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Abstract The genus Megalocytivirus, belonging to the family Iridoviridae, is one of the most detrimental virus groups to fish aquaculture. Megalocytivirus creates a virus-mock basement membrane (VMBM) on the surface of infected cells. This membrane provides attachment sites for lymphatic endothelial cells (LECs), disrupting fish's endothelial cell-extracellular matrix system. This disruption triggers injury to the vascular system and can result in death. Exploring the VMBM-cell interaction mechanism is crucial for uncovering the pathogenesis of Megalocytivirus and identifying therapeutic targets. Claudins, a class of tetra transmembrane proteins, play a key role in creating tight junctions between endothelial or epithelial cells. In this study, we demonstrated that the expression of Claudin2, a member of the Claudin family in fish, was significantly up-regulated by Megalocytivirus infection. Claudin2 was found in LECs attached to the surface of infected cells. It interacted with the VMBM viral components VP23R, VP08R, and VP33L at multiple binding sites through its two extracellular loops. However, it did not interact with the host basement membrane’s nidogen. Therefore, Claudin2 is involved in the interaction of LEC with VMBM and plays a role in the disturbed distribution of extracellular matrix and endothelial cells in Megalocytivirus-infected fish tissues. This study aims to uncover the molecular mechanisms by which Megalocytivirus infection leads to pathological changes in the vascular system.

The genus Megalocytivirus, belonging to the family Iridoviridae, is one of the most detrimental virus groups to fish aquaculture. Megalocytivirus creates a virus-mock basement membrane (VMBM) on the surface of infected cells. This membrane provides attachment sites for lymphatic endothelial cells (LECs), disrupting fish's endothelial cell-extracellular matrix system. This disruption triggers injury to the vascular system and can result in death. Exploring the VMBM-cell interaction mechanism is crucial for uncovering the pathogenesis of Megalocytivirus and identifying therapeutic targets. Claudins, a class of tetra transmembrane proteins, play a key role in creating tight junctions between endothelial or epithelial cells. In this study, we demonstrated that the expression of Claudin2, a member of the Claudin family in fish, was significantly up-regulated by Megalocytivirus infection. Claudin2 was found in LECs attached to the surface of infected cells. It interacted with the VMBM viral components VP23R, VP08R, and VP33L at multiple binding sites through its two extracellular loops. However, it did not interact with the host basement membrane's nidogen. Therefore, Claudin2 is involved in the interaction of LEC with VMBM and plays a role in the disturbed distribution of extracellular matrix and endothelial cells in Megalocytivirus-infected fish tissues. This study aims to uncover the molecular mechanisms by which Megalocytivirus infection leads to pathological changes in the vascular system. [ABSTRACT FROM AUTHOR]

The threespine stickleback iridovirus (TSIV), a double-stranded DNA virus, was the first megalocytivirus detected in wild North American fishes. We report a second occurrence of TSIV in threespine stickleback (Gasterosteus aculeatus) from Stormy Lake, Alaska, and assemble a nearly complete genome of TSIV. The 115-kilobase TSIV genome contains 94 open reading frames (ORFs), with 91 that share homology with other known iridoviruses. We identify three ORFs that likely originate from recent lateral gene transfers from a eukaryotic host and one ORF with homology to B22 poxvirus proteins that likely originated from a lateral gene transfer between viruses. Phylogenetic analysis of 24 iridovirus core genes and pairwise sequence identity analysis support TSIV as a divergent sister taxon to other megalocytiviruses and a candidate for a novel species designation. Screening of stickleback collected from Stormy Lake before and after a 2012 rotenone treatment to eliminate invasive fish shows 100% positivity for TSIV in the two years before treatment (95% confidence interval: 89–100% prevalence) and 0% positivity for TSIV in 2024 after treatment (95% confidence interval: 0 to 3.7% prevalence), suggesting that the rotenone treatment and subsequent crash and reestablishment of the stickleback population is associated with loss of TSIV. [ABSTRACT FROM AUTHOR]

The orange-spotted grouper (Epinephelus coioides) is an important mariculture fish in China. However, in recent years, with the rapid development of aquaculture activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing severe economic losses. In the present study, we isolated and identified a virus from diseased, orange-spotted groupers from an aquaculture farm in Hainan Province, China. The isolated virus was identified as orange-spotted grouper iridovirus, hence named the orange-spotted grouper iridovirus Hainan strain (OSGIV-HN-2018-001). OSGIV-HN-2018-001 induces a cytopathic effect after the infection of mandarin fish (Siniperca chuatsi) brain clonal passage (SBC) cells. In addition, the cytoplasm of the OSGIV-HN-2018-001-infected SBC cells was found to contain a large number of hexagonal virus particles with a diameter of approximately 134 nm. Using the Illumina NovaSeq system, we assembled the sequence data and annotated the complete genome of OSGIV-HN-2018-001 (GenBank accession number: PP974677), which consisted of 110,699 bp and contained 122 open reading frames (ORFs). Phylogenetic tree analysis showed that OSGIV-HN-2018-001 was most closely related to ISKNV-ASB-23. The cumulative mortality rate of groupers infected with OSGIV-HN-2018-001 reached 100% on day 8. The spleens were enlarged and blackened after the dissection of the dying groupers. These results contribute to the understanding of the molecular regulatory mechanism of the iridovirus infection and provide a basis for iridovirus prevention. [ABSTRACT FROM AUTHOR]

IntroductionThe prevalence of viral diseases has posed significant challenges to the sustainable development of large yellow croaker (Larimichthys crocea) aquaculture, with megalocytivirus being one of the primary viral pathogens affecting this species. There have been two proteomic and genomic studies regarding two members of the genus Megalocytivirus: the spotted knifejaw iridovirus (SKIV) and the infectious spleen and kidney necrosis virus (ISKNV). However, both studies were conducted more than 10 years ago. To further investigate the pathogenesis of megalocytivirus, we sequenced the genome of the viral strain FD201807 isolated from L. crocea, and conducted a proteomics analysis.MethodsViral DNA was sequenced using the Illumina HiSeq 2000 platform. Viral proteins from purified virions and supernatants of viral infected cells were subjected to LC-MS/MS analysis, and the expression of four viral proteins was further confirmed by Western blotting. The entire viral genome was subjected to phylogenetic and bioinformatic analyses.ResultsThe FD201807 genome comprises 112,214 bp of double-stranded DNA with a G + C content of 53.53%. It contains 130 potential open reading frames, with coding capacities ranging from 41 to 1,293 amino acids. Phylogenetic analysis of the whole-genome sequence indicated that the closest known megalocytivirus related to FD201807 is Pompano iridovirus, with a sequence identity of 98.98%. Label-free proteomics analysis identified 27 viral proteins in the viral-infected cell culture supernatants and 46 viral proteins in the purified virus of FD201807. Among these, 19 viral proteins were detected in both the viral-infected cell culture supernatants and the purified virus samples, while 8 viral proteins were exclusively identified in the viral-infected cell culture supernatants. Notably, there were two proteins derived from the cultured cell line MFF-1 (mandarin fish fry cell line-1), namely cytochrome c and ubiquitin-activating enzyme E1, present in both the purified virus samples and the culture supernatant of infected cells. These cellular proteins may be associated with virus-host protein interactions and/or host cell apoptosis.DiscussionWe present the most comprehensive proteomic analysis to date of the megalocytivirus isolated from L. crocea, and help identify highly expressed proteins that may serve as future targets for immunotherapy and biochemical analysis.

