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.

Molecular epidemiology of Megalocytivirus in freshwater angelfish (Pterophyllum scalare) from Johor, Malaysia.

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.

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.

Assessing the transmission risk of red sea bream iridovirus (RSIV) in environmental water: insights from fish farms and experimental settings.

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.

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

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

Impact of Red Sea Bream Iridovirus Infection on Rock Bream (Oplegnathus fasciatus) and Other Fish Species: A Study of Horizontal Transmission.

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.

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.

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.

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.

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.

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.

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.

Isolation, Identification and Characterization of a Novel Megalocytivirus from Cultured Tilapia (Oreochromis spp.) from Southern California, USA.

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.

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

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

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.

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.

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.