Infectious Spleen and Kidney Necrosis Virus

Overview and Taxonomy of Infectious Spleen and Kidney Necrosis Virus

Infectious spleen and kidney necrosis virus (ISKNV) stands as the type species of the genus Megalocytivirus within the family Iridoviridae, a lineage of large, double-stranded DNA (dsDNA) viruses that has emerged as one of the most significant pathogens threatening global finfish aquaculture and ornamental fish trade [1, 8, 12]. First documented in the early 1990s during epizootics in farmed mandarin fish (Siniperca chuatsi) in China, ISKNV has since been recognized as the aetiological agent of infectious spleen and kidney necrosis disease (ISKNVD), a systemic, highly lethal condition that has spread across Asia, Africa, the Americas, and Oceania, causing catastrophic economic losses routinely exceeding 60–90% mortality in affected populations [9, 13, 19, 21]. The virus is classified under the Iridoviridae family, which encompasses five genera, Ranavirus, Megalocytivirus, Lymphocystivirus, Iridovirus, and Chloriridovirus, with Megalocytivirus being the only genus whose members exclusively infect teleost fish [1, 12]. ISKNV particles are icosahedral, enveloped or non-enveloped depending on the maturation stage, approximately 150–160 nm in diameter, and possess a linear dsDNA genome of roughly 111 kb encoding at least 122 predicted open reading frames (ORFs) [12, 25, 26]. The major capsid protein (MCP), encoded by ORF006, is the most abundant structural component and serves as the principal target for molecular diagnostics, phylogenetic classification, and vaccine development due to its high conservation across megalocytiviruses [2, 3, 7, 22, 28].

At the taxonomic level, ISKNV belongs to the species Megalocytivirus pagrus1 according to the International Committee on Taxonomy of Viruses (ICTV), a species that also includes red seabream iridovirus (RSIV) and turbot reddish body iridovirus (TRBIV) as distinct genogroups or genotypes [5, 12, 18]. The species Megalocytivirus pagrus1 thus comprises three major genotypes: ISKNV (genotype I, often referred to as ISKNV-I), RSIV (genotype II), and TRBIV (genotype III) [12, 18]. Within the ISKNV genotype itself, further subdivision into Clade 1 and Clade 2 has been proposed based on single-nucleotide polymorphisms (SNPs) and phylogenetic analysis of the MCP and ATPase genes [4, 12, 26]. Clade 1 isolates encompass the vast majority of strains recovered from freshwater and marine hosts across Asia, Africa, and the Americas, including those from mandarin fish, tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer), gourami species (Trichopodus spp., Trichogaster spp.), grouper (Epinephelus spp.), and ornamental cyprinids [4, 12, 15, 23, 25, 26, 31]. Clade 2 (ISKNV-II) has been reported less frequently, primarily from marine fish such as Asian seabass in southern China and red seabream, and exhibits 20–21 same-sense nucleotide differences relative to Clade 1, with no evidence of amino acid changes in MCP [12, 26]. Remarkably, whole-genome comparisons of 14 Clade 1 ISKNV genomes from diverse hosts and geographic origins revealed >98.81% nucleotide identity and 100% amino acid identity for 92–105 of 122 predicted genes, underscoring the extraordinary genetic stability of this virus even across host species barriers and temporal scales [12]. This high conservation suggests a recent common ancestor and strong purifying selection, yet even low mutation rates (e.g., 20 SNPs accumulated over 3.5 years in Lake Volta, Ghana) are sufficient for genomic epidemiology to trace transmission pathways [13, 14].