Background and Aim: Malaysia has more than 630 culturists who are involved in the ornamental fish industry and culture 250 species, including local and exotic species. Among these viruses, megalocytiviruses have been associated with severe systemic diseases and economic losses in ornamental fish. The intensity of Megalocytivirus infection in Pterophyllum scalare in Malaysia remains unknown. Thus, this study aimed to investigate the occurrence of Megalocytivirus while discovering its associated risk factors and the genotypes of its causative agents in an ornamental fish farm in Malaysia. Materials and Methods: Seven broodstock pairs of P. scalare were used in this study to follow the life stages of fish, from egg to market size. Water samples and other samples, such as mucus swabs, gill swabs, P. scalare eggs, fries, juveniles, snails, snail eggs, live feed (Tubifex worms and Moina spp.), sediment samples, and wild fish, were collected periodically for initial environmental sampling from day 0 to day 60. Nested polymerase chain reaction amplifications were performed for megalocytivirus-related sequences. The phylogenetic tree, including the sampled causative agents of megalocytiviruses, was inferred from the major capsid protein genes of all known Iridoviridae species. Pearson’s correlation coefficients were calculated to determine the strength of the correlation between the presence of megalocytiviruses in P. scalare samples and the associated risk factors. Results: A total of 312 out of 935 pooled and individual samples tested positive for the presence of Megalocytivirus-related sequences, except snail eggs and wild fish (Poecilia reticulata). No clinical symptoms were observed in any fish samples. Megalocytivirus-associated viruses detected in water samples indicate horizontal transmission of the virus. All the nucleotide sequences found in this study had high nucleotide identities of 95%–99 % and were closely related to Megalocytivirus genotype I infectious spleen and kidney necrosis virus. Risk factors associated with Megalocytivirus include water temperature, dissolved oxygen (DO), pH, ammonia, nitrate, nitrite, and the life stages of P. scalare. High Megalocytivirus infection was detected when the water temperature, DO, and pH were high in P. scalare, high water temperature and nitrate in the water samples, and the same rate of Megalocytivirus infection in P. scalare fry and juveniles. Conclusion: This is the first study to confirm the existence of different possible routes of megalocytivirus distribution in ornamental fish farms in Malaysia. Nevertheless, the connection between the mode of transmission and the risk factors for this virus needs to be explored further to recognize the evolution and potential new host species.

The Megalocytivirus genus, family Iridoviridae, is a group of large DNA viruses that pose significant threats to marine and freshwater fishes. In tissues, the Megalocytivirus-infected cells are attached by a layer of lymphatic endothelial cells (LECs), constituting a unique histopathological phenomenon. Megalocytivirus induces the formation of an extracellular structure on the surface of infected cells that is functionally similar to the host basement membrane (BM), known as the virus-mock basement membrane (VMBM). VMBM is composed of host BM component nidogen-1 and viral proteins VP23R and VP08R and specifically provides adhesive support for LECs. In the current study, the viral protein VP33L was identified as a novel component of VMBM, and the way it participates in VMBM assembly was investigated. The interactions between virus-encoded VMBM components and LEC-specific markers lymphatic vessel endothelial receptor-1 (LYVE-1) and vascular endothelial growth factor receptor 3 (VEGFR-3), which contribute to the selective adhesion of LECs on VMBM, were further analyzed. Moreover, the viral components of VMBM could regulate the proliferation and migration of LECs via the VEGFR-3 signaling, which partially explains the origin of LECs attached to infected cells. This study is important for revealing the pathogenesis of Megalocytivirus and potentially provides reference clues for exploring the interaction mechanism between endothelial cells and extracellular matrix in animals. [ABSTRACT FROM AUTHOR]

Tilapia Lake virus and megalocytivirus are two major viruses that are causing global fish declines. The detection of these viruses in nearby water samples suggests a non-invasive and consistent method for identifying fishviral infections and forecasting early viral illnesses in aquaculture systems. The goal of this study was to examine the prevalence of these viruses in water samples taken from Qarun Lake in Egypt's Fayoum Governorate. 72 water samples were collected regularly from two sites from May 2021 to November 2022. The organic flocculation concentration method was combined with molecular analysis to identify fish viral infections. Only 4.1% of the 72 were infected with tilapia lake virus, whereas 9.7% were infected with megalocytivirus. Finally, our results demonstrated that organic flocculation combined with PCR environmental nucleic acid detection is effective for identifying tilapia Lake virus and megalocytivirus in water. [ABSTRACT FROM AUTHOR]

ABSTRACT Aquatic animal viruses are considered to be transmitted via environmental water between fish farms. This study aimed to understand the actual transmission risk of red sea bream iridovirus (RSIV) through environmental water among fish farms. An environmental DNA (eDNA) method using iron-based flocculation coupled with large-pore filtration was used to monitor RSIV DNA copies in seawater from fish farms and from an experimental infection model. RSIV dispersion in seawater from a net pen where the disease outbreak occurred was visualized by the inverse distance weighting method using multiple-sampling data sets from a fish farm. The analysis demonstrated that the center of the net pen had a high viral load, and RSIV seemed to be quickly diluted by the tidal current. To evaluate the transmission risk of RSIV in environmental water, the red sea bream Pagrus major (approximately 10 g) was exposed to RSIV-contained seawater (103, 104, 105, 106, and 107 copies/L) for 3 days, which mimicked field exposure. A probit analysis of the challenge test indicated that the inferred infection rates of seawater containing 105.9 copies/L and 103.1 copies/L of RSIV were 50% and 0.0001%, respectively. In the surveillance for 3 years at 10 fixed points (n = 306), there were only seven samples in which the viral load exceeded 104 copies/L in seawater. These results suggest that the transmission of RSIV among fish farms via seawater is highly associated with the distance between the net pens, and the environmental water is not always an infection source for the transmission of RSIV between fish farms. IMPORTANCE Our surveillance of viral loads for red sea bream iridovirus (RSIV) by monitoring environmental DNA in fish farms suggested that the viral loads in the seawater were low, except for the net pens where RSIV outbreaks occurred. Furthermore, our experimental infection model indicated that the infection risk of RSIV-contained seawater with less than 103 copies/L was extremely low. The limited risk of environmental water for transmission of RSIV gives an insight that RSIV could be partly transmitted between fish farms due to the movement of equipment and/or humans from the fish farm where the disease outbreaks. Since our data suggest that seawater can function as a potential wall to reduce the transmission of RSIV, biosecurity management, such as disinfection of equipment associated with fish farms could be effective, even in the semi-open system aquaculture that the environmental water can be freely transferred, to reduce the risk of RSIV outbreaks.

