Molecular mechanism of the interaction between Megalocytivirus-induced virus-mock basement membrane (VMBM) and lymphatic endothelial cells.

IMPORTANCE: Viruses are able to mimic the physiological or pathological mechanism of the host to favor their infection and replication. Virus-mock basement membrane (VMBM) is a Megalocytivirus-induced extracellular structure formed on the surface of infected cells and structurally and functionally mimics the basement membrane of the host. VMBM provides specific support for lymphatic endothelial cells (LECs) rather than blood endothelial cells to adhere to the surface of infected cells, which constitutes a unique phenomenon of Megalocytivirus infection. Here, the structure of VMBM and the interactions between VMBM components and LECs have been analyzed at the molecular level. The regulatory effect of VMBM components on the proliferation and migration of LECs has also been explored. This study helps to understand the mechanism of LEC-specific attachment to VMBM and to address the issue of where the LECs come from in the context of Megalocytivirus infection.

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

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

Genetic differentiation of Megalocytivirus by real time PCR and sequencing.

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.

Deleting ORF71L of infectious spleen and kidney necrosis virus (ISKNV) resulted in virulence attenuation in Mandarin fish.

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.

Efficacy of saponin-based inactivated rock bream iridovirus (RBIV) vaccine in rock bream (Oplegnathus fasciatus).

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-1ß, 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.

Characterization of three novel cell lines derived from the brain of spotted sea bass: Focusing on cell markers and susceptibility toward iridoviruses.

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/ß-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.

Comparative transcriptome analysis reveals potential anti-viral immune pathways of turbot (Scophthalmus maximus) subverted by megalocytivirus RBIV-C1 for immune evasion.

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.

Neomycin inhibits Megalocytivirus infection in fish by antagonizing the increase of intracellular reduced glutathione.

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.

Detection of megalocytivirus in Oreochromis niloticus and Pseudoplatystoma corruscans in Brazil.

The infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus (MCV), a group of double-stranded DNA genome viruses. The aim of this study was to retrospectively analyze samples from suspected foci of MCV infection in freshwater fish in Brazil. Samples were collected from infected fish between 2017 and 2021. Phylogenetic analysis revealed 2 groups of MCV circulating in the country. A genetically homogeneous group formed a clade with ISKNV samples from different parts of the world. Only 2 of the sequences from the state of Goiás showed a small genetic distance when compared to the larger group in the same clade. This study describes the validation of 3 qPCR methods and the presence of MCV in Brazil since 2017, including a genotype not previously described.

Complete genome sequences of infectious spleen and kidney necrosis virus isolated from farmed albino rainbow sharks Epalzeorhynchos frenatum in the United States.

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.

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

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

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 (115-125 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.

Megalocytivirus Induces Complicated Fish Immune Response at Multiple RNA Levels Involving mRNA, miRNA, and circRNA.

Megalocytivirus is an important viral pathogen to many farmed fishes, including Japanese flounder (Paralichthys olivaceus). In this study, we examined megalocytivirus-induced RNA responses in the spleen of flounder by high-throughput sequencing and integrative analysis of various RNA-seq data. A total of 1327 microRNAs (miRNAs), including 368 novel miRNAs, were identified, among which, 171 (named DEmiRs) exhibited significantly differential expressions during viral infection in a time-dependent manner. For these DEmiRs, 805 differentially expressed target mRNAs (DETmRs) were predicted, whose expressions not only significantly changed after megalocytivirus infection but were also negatively correlated with their paired DEmiRs. Integrative analysis of immune-related DETmRs and their target DEmiRs identified 12 hub DEmiRs, which, together with their corresponding DETmRs, formed an interaction network containing 84 pairs of DEmiR and DETmR. In addition to DETmRs, 19 DEmiRs were also found to regulate six key immune genes (mRNAs) differentially expressed during megalocytivirus infection, and together they formed a network consisting of 21 interactive miRNA-messenger RNA (mRNA) pairs. Further analysis identified 9434 circular RNAs (circRNAs), 169 of which (named DEcircRs) showed time-specific and significantly altered expressions during megalocytivirus infection. Integrated analysis of the DETmR-DEmiR and DEcircR-DEmiR interactions led to the identification of a group of competing endogenous RNAs (ceRNAs) constituted by interacting triplets of circRNA, miRNA, and mRNA involved in antiviral immunity. Together these results indicate that complicated regulatory networks of different types of non-coding RNAs and coding RNAs are involved in megalocytivirus infection.

