rubripes (Temminck et. Schlegel) as a marine fish model of aquaculture importance, we established cell lines called TSOC1 and TSOC2 from a juvenile ovary of this organism. TSOC1 is composed of spindle epithelial-like cells, while the other is cobblestone-like cells. Therefore, TSOC1 and TSOC2 appear to consist of ovarian somatic cells. Growth requirement condition was investigated including temperature, concentration of FBS and pH. Significant fluorescent signals were observed after TSOC1 and TSOC2 cells were transfected with pEGFP-N3 vector, indicating its potential utility for genetic manipulation such as gene function studies. It is shown that these cell lines are effective for infection by the turbot reddish body iridovirus and flounder lymphosystis disease virus as evidenced by the appearance of cytopathic effect and virus propagation in the virus-infected cells, and most convincingly, the observation of viral particles by electron microscopy, demonstrating that TSOC1 and TSOC2 are suitable to study interactions between virus and host cells. It is believed that TSOC1 and TSOC2 will be useful tools to study sex-related events and interactions between primordial germ cells and oogonia cells during oogenesis. Therefore, establishment of ovary cell lines from Fugu rubripes seems to be significant for those research areas.