Simple Summary: Red sea bream iridovirus (RSIV) has been reported to be susceptible to various marine fish species in Asian countries such as South Korea, and it causes significant economic losses due to massive mortalities in rock bream (Oplegnathus fasciatus) in particular. However, studies on the impact of the virus shed from RSIV-infected rock bream on other fish species are limited. Here, we investigated the dynamics of the virus by simulating the natural conditions of RSIV infection in rock bream and examining its release into seawater and the risk of horizontal transmission through cohabitation with other fish species. Our data demonstrated that the virus shed from RSIV-infected rock bream caused high mortality rates in surrounding fish species through horizontal transmission. Our findings suggest the potential for RSIV-infected rock bream in farms to spread the disease to neighboring farms. Additionally, our results demonstrated a high correlation between RSIV load and viral shedding ratio in rock breams, as well as between RSIV load and histopathological infection grades. Red sea bream iridovirus (RSIV) causes significant economic losses in aquaculture. Here, we analyzed the pathogenicity, viral shedding, and transmission dynamics of RSIV in rock bream (Oplegnathus fasciatus) by employing immersion infection and cohabitation challenge models. Rock bream challenged by immersion exposure exhibited 100% mortality within 35 days post RSIV exposure, indicating that the viral shedding in seawater peaked after mortality. At 25 °C, a positive correlation between the viral loads within infected rock bream and virus shedding into the seawater was observed. Specific RSIV lesions were observed in the spleen and kidney of the infected rock bream, and the viral load in the spleen had the highest correlation with the histopathological grade. A cohabitation challenge mimicking the natural transmission conditions was performed to assess the virus transmission and determine the pathogenicity and viral load. The RSIV-infected rock breams (donors) were cohabited with uninfected rock bream, red sea bream (Pagrus major), and flathead grey mullet (Mugil cephalus) (recipients) at both 25 °C and 15 °C. In the cohabitation challenge group maintained at 15 °C, no mortality was observed across all experimental groups. However, RSIV was detected in both seawater and the recipient fish. Our results provide preliminary data for further epidemiological analyses and aid in the development of preventive measures and management of RSIVD in aquaculture. [ABSTRACT FROM AUTHOR]

Background: Megalocytiviruses (MCV) are double-stranded DNA viruses that infect fish. Two species within the genus are epidemiologically important for fish farming: red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV). The objective of this work was to study regions that allow the differentiation and correct diagnosis of RSIV and ISKNV. Methods: The regions ORF450L, ORF342L, ORF077, and the intergenic region between ORF37 and ORF42R were sequenced and compared with samples from the database. Results: The tree constructed using the sequencing of the PCR product Megalocytivirus. ORF077 separated the three major clades of MCV. RISV genotypes were well divided, but not ISKNV. All qPCRs tests showed acceptable repeatability values, that is, less than 5%. Conclusion: Two qPCRs for ISKNV detection and two for RSIV were considered suitable for use in the diagnosis and typing of MCV. The results of this study demonstrate the importance of an accurate evaluation of methodologies for the differentiation of MCV. [ABSTRACT FROM AUTHOR]

The study presents the first report of the ISKNV whole genome sequence isolated from angel fish (Pterophyllum scalare) in India, offering insights into gene composition, function, phylogeny, and potential vaccine development. The complete genome of AK-ISKNV was found to be of 110,460 bp with the GC content of 54.79 %. The complete genome has 122 open reading frames (ORFs) and 48 ORFs were predicted as functional proteins while the rest were hypothetical proteins. Phylogenetic tree constructed based on the major capsid protein and ATPase genes revealed AK-ISKNV in this study belong to the genus Megalocytivirus of the ISKNV-Genotype I. The whole genome exhibits a 99.81 % similarity, and the phylogenetic analysis indicates a close association with Angel Fish Iridovirus (AFIV), specifically identified as MK689685.1, originating from Australia. Further, phylogenetic analysis confirms the ISKNV genotype and its distinction from RSIV. The present study provides the first report of the complete genome sequence and annotation of the ISKNV strain (AK-ISKNV) isolated from India. Four proteins were selected as prospective vaccination candidates based on their high B-cell epitope score and antigenic locations. These proteins include myristylated membrane protein (ORF 105), main capsid protein (ORF 104), and two hypothetical proteins (ORF 11 and ORF 14). In conclusion, this research contributes vital genomic information essential for understanding and mitigating the impact of ISKNV, providing a foundation for targeted disease control measures in aquaculture. • First complete ISKNV strain (AK-ISKNV) genome sequenced from India (110,460 bp, 54.79 % GC). • AK-ISKNV closely related to Australian angel fish iridovirus (AFIV). • AK-ISKNV belongs to Megalocytivirus genus (ISKNV-Genotype I). • Four potential vaccine candidates identified (ORFs 105, 104, 11, 14). [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

Infectious spleen and kidney necrosis virus (ISKNV) has a broad host range and poses a significant threat to aquaculture species. Herein, we report two disease outbreaks in snakeskin gourami (Trichopodus pectoralis) in Thailand, marked by skin haemorrhage, scale loss, internal organ discoloration and degeneration and a mortality exceeding 60%. Additionally, three spot gourami (Trichopodus trichopterus) cultured in the same ponds along with snakeskin gourami were found to be affected and tested positive for the virus. Histopathological examination revealed extensive necrosis of haematopoietic tissues in the kidneys and spleen, accompanied by the presence of numerous megalocytic cells in both fish species. Real-time PCR assays, using protocols targeting the major capsid protein (MCP) gene in a broad range of Megalocytivirus genotypes, confirmed the presence of ISKNV in the infected fish. Furthermore, molecular and phylogenetic analyses based on the MCP gene (1362 bp) and the adenosine triphosphatase (ATPase) gene (720 bp) revealed that the ISKNV strains in gourami and other fish hosts were closely related, suggesting possible cross-species transmission. This report extends the host range of ISKNV and highlights the need to prevent the spread of this virus across species boundaries. Further research is needed to understand the pathogenicity and transmission routes of the virus, gain insights into its epidemiology and develop strategies to mitigate disease outbreaks., (© 2025 John Wiley & Sons Ltd.)