The host range of ISKNV is remarkably broad and continues to expand with intensified surveillance. While initial reports focused on mandarin fish and red seabream, ISKNV has now been detected in over 50 species of freshwater, brackish, and marine fish spanning at least 20 families, including Cichlidae (tilapia, peacock bass), Osphronemidae (gourami), Latidae (barramundi, Asian seabass), Serranidae (grouper), Cyprinidae (zebrafish, rainbow shark), and Poeciliidae (mollies) [8, 11, 16, 23, 30]. Natural infections have been documented in both food-fish aquaculture and the ornamental fish trade, with subclinical carriers posing a particularly insidious risk for transboundary spread [6, 23, 32, 35]. For example, apparently healthy dwarf gourami (Trichogaster lalius) and angelfish (Pterophyllum scalare) imported into India and Malaysia were found to harbor ISKNV without overt clinical signs, yet the virus retained full infectivity and virulence upon passage to naïve hosts [6, 24, 35]. Experimental transmission studies have further demonstrated that ISKNV can move between freshwater ornamental fish and marine finfish (e.g., from dwarf gourami to silver sweep Scorpis lineolata), confirming a feasible pathway for pathogen exchange across aquatic environments [36]. The World Organisation for Animal Health (WOAH) has accordingly listed “red seabream iridoviral disease” (RSIVD) as a notifiable disease, recognizing that ISKNV and RSIV strains both cause identical clinical and pathological presentations and are not reliably distinguished without molecular characterization [5, 16, 33].

Epidemiologically, ISKNV has transitioned from an enzootic pathogen in East and Southeast Asia to a panzootic threat. The virus was first reported outside Asia in Australia in 2003 in ornamental fish, and subsequently caused massive epizootics in farmed tilapia in Lake Volta, Ghana, in 2018, with mortality rates of 60–90% and losses exceeding 10 metric tons of fish per day [13, 19, 32]. In the Americas, ISKNV was first confirmed in Nile tilapia in Brazil in 2020, and later in native South American cichlids such as peacock bass (Cichla ocellaris) and pearl cichlid (Geophagus brasiliensis), indicating spillover into wild populations [11, 21]. The virus’s ability to persist as a subclinical infection in multiple ornamental species, combined with its environmental stability, remaining infectious for at least 48 hours in aquarium water at 25°C and surviving freezing at –20°C for seven days in fish fillets, highlights the multiple routes of introduction via international trade of live fish, frozen seafood products, and possibly contaminated water [5, 33]. Indeed, a challenge model using albino rainbow shark demonstrated that the median infectious dose (ID₅₀) of ISKNV is only 42 genome equivalents, and frozen skin-on fillets remained infectious via both intraperitoneal injection and immersion [5]. This evidence has profound implications for import risk assessments and biosecurity protocols, as the virus can be carried undetected in commodity seafood.

Taxonomically, the Megalocytivirus genus is distinguished from other iridoviruses by its unique clinical presentation, characterized by splenomegaly, renomegaly, and the presence of pathognomonic megalocytes (hypertrophied, basophilic cells with intracytoplasmic inclusion bodies) in hematopoietic tissues, and by phylogenetic clustering of the MCP and ATPase genes [4, 26, 27]. The high degree of genomic homogeneity among ISKNV clade 1 isolates, even across continents and host species, suggests that a single introduction event can rapidly become established in a new region, as witnessed in Ghana and Brazil [12, 19, 21]. However, co-infection with different genotypes (e.g., RSIV and ISKNV) has been documented in the same fish and farm, indicating that multiple lineages can circulate sympatrically [12]. The development of robust diagnostic tools, including TaqMan qPCR assays targeting the ISKNV104R gene, droplet digital PCR with a limit of detection of 1.5 copies/μL, recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a or lateral flow dipsticks, and whole-genome sequencing via tiled-PCR, now enables sensitive and specific detection of all three genotypes [7, 10, 18, 28, 29]. These advances are critical for genomic surveillance, outbreak investigation, and the eventual deployment of control measures such as gene-deleted live attenuated immersion vaccines that have shown >95% relative percent survival in mandarin fish [9, 16, 17, 20, 34].

In summary, the taxonomy of ISKNV is well resolved within the Megalocytivirus genus, yet its dynamic host range and geographic expansion underscore the need for continued phylogenetic and epidemiological monitoring. The virus’s genetic stability and low mutation rate paradoxically facilitate both vaccine development and the risk of rapid transboundary spread, as genomic epidemiology can track strains but also reveals that a single lineage can dominate across vast distances. The WOAH listing and the accumulation of field and experimental evidence firmly establish ISKNV as a pathogen of paramount concern for global aquaculture security.

