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The infectious spleen and kidney necrosis virus (ISKNV) is a major pathogen causing infectious spleen and kidney necrosis disease in largemouth bass. As its major capsid protein (MCP) is considered a candidate subunit vaccine, its protective efficacy in practical applications remains suboptimal, failing to meet clinical prevention and control demands. This study employed bioinformatics techniques to analyze the sequences and predict the functions of MCP and candidate protein 051 L of ISKNV. Two truncated proteins (designated tMCP and t051L) with rich antigenic epitope were screened, which were linked via a rigid linker peptide to construct the fusion truncated protein t051L-tMCP. tMCP, t051L, and t051L-tMCP were induced and purified by using the prokaryotic expression system. The immunogenicity and protective efficacy of the different proteins were evaluated by intraperitoneal injection. Serum ELISA results showed that serum antibody titers in all immunized groups gradually increased with time within 28 days post-immunization. Simultaneously, optimal protection was achieved at an immunization dose of 1 mg/mL: The survival percentages of fish in the tMCP group and in the t051L group were 85.01 % and 79.01 %, while the t051L-tMCP group demonstrated a significantly survival percentages of 95.50 %, superior to the tMCP and t051L groups. Furthermore, viral load detection 7 days post-challenge revealed significantly reduced viral replication in the head kidney liver, and spleen tissues of vaccinated fish compared to the PBS group. Above all, a clear dose-dependent relationship was also observed in the immunoassay results. Consequently, immunological studies demonstrate that the fusion protein t051L-tMCP exhibits excellent immunogenicity. Furthermore, t051L-tMCP significantly enhances the immune protective capacity compared to tMCP and t051L. Our study provides an efficient and viable recombinant fusion truncated protein candidate vaccine for the prevention of infectious spleen and kidney necrosis virus disease. •Bioinformatics analysis identified rich antigenic epitope regions in MCP and 051L of ISKNV.•The truncated MCP was linked to the truncated 051L via a rigid peptide (EAAAK), forming the fusion protein t051L-tMCP.•The t051L-tMCP subunit vaccine demonstrated superior protective efficacy compared to tMCP or t051L vaccines alone.•The protective efficacy of the three subunit vaccines exhibited a dose-dependent relationship with protein concentration.

Asian seabass ( Lates calcarifer ) is an economically important fish species in Southeast Asia. Iridoviral disease and streptococcosis are two of the major diseases in farmed Asian seabass. Here, we studied the efficacy of PISCIVAC™ Irido Si, a commercially available inactivated vaccine containing red seabream iridovirus (RSIV genotype II) and Streptococcus iniae antigens, against infectious spleen and kidney necrosis virus (ISKNV) genotype I and S. iniae infections in Asian seabass. In laboratory settings, the challenge of vaccinated fish with Vietnamese ISKNV genotype I strain showed 70–83% relative percent survival (RPS) at 21 days post-vaccination (dpv) and S. iniae challenge showed 75% or higher RPS at 7, 14, and 32 dpv. Also, the vaccine efficacy was confirmed at early vaccination stages (3 and 7 dpv) with an RPS of 85–97% against both pathogens and lasted for at least 74 and 348 dpv against ISKNV genotype I and S. iniae challenges, respectively. Furthermore, the vaccine was highly effective against challenges of ISKNV genotype I and S. iniae strains isolated in Thailand and Indonesia. These results suggest that the PISCIVAC™ Irido Si is useful for the prevention of iridoviral disease and streptococcosis in aquaculture of Asian seabass in ASEAN countries.

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.

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch’s postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

Vaccination is an effective method to prevent viral diseases. However, the biological barrier prevents the immersion vaccine from achieving the best effect without adding adjuvants and carriers. Researches on the targeted presentation technology of vaccines with nanocarriers are helpful to develop immersion vaccines for fish that can break through biological barriers and play an effective role in fish defense. In our study, functionally modified single-walled carbon nanotubes (SWCNTs) were used as carriers to construct a targeted immersion vaccine (SWCNTs-M−MCP) with mannose modified major capsid protein (MCP) to target antigen-presenting cells (APCs), against iridovirus diseases. After bath immunization, our results showed that SWCNTs-M−MCP induced the presentation process and uptake of APCs, triggering a powerful immune response. Moreover, the highest relative percent survival (RPS) was 81.3% in SWCNTs-M−MCP group, which was only 41.5% in SWCNTs-MCP group. Altogether, this study indicates that the SWCNTs-based targeted immersion vaccine induces strong immune response and provided an effective protection against iridovirus diseases.