This is a case study that assessed risk factors for transmission of red sea bream iridovirus (RSIV) focused on cross-contamination of aquaculture equipment in semi-open system aquaculture. Our investigation during the RSIV outbreak in the fish farm demonstrated that equipment used for collecting dead fish, such as landing nets and gloves, was highly contaminated with RSIV. Based on the results, a daily operation for collecting dead fish was implemented, starting with net pens where no disease occurred, followed by the net pen where the RSIV outbreak occurred to prevent fomite transmission via RSIV-contaminated equipment. In addition, the landing nets used for collecting dead fish were disinfected at the end of each day to avoid carryover of the virus to the following day. RSIV was not transmitted to the other net pens in the fish farm for more than 30 days. However, once an RSIV outbreak occurred in the net pen that was upstream in the operation for collecting dead fish, RSIV was transmitted to all net pens in approximately 1 week, implying that the transmission was caused by cross-contamination. This study suggested that appropriate hygiene management is important to prevent fomite transmission between net pens, even in semi-open system aquaculture., (© 2025 John Wiley & Sons Ltd.)

Despite tens of cell lines originating from fish brain tissue have been constructed, little is known about the definite cell types they belong to. Whether fish cell lines derived from the brain shares similar characteristics is not well-answered yet. Here, we constructed three cell lines designated as LMB-S, LMB-M, LMB-L using brain tissue of spotted sea bass (Lateolabrax maculatus). Among them, LMB-L was identified as astroglia-like cells considering the high expression of GFAP, DCX, PTX, S100b, which are regarded as astrocyte-specific or astrocyte-associated cell markers. LMB-M exhibited smooth muscle-like features showing strong expression of LMOD1, SLAMP, M-cadherin, MGP, which are confirmed as muscle-restricted or myogenesis-involved cell markers. Although LMB-S was not definitely identified, it appeared an activation of WNT/β-catenin pathway. Besides the distinct expression profiles of cell markers, the three cell lines also presented differences in transfection efficiency and susceptibility to iridovirus infection. Relying on the established cell lines, a novel megalocytivirus, named LMIV (Lateolabrax maculatus iridovirus), was first isolated from diseased spotted sea bass. Genetic analysis of major capsid protein (MCP) and adenosine triphosphatase (ATPase) manifested that LMIV was clearly distinguishable from other representative teleost iridoviruses. Further investigations revealed that LMIV could replicate most efficiently in LMB-L cells obtaining the highest viral load (2.16 × 1010 copy/mL). By contrast, LMB-S cells gave rise to the highest viral load up to 3.86 × 108 copy/mL, when the three cell lines were infected with MRV, a newly emerged ranavirus. Moreover, LMIV infection caused lots of cells to be detached from monolayers, generating adherent and non-adherent cells. An opposite expression profiling of type I IFN pathway-related genes (JAK1, STAT1, STAT2, IRF9, Mx1) was found between adherent and non-adherent cells. Combined with the analysis of MCP gene expression, it is speculated that inhibiting type I IFN pathway in non-adherent cells allowed the facilitation of virus duplication. Taken together, the present study broadens our understanding about the diversity of cell lines derived from fish brain tissue and screening cells more susceptible to virus is not only meaningful for the development of vaccine, but also provide clues for further clarification of cell-iridovirus interactions. • Three cell lines from the brain of spotted sea bass were developed. • Two of cell lines were identified as astroglia-like and smooth muscle-like cells. • A novel megalocytivirus designated as LMIV was first isolated. • Inhibiting Type I IFN pathway probably facilitated LMIV duplication. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus (ISKNV) was first detected in imported ornamental fish in Korea in 2004. Herein, ISKNV was re-detected in ornamental fish imported to Korea from Singapore. Diseased dwarf gourami (Trichogaster lalius) imported from Singapore exhibited typical clinical signs associated with ISKNV infection, including darkened body coloration and splenomegaly. Through conventional PCR, ISKNV positivity was identified in 26.7% (4/15) of the cases, which differed from the 60% positivity (9/15) determined by real-time PCR. Furthermore, histopathological lesions exhibited basophilic dysplastic hypertrophied cells and melano-macrophage center (MMC) formation in the spleen and kidney tissues, indicating chronic manifestations of ISKNV infection. Thus, it appears that the dwarf gourami suspected of being infected with ISKNV were imported from Singapore. Furthermore, homogenate-inoculated dwarf gourami fin (DGF) cells (a mixture of spleen and kidney tissues) displayed the typical cytopathic effects of ISKNV infection, including enlarged and rounded cells, and the newly identified ISKNV (DGIV-SAY-23) was successfully isolated. Moreover, virions with icosahedral capsid symmetry, a general morphological feature of the genus Megalocytivirus , were identified using transmission electron microscopy. Based on the phylogeny of the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes, DGIV-SAY-23 belongs to ISKNV subtype I. The MCP gene showed 99.85% identity with DGA 10/8 isolated in Singapore in 2000. Mortality was observed in artificially infected ornamental fish for 14 days. The cumulative mortality rate of artificially infected pearl gourami (Trichogaster leeri) was 100%. The recurrent detection of ISKNV in imported ornamental fish in Korea supports the need for enhanced quarantine measures to prevent the spread of infectious diseases through international trade. To achieve an enhanced quarantine system, an appropriate detention period for live ornamental fish should be applied in the isolation facility based on the disease incubation period. • ISKNV was redetected in dwarf gourami (Trichogaster lalius) imported from Singapore to Korea. • ISKNV was successfully isolated from the DGF cells and its virions were identified using transmission electron microscopy. • Based on the phylogeny of the MCP and ATPase genes, the isolated ISKNV was classified into ISKNV subtype I. [ABSTRACT FROM AUTHOR]