Transcriptome analysis reveals seven key immune pathways of Japanese flounder (Paralichthys olivaceus) involved in megalocytivirus infection.

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.

Transcriptional programs of infectious spleen and kidney necrosis virus (ISKNV) in vitro and in vivo.

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.

Isolation and characterisation of an ISKNV-genotype megalocytivirus from imported angelfish Pterophyllum scalare.

Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.

Development of primary cell culture from spleen of giant gourami Osphronemus goramy for propagation of giant gourami iridovirus (GGIV).

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.

First report of megalocytivirus (iridoviridae) in cultured bluegill sunfish, Lepomis macrochirus, in China.

The bluegill sunfish, Lepomis macrochirus, is an important aquacultural and recreational species in southern China because of its excellent taste, rapid growth rate, and good looks. At present, few pathogens are known to affect the bluegill sunfish. However, an iridovirus-like disease recently caused heavy losses to the bluegill sunfish aquaculture industry in Guangdong, China. We report that a virus, designated BSMIV-SD-20171020, was isolated from diseased bluegill sunfish in China. The isolate was efficiently propagated in a Chinese perch brain (CPB) cell line. The cytopathic effect was observed, the MCP gene PCR amplified, and the virus observed with electron microscopy. Its viral titer in CPB cells reached 104.13 TCID50 mL-1. The mortality rate was 100% when bluegill sunfish were challenged with BSMIV-SD-20171020 at a dose of 103.13 TCID50/fish. A histopathological examination revealed basophilic hypertrophied cells in the intestine, liver, and spleen. A nucleotide sequence alignment and phylogenetic analysis of the major capsid protein revealed that isolate BSMIV-SD-20171020 is the species Infectious spleen and kidney necrosis virus (ISKNV), in the genus Megalocytivirus.

Australian bass Macquaria novemaculeata susceptibility to experimental megalocytivirus infection and utility as a model disease vector.

Megalocytiviruses, particularly red seabream iridovirus, infect a broad range of fish including both freshwater and marine species. Although a limited number of infectious spleen and kidney necrosis virus (ISKNV) strains have been reported in association with mortality events in marine aquaculture species, the potential host range for ISKNV strains, particularly of those that have been detected in ornamental fish, has not been well characterised. There have also been few reports on the susceptibility of euryhaline fish species that could potentially transmit megalocytiviruses between freshwater and marine environments. We found that the euryhaline Australian native percichthyid fish, Australian bass Macquaria novemaculeata, is susceptible experimentally to ISKNV (strain DGIV-10), obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. Australian bass developed clinical disease following direct inoculation and also following cohabitation with infected fish, and were able to transmit DGIV-10 to naïve Murray cod Maccullochella peelii. This study demonstrated the potential for a euryhaline species to become infected with, and transmit, the megalocytivirus ISKNV between fish populations.

Experimental transmission of infectious spleen and kidney necrosis virus (ISKNV) from freshwater ornamental fish to silver sweep Scorpis lineolata, an Australian marine fish.

The Australian native marine fish species, silver sweep Scorpis lineolata, is susceptible to the megalocytivirus Infectious spleen and kidney necrosis virus (strain DGIV-10) obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. This was demonstrated by direct inoculation and through cohabitation. Transmission by cohabitation was also demonstrated from inoculated freshwater Murray cod Maccullochella peelii to euryhaline Australian bass Macquaria novemaculeata and to marine silver sweep. The virus was also transmitted from infected marine silver sweep to euryhaline Australian bass and then to freshwater Murray cod. This study is the first to demonstrate the virulence of a megalocytivirus derived from ornamental fish in an Australian marine species and the first to show a feasible pathway for the exchange of megalocytiviruses between freshwater and marine finfish hosts. These results demonstrate that megalocytiviruses from freshwater ornamental fish have the potential to spread to diverse aquatic environments.