The ornamental fish trade plays a critical role in global aquaculture, with endemic species like the Canara pearlspot ( Etroplus canarensis ) contributing to the industry’s economic and ecological significance. This study reports the first documented case of co-infection of infectious spleen and kidney necrosis virus (ISKNV) with Aeromonas hydrophila and Aeromonas dhakensis in Canara pearlspot from the Western Ghats, India. Clinical signs observed in the infected fish included skin darkening, hemorrhages, fin rot, splenomegaly, and nephromegaly. Histopathological analysis revealed necrosis and vacuolar degeneration in the liver, spleen, and brain tissues. Molecular diagnostics confirmed ISKNV and bacterial infections, with ISKNV showing 100% genetic similarity to isolates from ornamental fish in Australia, Ghana, Japan, and India. Phylogenetic analyses further classified A. hydrophila and A. dhakensis as highly virulent and moderately virulent, respectively, with antibiotic resistance profiles revealing multi-drug resistance (MDR) in both bacterial isolates. In vivo challenge studies demonstrated significant mortality due to ISKNV, while the lethal dose (LD 50 ) for A. hydrophila and A. dhakensis was calculated to be 7.1 × 10 5 CFU/mL and 2.37 × 10⁷ CFU/mL, respectively. These findings highlight the vulnerability of native endemic species to emerging pathogens, emphasizing the urgent need for enhanced biosecurity measures and targeted interventions to mitigate the risks associated with co-infections in aquaculture and ornamental fish systems.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Largemouth bass (Micropterus salmoides) is an important commercial fish farmed in China. Challenges related to diseases caused by pathogens, such as iridovirus, have become increasingly serious. In 2017, we detected iridovirus-infected diseased largemouth bass in Zunyi, Guizhou Province. The isolated virus was identified as an infectious spleen and kidney necrosis virus (ISKNV)-like virus (ISKNV-ZY). ISKNV-ZY induces a cytopathic effect after infecting mandarin fish brain (MFB) cells. Abundant hexagonal virus particles were observed in the cytoplasm of ISKNV-ZY-infected MFB cells, using electron microscopy. The whole genome of ISKNV-ZY contained 112,248 bp and 122 open reading frames. Phylogenetic tree analysis showed that ISKNV-ZY was most closely related to BCIV, indicating that it is an ISKNV-like megalocytivirus. ISKNV-ZY-infected largemouth bass started to die on day six and reached a death peak on days 7–8. Cumulative mortality reached 100% on day 10. Using RNA sequencing-based transcriptome analysis after ISKNV-ZY infection, 6254 differentially expressed unigenes (DEGs) were identified, of which 3518 were upregulated and 2673 downregulated. The DEGs were associated with endocytosis, thermogenesis, oxidative phosphorylation, the JAK-STAT signaling pathway, the MAPK signaling pathway, etc. These results contribute to understanding the molecular regulation mechanism of ISKNV infection and provide a basis for ISKNV prevention.

Infectious spleen and kidney necrosis virus (ISKNV) is an emerging viral pathogen with an expanding host range, posing a significant threat to economically important fish species. In this study, we isolated the ISKNV strain responsible for disease outbreaks in Asian seabass (Lates calcarifer) and analyzed the transcriptomic profile of spleen tissues from experimentally infected fish. The phylogenetic analysis confirmed that the virus belongs to clade I of ISKNV. Next-generation sequencing identified differentially expressed genes, providing a comprehensive overview of the transcriptional landscape in the spleen of ISKNV-infected fish. The pathway analysis revealed complex host–virus interactions, impacting immune regulation, endocytosis, cell communication, cell cycle arrest, and programmed cell death. To further investigate these interactions, we analyzed relevant pathways in the Reactome database for Asian seabass, humans, and zebrafish, constructed a protein–protein interaction (PPI) network using STRING database, and identified hub genes using six different algorithms. This analysis revealed 69 key genes, including 41 hub genes and 28 key genes that connect different pathways or clusters within the PPI network. These findings provide new insights into the molecular mechanisms driving ISKNV infection in Asian seabass. Future research should focus on elucidating the regulatory functions of these key genes and their roles in ISKNV pathogenesis.

Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981–66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.