The Chinese bahaba (Bahaba taipingensis) is an endangered fish species with high economic value, found in the southeastern sea of China. In this study, we established the first Chinese bahaba cell line, named BTF, derived from fin tissue. The primary cells were cultured for over 55 passages in L-15 medium with 15% FBS at 27 °C. These cells exhibited a fibroblast-like morphology with multipolar shapes. Karyotype testing revealed a chromosome number of 2n = 48. After thawing the cells that had been stored in liquid nitrogen for 10 months, their viability was >80%. The cell was separately transfected with EGFP-N3 plasmid and cy3 labeled scramble siRNA and clear fluorescence were observed. Moreover, the cells displayed sensitivity to megalocytivirus SKIV-SD strain, demonstrating a pronounced cytopathic effect (CPE) 24 h after infection. Transcriptome sequencing analysis of SKIV-SD infected cells revealed highly expressed genes significantly enriched in nucleic acid replication and protein translation-related pathways, such as Ribosome biogenesis, RNA polymerase, Nucleocytoplasmic transport, and DNA replication. In addition, the infected cells showed differential expression of immune-related genes such as cxcr4 and dr5 , which are involved in virus infection. In conclusion, the BTF cell line, derived from the fin tissue of the Chinese bahaba, exhibits stable growth and can be stored in liquid nitrogen for extended periods. This cell line plays a crucial role in conserving the germplasm resources of Chinese bahaba and will serve as a valuable reference for establishing cell lines from other tissues of the Chinese bahaba. Furthermore, our findings suggest that this cell line holds significant potential for virology research, foreign protein expression, and in vitro biological studies of Chinese bahaba. • This study establishes the first Chinese bahaba cell line, BTF. • The BTF cell line is capable of efficient plasmid and siRNA transfection, enabling the expression of foreign gene. • The BTF cell line serves as a substrate in vitro for megalocytivirus amplification. • Megalocytivirus infected BTF cells revealed highly expressed genes significantly enriched in nucleic acid replication and protein translation-related pathways. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus that infects a number of marine and freshwater fishes, causing huge economic losses in aquaculture. The ISKNV infection leads to increase of reducing power in cells. As the antibiotic neomycin can promote the production of reactive oxygen species (ROS) in animal cells, in the current study, the potential therapeutic effect of neomycin on ISKNV infection was explored. We showed that neomycin could decrease the reducing power in cultured MFF-1 cells and inhibit ISKNV infection by antagonizing the shift of the cellular redox balance toward reduction. In vivo experiments further demonstrated that neomycin treatment significantly suppresses ISKNV infection in mandarin fish. Expression of the major capsid protein (MCP) and the proportion of infected cells in tissues were down-regulated after neomycin treatment. Furthermore, neomycin showed complex effects on expression of a set of antiviral related genes of the host. Taking together, the current study suggested that the viral-induced redox imbalance in the infected cells could be used as a target for suppressing ISKNV infection. Neomycin can be potentially utilized for therapeutic treatment of Megalocytivirus diseases by antagonizing intracellular redox changes. • The improvement of reducing power in cells is important for Megalocytivirus replication. • The anti- Megalocytivirus effect can be achieved by antagonizing intracellular redox imbalance. • Neomycin inhibits ISKNV infection in vitro and in vivo. • Immersion with neomycin increases survival rate of ISKNV-infected mandarin fish. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus , infects a variety of teleost fish species and causes substantial losses in the aquaculture industry worldwide. ISKNV ORF71L is 1611 bp in length, encodes a 537-amino-acid peptide and was previously identified as a viral structural protein in the ISKNV virion. In this study, the ORF71L deletion mutant virus strain ISKNV-Δ71 was obtained through a homologous recombination approach. The multistep growth curves showed that ISKNV-Δ71 replication was faster than ISKNV-WT replication in mandarin fish fry cells (MFF-1 cells) before 48 h post-infection (hpi). The cumulative mortality of ISKNV-Δ71-infected mandarin fish (Siniperca chuatsi) was lower than that of fish infected with ISKNV-WT. The copy numbers of viral genome equivalents (GEs) in ISKNV-Δ71-infected mandarin fish spleens were also lower than those in ISKNV-WT-infected spleens. Deletion of ORF71L resulted in ISKNV virulence attenuation in mandarin fish. Furthermore, we found that the number of melanomacrophage centers (MMCs) in ISKNV-Δ71-infected mandarin fish spleens was higher than that in ISKNV-WT-infected mandarin fish spleens. Transcriptomic analysis showed that the cytokine–cytokine receptor interaction pathway had the most significant change between ISKNV-Δ71- and ISKNV-WT-infected MFF-1 cells. These results indicated ORF71L is a virulence-related gene of ISKNV. ORF71L could be considered as a potential target for the development of engineered attenuated live vaccines via multigene deletion or as a potential insertion site for exogenous protein expression. • ORF71L deletion mutant strain of ISKNV (ISKNV-Δ71) was obtained through a homologous recombination approach. • ISKNV-Δ71 replication was faster than ISKNV-WT in MFF-1 cells. • ISKNV ORF71L was a virulence-related gene. • The expression of cxcl12a was upregulated in ISKNV-Δ71-infected liver and spleen compared with those by ISKNV-WT. [ABSTRACT FROM AUTHOR]

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.

Successful viral infection and multiplication chiefly rely on virus subversion mechanisms against host anti-viral immune responses. In this study, in order to reveal the anti-viral immune-related pathways suppressed by megalocytivirus infection, transcriptome analysis was performed on the head-kidney of turbot (Scophthalmus maximus) infected with lethal dose of RBIV-C1 at 3, 6 and 9 days post challenge (dpc). The results showed that, compared to unchallenged groups, 190, 1220, and 3963 DEGs were detected in RBIV-C1 infected groups at 3, 6 and 9 dpc, respectively, of which, DEGs of complement components and pattern recognition proteins were up-regulated at 3 dpc and down-regulated at 6 and 9 dpc, DEGs of cytokines were up-regulated at 6 dpc and down-regulated at 9 dpc. Expression trend analysis revealed that DEGs of profiles 9 and 13 featured decreased expression patterns and were significantly enriched into 10 immune-related pathways, i.e., complement and coagulation cascades, cytokine-cytokine receptor interaction, chemokine signaling pathway, B/T cell receptor signaling pathway, antigen processing and presentation, and so on. Further co-expression network analysis (WGCNA) revealed positive correlated innate immune related pathways at 3 and 6 dpc, and negative correlated innate and adaptive immune related pathways at 9 dpc. This study revealed a set of anti-viral immune genes/pathways that would also be potential targets subverted by RBIV-C1 for immune evasion, which can serve as a valuable resource for future studies on the molecular mechanisms of anti-viral immune defense of turbot and immune escape of megalocytivirus. • Transcriptome analysis was performed on the head-kidney of turbot infected with RBIV-C1. • Innate immune related DEGs exhibited an earlier increasing and later decreasing expression trend. • Adaptive immune related DEGs exhibited a trend toward down-regulation. • Down-regulated immune pathways would be potential targets suppressed by virus for immune evasion. [ABSTRACT FROM AUTHOR]

Rock bream iridovirus (RBIV) causes severe mortality in rock bream (Oplegnathus fasciatus) for last two decades. In view of this constant threat of RBIV to the rock bream industry, we conducted the present study with the aim to develop a safe and efficient remedial measure against the virus. In this study, we evaluated the safety and potentiality of squalene, aluminium hydroxide and saponin adjuvants, singly or in combinations, which can be used for developing an efficient inactivated (IV) vaccine to protect rock bream from RBIV infection. The evaluation results demonstrated that saponin (Sa) has the required potential in enacting the antiviral immune response in the host and in providing protection against virus mediated lethality, without causing any adverted side-effects. The study further, showed that a single primary dose of Sa-adjuvanted IV vaccine can confer moderate protections in short (60.04% relative percent mortality (RPS) at 4 wpv) and medium (53.38% RPS at 8 wpv) term post RBIV challenge; whereas, the same vaccine when administered in a prime-boost strategy, it resulted enhanced 93.34% RPS post virus challenge at 4 and 8 wpv. The moderate to high survivability demonstrated by the Sa-adjuvanted IV vaccine, was substantiated by the significant (p < 0.05) upregulation of IL-1β, Mx and PKR gene transcript. All surviving fish from the Sa-adjuvanted IV vaccine groups were strongly protected from re-infection with RBIV (1.1 × 107) at 70 days post infection (dpi). In conclusion, it can be inferred that, Sa-adjuvanted IV RBIV vaccine can be an efficient control measure to protect the rock bream aquaculture industry against the lethal RBIV virus. • Administration of saponin adjuvant-based inactivated RBIV vaccine offered significantly high protection. • Boost immunization with vaccine provided significantly higher protection than one time immunization. • Saponin adjuvant-based inactivated RBIV vaccine upregulated Il-1β, Mx and PKR gene expression. [ABSTRACT FROM AUTHOR]

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.

ABSTRACT 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.

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 (115–125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) 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. [ABSTRACT FROM AUTHOR]

Native freshwater fish of Australia have a diverse but largely undescribed endemic pathogen and parasite fauna. However, due to long-shared evolutionary histories and virulence/transmissibility trade-offs, effects of these endemic pathogens and parasites appear to be subtle: significant impacts are rarely observed and epizootics have not been recorded. In contrast, a number of alien pathogens and parasites are now established across southern Australia, causing manifestly harmful effects to native fish species and known or suspected epizootics in native fish populations. Undetected and/or undescribed alien viral pathogens are also suspected of being present. Alien pathogens and parasites were introduced to Australia with imports of live alien fish or their fertilised eggs. A review of the scientific and historical evidence indicates that they have had, and continue to have, greater impacts on native fish species than previously realised—especially for freshwater species. This review also documents a previously unknown, Murray-Darling-Basin-wide epizootic of Murray cod Maccullochella peelii in 1929–30, which may have contributed to strong declines in the related eastern freshwater cod Maccullochella ikei. A serious Chilodonella epizootic of M. peelii in 1982 is also examined. In addition, a possible role for alien viruses of the family Iridoviridae (Ranavirus and Megalocytivirus) in the general decline of the critically endangered silver perch Bidyanus bidyanus, and the rapid collapse of two specific native fish populations—upper Murrumbidgee River B. bidyanus and Shoalhaven River Macquarie perch Macquaria australasica—is suggested. It is argued that the severity of the impact of the virulent alien oomycete Saprolegnia parasitica, both historical and present day, has been underestimated. Finally, action is recommended against emerging new pathogen and parasite threats, and the extreme risk current alien fish importations pose in introducing them. These will further threaten already stressed native fish populations in southern Australia, particularly across the Murray-Darling Basin. [ABSTRACT FROM AUTHOR]

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus (ISKNV), a member of family iridoviridae, reported for the first time in a wide range of ornamental fish species in India. Significant mortalities during the year 2018–19 were reported from a number of retailers in the region with various clinical signs. The samples of moribund, dead and apparently healthy ornamental fishes were collected from retailers, located in three districts of Karnataka, India. Out of 140 fish samples, 16 samples (11.42%) representing 10 different fish species were found positive to ISKNV by OIE listed primers and same samples were reported to amplify the major capsid protein (MCP) gene of ISKNV. Further, sequence analysis of MCP gene showed that all strains detected in this study were closely related to other documented isolates from different countries with an identity ranging from 98.76% to 100%. Further, they clustered in the clade of ISKNV, during the phylogenetic analysis. The sequence similarity was high (99.94%) to ISKNV strains from Japan, Australia and Malaysia. This is the first report of an ISKNV infection in India. Moreover, out of 10 ISKNV‐positive fish species, three species were reported positive to ISKNV for the first time in the world. Further, the in vitro experiment showed the growth of virus in Asian sea bass cell line, which is a natural host of ISKNV. Therefore, considering the lethal nature of megalocytiviruses to infect a vast range of species, proper biosecurity measures need to be taken to control these emerging pathogens. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus (ISKNV), causing serious infectious diseases to marine and freshwater fishes, is the type species of the genus Megalocytivirus, family Iridoviridae. In this study, the transcriptional programs of ISKNV in vitro (MFF-1 cells) and in vivo (spleens from mandarin fish) were investigated using real-time PCR. Transcription of all the putative open reading frames (ORFs) of ISKNV was verified. The temporal expression patterns of ISKNV ORFs in vitro and in vivo, including peak expression times (PETs) and relative maximal expression levels, were determined and compared. The K-means clustering with Spearman rank correlation was generated in heat maps constructed based on ISKNV ORF expression profiles in vivo and in vitro. The current study may provide a global picture of ISKNV infection at the transcription level and help better understand the molecular pathogenic mechanism of megalocytiviruses. [ABSTRACT FROM AUTHOR]

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Megalocytivirus is a serious viral pathogen to many farmed fish including Japanese flounder (Paralichthys olivaceus). In this study, in order to systematically identify host immune genes induced by megalocytivirus infection, we examined the transcription profiles of flounder infected by megalocytivirus for 2, 6, and 8 days. Compared with uninfected fish, virus-infected fish exhibited 1242 differentially expressed genes (DEGs), with 225, 275, and 877 DEGs occurring at 2, 6, and 8 days post infection, respectively. Of these DEGs, 728 were upregulated and 659 were downregulated. The majority of DEGs were time-specific and formed four distinct expression profiles well correlated with the time of infection. The DEGs were classified into diverse Gene Ontology (GO) functional terms and enriched in 27 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, approximately one third of which were related to immunity. Weighted co-expression network analysis (WGCNA) was used to identify 16 key immune DEGs belonging to seven immune pathways (RIG-I-like receptor signaling pathway, JAK-STAT signaling pathway, TLR signaling pathway, cytokine-cytokine receptor interaction, phagosome, apoptosis, and p53 signaling pathway). These pathways interacted extensively and formed complicated networks. This study provided a global picture of megalocytivirus-induced gene expression profiles of flounder at the transcriptome level and uncovered a set of key immune genes and pathways closely linked to megalocytivirus infection. These results provided a set of targets for future delineation of the key factors implicated in the anti-megalocytivirus immunity of flounder. • A total of 1242 differentially expressed genes were induced by megalocytivirus. • The expressions of the DEGs were highly time-dependent and clustered into 4 groups. • Sixteen key immune genes forming complex networks with each other were identified. • The interactive key genes belonged to 7 immune pathways vital to antiviral immunity. [ABSTRACT FROM AUTHOR]

The severe mortality of fish due to the infection of megalocytivirus caused significant economic losses. Since 2011, megalocytivirus (giant gourami iridovirus (GGIV)) has become the main pathogen in giant gourami (Osphronemus goramy), particularly in West Java, Central Java and Bali. This study aimed to develop primary cell culture from spleen as the target organ for propagating megalocytivirus in vitro, which was developed by explant method with enzymatic dissociation. Optimization was carried out at incubation temperature, medium and serum concentrations. The origin of the primary cell, cell susceptibility and GGIV pathogenicity were observed. The results showed that the primary cell (GP cells) can grow well in 10% foetal bovine serum L‐15 medium at 27°C, which was sufficient for cell growth. PCR and BLAST analyses showed the primary cell was originated from giant gourami. In infected GP cells, cell enlargement and cell rounding were observed. Virus propagated in GP cells was highly virulent when injecting giant gourami in an artificial infection experiment. Intraperitoneal injection of diluted virus supernatant showed 100% mortality in 7–11 days post‐injection and 97% mortality in 21 days post‐cohabitation, with abnormalities observed in spleen and kidney. In conclusion, GP cell was successfully subcultured for more than 30 passages and susceptible to GGIV. [ABSTRACT FROM AUTHOR]

Megalocytivirus of family Iridoviridae has been identified as a pathogen that caused the fatal systemic infection to lead to massive death of numerous fish species in both ornamental and food fish industries. It caused significant economic losses due to severe mortality of infected fish. Since 2011 in Indonesia, megalocytivirus in giant gourami (Osphronemus goramy) had been found as the main pathogen in some cases of giant gourami outbreak, especially in West Java, Central Java, and Bali. The aim of this study is to identify and characterize the pathogen known as a megalocytivirus infection in freshwater-cultured giant gourami. We identified the virus using universal and specific primers for megalocytivirus and infectious spleen and kidney necrosis virus (ISKNV). Sequencing and BLAST analysis were used to develop a phylogenetic tree. The result showed that phylogenetic analysis of major capsid protein (MCP) gene unveiled a new member of megalocytivirus, designated as giant gourami iridovirus (GGIV). GGIV-formed cluster belonged to ISKNV and has 100% homology to ISKNV complete genome. Artificial infection by intraperitoneal injection with supernatant homogenate from spleen and kidney of naturally infected fish showed 93% cumulative mortality in 12 days. Fish showed a clinical sign of infection as lethargic, loss of appetite, pale or darken body color, and hemorrhages. Internal organ on dead fish showed swollen spleen and kidney and also a pale liver. Quantitative PCR analysis on internal organs showed spleen had the highest viral DNA copy number followed by kidney, gill, and liver. Histopathological analysis showed many abnormally hypertrophied cells in spleen which is typical histopathological characteristic of megalocytivirus infection. In conclusion, GGIV belonged to ISKNV from genus megalocytivirus. [ABSTRACT FROM AUTHOR]

A successful viral infection is primarily dependent on interactions between the viral pathogenesis and the host's antiviral immunity. MicroRNAs (miRNAs) are non-coding regulatory RNAs that play significant roles in modulating the host's antiviral immune responses. To elucidate the functional mechanism of miRNAs during megalocytivirus infection, in this study, micro-transcriptome analysis was performed on the head-kidney of turbot (Scophthalmus maximus) infected with RBIV-C1 or not at 3, 6 and 9 days after infection. A total of 247 differentially expressed miRNAs (DEmiRs) were identified, among which 125 were known miRNA and 122 were newly identified. Based on an integrated negative correlation analysis of miRNA-mRNA, a total of 1173 up-regulated and 644 down-regulated target genes (DEmRs) were identified. These DEmRs were found to be enriched in different KEGG pathways associated with signal transduction, endocrine system, immune system, cancer, and infectious diseases. Moreover, networks of immune-related DEmiRs-DEmRs were constructed and analyzed, revealing significant associations between RBIV-C1 infection and immune-related pathways, such as pattern recognition receptor, apoptosis, phagolysosome, autophagy, TNF signaling, adaptive immunity, cytotoxicity, and NF-kappa B signaling. Additionally, the negative correlation network analysis unveiled numerous known and novel DEmiRs that directly target RBIV-C1 genes. This study presents, for the first time, a comprehensive micro-transcriptome dataset of turbot following RBIV-C1 infection, offering valuable insights into the underlying survival mechanisms that involve immune evasion and antiviral immunity. • Micro-transcriptome analysis was performed on RBIV-C1 infected turbot. • DEmiRs exhibit a time-dependent expression pattern upon RBIV-C1 infection. • MiRNA-mRNA network analysis revealed key anti-viral immune-regulated pathways. • RBIV-C1 break through antiviral system via regulating the host's miRNA level. [ABSTRACT FROM AUTHOR]

In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6–10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues.

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.

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. [ABSTRACT FROM AUTHOR]

The genus Megalocytivirus (MCV) includes Infectious spleen and kidney necrosis virus (ISKNV) and red seabream iridovirus (RSIV) which are listed by the World Organization for Animal Health (OIE). MCV has a broad host range and can be present as a persistent subclinical infection in some fish. Australia is considered free from MCV and important aquaculture and wild fisheries are at risk. Consequently, biosecurity measures are implemented to ameliorate transmission pathways through imported ornamental fish which include certification of freedom from infection with MCV. Evaluation of practical and cost effective disinfection protocols suitable for recirculating aquaculture facilities is an important aspect of the biosecurity plan to facilitate eradication in the event of early detection of an outbreak. An authentic sample matrix was prepared by in vivo amplification of ISKNV in Murray cod (Maccullochella peelii) and the biological load of a clarified tissue homogenate was standardized by addition of 10% v/v foetal bovine serum. In the absence of a cell culture system for ISKNV, a bioassay was used to assess infectivity after disinfection. A cellulose membrane buffer exchange device was used to remove residual disinfectants before intraperitoneal injection of juvenile Murray cod. Fish were maintained in 100 L aquaria at 23 °C and observed for clinical signs over 14 days. Each bioassay was conducted in duplicate tanks with 6 to 19 fish per tank. A positive assay was defined by an increase in ISKNV DNA quantified by qPCR in any fish from a subsample of challenged fish collected at 7 d or those remaining at 14 d. Negative bioassays were defined by the absence of ISKNV DNA in all fish at the both sampling times. Clinical disease and amplified viral DNA was detected after injection of a dilute positive control, indicating greater analytical sensitivity compared to qPCR (with a detection limit of 100 copies per reaction). Further, the system was used to demonstrate that ISKNV can remain infectious in aquaria without fish for at least 48 h at 25 °C. Effective disinfection measures included: heating to 65 °C for 20 min; pH 3; pH 11; 1% Virkon™; 1000 ppm sodium hypochlorite and benzalkonium chloride at the recommended concentration and contact time. These data can be interpreted to provide effective disinfection protocols for MCV in a wide variety of disease control scenarios. • Six methods for effective disinfection of ISKNV were identified. • Bioassay for ISKNV was more sensitive than the qPCR. • ISKNV contaminated water can remain infectious for at least 48 h after diseased fish are removed. [ABSTRACT FROM AUTHOR]

Megalocytiviruses are responsible for severe mortality in various marine and freshwater fish species worldwide. Specifically, the red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV) genotypes are known to cause the red sea bream iridoviral disease (RSIVD). Although several quantitative polymerase chain reaction (qPCR) assays have been developed for diagnosing RSIVD, these methods fail to differentiate between viral genotypes within Megalocytivirus. The present study introduces an innovative multiplex qPCR assay for the precise detection of RSIVD. This diagnostic method targets the histidine triad motif (HIT)-like protein and ORF080L genes, which are unique markers for the RSIV and ISKNV genotypes. The assay exhibited high analytical sensitivity, expressed as the 95% limit of detection (LoD 95%), of 8.69 copies/5 μL for RSIV and 19.07 copies/5 μL for ISKNV. Notably, the assay exhibited no cross-reactivity and was unaffected by common inhibitory substances. The diagnostic sensitivity and specificity were 100% for both RSIV and ISKNV, demonstrating the reliability of the assay. The multiplex qPCR assay was 100–1000 times more sensitive than conventional PCR methods, emphasizing its importance in early-stage infection detection. The repeatability and reproducibility of the assay were confirmed through extensive testing, showing low variation and high correlation across different technicians and laboratory settings. Furthermore, the ability of the assay to selectively detect and differentiate between RSIVD genotypes represents a significant advancement in aquatic animal disease diagnostics. This multiplex qPCR assay offers a rapid, specific, sensitive, and quantitative diagnostic tool for global RSIVD surveillance efforts and contributes to the prevention and understanding of disease progression in the aquaculture industry. • This study presents a multiplex qPCR assay for red sea bream iridoviral disease (RSIVD) detection • The assay detects RSIV and ISKNV, causative agents of RSIVD • The assay showed high analytical performance, with 100% diagnostic sensitivity and specificity for both RSIVD subtypes • This diagnostic tool helps prevent and understand disease progression in the aquaculture industry [ABSTRACT FROM AUTHOR]

A cross-sectional survey was used to estimate the prevalence of infections with the Infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), nervous necrosis virus (NNV, Betanodavirus), and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0–12) to 73% (95% CI: 54–88). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4–31%). Trichodina sp., Cryptocaryon irritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p < 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia.

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.

Abstract Megalocytivirus is a DNA virus with a broad host range among farmed fish including tongue sole (Cynoglossus semilaevis). In this study, label-free proteomics was performed to examine protein expression in tongue sole spleen induced by megalocytivirus at 8 and 12 days post infection (dpi). Compared to uninfected control fish, virus-infected fish displayed 315 up-regulated proteins and 111 down-regulated proteins at 8 dpi, and 48 up-regulated proteins and 43 down-regulated proteins at 12 dpi. The expressions of five differentially expressed proteins were confirmed by Western blot. The differentially expressed proteins were enriched in the pathways and processes associated with innate immune response and viral infection. Interference with the expression of two up-regulated proteins of the ubiquitin proteasome system (UPS), i.e. proteasome assembly chaperone 2 and proteasome maturation protein, significantly reduced viral propagation in fish, whereas overexpression of these two proteins significantly enhanced viral propagation. Consistently, inhibition of the functioning of proteasome significantly impaired viral replication in vivo. This study provided the first global protein profile responsive to megalocytivirus in tongue sole, and revealed an essential role of UPS in viral infection. Graphical abstract Image 1 Highlights • The protein expression profile in megalocytivirus-infected tongue sole was investigated. • A total of 517 differentially expressed proteins (DEPs) were identified. • The DEPs were enriched in pathways of immune response and viral infection. • Factors of the ubiquitin proteasome system (UPS) were among the DEPs. • Normal expression and functioning of the UPS were essential to viral infection. [ABSTRACT FROM AUTHOR]

Malaysia is the 8th largest world producer of freshwater ornamental fish. Ornamental fish are commonly associated with Megalocytiviruses infection, which has led to severe diseases and economic loss. The purpose of this study was to detect the presence or absence of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in four ornamental fish species namely Xiphophorus maculatus, Poecilia reticulata, Trichogaster leeri and Apistogramma ramirezi from Malaysia. A total of 175 samples were analysed using PCR analysis for detection of ISKNV. The PCR analysis demonstrated 22 positive pooled samples (n = 110) for the presence of ISKNV. No clinical signs were observed in positive samples except darkened body in X. maculatus. Sequencing analysis of Megalocytivirus major capsid protein (MCP) revealed that the ISKNV strains in this study demonstrated high nucleotide identity to each other and reference ISKNV (96% to 100%). Based on the phylogenetic tree, the ISKNV strains were closely related to reference ISKNV and can be classified into Megalocytivirus genotype I. [ABSTRACT FROM AUTHOR]

Abstract Infectious spleen and kidney necrosis disease (ISKND) is one of major viral diseases in marine fish species. Here, we isolated infectious spleen and kidney necrosis virus (ISKNV) from diseased barramundi in Thailand and evaluated its pathogenicity with intramuscular challenge in naïve barramundi. ISKNV infection in grunt fin (GF) cells was observed within 14 days post inoculation (dpi) by immunofluorescence assay using a monoclonal antibody against red seabream iridovirus (M10). Phylogenetic analysis of the major capsid protein amino acid sequences revealed that two ISKNV isolates belong to genotype I ISKNV. In the challenge test, 35% mortality was observed in fish intramuscularly injected with 10–0.55 TCID 50 /fish, whereas higher mortalities, 44% and 77%, were observed after injection with 100.55 and 101.55 TCID 50 /fish, respectively. These results demonstrate dose dependent virulence of ISKNV in barramundi. This study is the first report of ISKNV in farmed barramundi in Thailand. A high susceptibility of barramundi to the infection may suggest an immediate need for control of the emerging disease in barramundi culture including effective vaccine. Highlights • This is the first isolation of ISKNV in barramundi cultured in Thailand. • ISKNV isolates belong to genotype I ISKNV. • The virulence of ISKNV infection in barramundi is dose dependent. [ABSTRACT FROM AUTHOR]

Ornamental fishes were collected from 173 locations in nine provinces of Thailand during 2016-2018. Using real-time PCR analysis, twelve fish species of three families (Poeciliidae, Cichlidae and Osphronemidae) were found positive for Megalocytivirus, a pathogenic agent which causes a significantly high mortality in a wide range of aquatic animal hosts. A geographic information system (GIS) displayed the distribution of megalocytivirus to the northern, central and western regions of Thailand. Nevertheless, one sampling province showed megalocytivirus-free status and the overall rate of virus occurrence declined by 10% during the study period. The transport and translocation of ornamental fish could be a key route for megalocytivirus introduction to new geographical areas. Molecular phylogenetic analysis based on the major capsid protein (MCP) confirmed that Megalocytivius in the current study belongs to members of an infectious spleen and kidney necrosis virus (ISKNV) cluster. Although most infected fish were asymptomatic, non-specific symptoms such as haemorrhages, loss of coloration on the fish body and pale liver were observed without substantial mortality. Cytomegalic cells with inclusion bodies were examined. Issues associated with host molecular defense mechanism, persistent infection, and mortality risk factors will await further study. [ABSTRACT FROM AUTHOR]