Molecular Pathogenesis and Host-Virus Transcriptomic Interactions

Infectious Spleen and Kidney Necrosis Virus (ISKNV), the type species of the genus Megalocytivirus within the family Iridoviridae, represents a paradigm of viral subversion of host cellular machinery. As a WOAH-notifiable pathogen [5, 16] with a rapidly expanding host range encompassing over 50 species of freshwater and marine fish [1, 8], ISKNV orchestrates a complex and multi-layered pathogenesis that is fundamentally rooted in the reprogramming of host metabolic pathways, the manipulation of critical signaling cascades, and the induction of specific programmed cell death modalities to create a permissive environment for virion production. The virus’s large double-stranded DNA genome (≈111 kb) encodes a repertoire of proteins that directly interface with host factors, effectively commandeering the cellular infrastructure from the point of entry through to egress. Recent transcriptomic and proteomic investigations have elucidated a sophisticated network of host-virus interactions, revealing that ISKNV pathogenesis is not a passive lytic event but an active, highly regulated process driven by specific viral effectors.

Viral Subversion of Host Signaling and Stress Pathways

A hallmark of ISKNV molecular pathogenesis is its capacity to manipulate the cellular response to environmental stress, most notably the hypoxia-inducible factor (HIF) pathway. Hypoxia is a frequent occurrence in intensive aquaculture systems, and ISKNV has evolved to exploit this environmental stressor to its advantage. Mechanistic studies have demonstrated that ISKNV infection actively activates the HIF pathway [8]. The virus encodes a key protein, VP077R, which acts as a master manipulator of this pathway. VP077R interacts directly with the Von Hippel-Lindau (VHL) protein at the HIF-binding region, competitively inhibiting the interaction of HIF-1α with VHL. This action prevents the normal ubiquitination and proteasomal degradation of HIF-1α under normoxic conditions, leading to its stabilization and accumulation [8]. Furthermore, VP077R interacts with factor-inhibiting HIF (FIH), recruiting FIH and S-phase kinase-associated protein 1 (Skp1) to form an FIH–VP077R–Skp1 complex, which promotes FIH degradation via ubiquitination [8]. By simultaneously disabling both "brakes" on the HIF pathway (VHL and FIH), ISKNV ensures constitutive activation of HIF signaling. This activation is not merely a byproduct of infection; it is essential for viral replication. The ISKNV genome contains 15 viral hypoxia response elements (HREs), and the promoter of the orf077r gene itself is highly responsive to HIF [44]. This creates a potent positive feedback loop: infection activates HIF, which then drives the expression of viral genes, including VP077R, which further stabilizes HIF [8, 44]. This mechanism directly links environmental hypoxia to the outbreak of ISKNV disease, explaining the correlation between low dissolved oxygen in aquaculture ponds and severe epizootics.

Concurrently, ISKNV profoundly alters the host cell's redox balance. Infection triggers a robust oxidative stress response characterized by a significant elevation of reactive oxygen species (ROS), increasing up to 60-80% in infected cells from day 3 to day 5 post-infection [46]. This oxidative burst activates the Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated stress response, leading to the upregulation of antioxidant enzymes like catalase, SOD1, and SOD2 [46]. This Nrf2-mediated response, while a host defense mechanism, appears to be co-opted by the virus to regulate downstream cell death pathways without causing early catastrophic cell lysis. The interplay between ROS, Nrf2 signaling, and mitochondrial function is critical, as antioxidants like glutathione (GSH) and N-acetylcysteine (NAC) can block ISKNV-induced apoptosis and significantly reduce viral replication [46].

Metabolic Reprogramming: Fueling Viral Replication

A central tenet of ISKNV pathogenesis is the extensive metabolic reprogramming of the host cell to satisfy the enormous bioenergetic and biosynthetic demands of viral replication. ISKNV induces a classical "Warburg effect," shifting cellular metabolism toward aerobic glycolysis even in the presence of oxygen. Transcriptomic, proteomic, and metabolomic analyses have revealed that ISKNV infection enhances the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle at early stages, while strongly upregulating aerobic glycolysis at late stages of the replication cycle [49]. The PPP is critical for generating ribose-5-phosphate for nucleotide biosynthesis, a necessity for dsDNA genome replication. The reliance on glycolysis is absolute; inhibition of aerobic glycolysis severely impairs ISKNV multiplication [49].

Beyond glucose, glutamine metabolism is another critical node hijacked by ISKNV. Glutamine serves as a key carbon and nitrogen source, and its metabolism is essential for virus replication [39]. The virus orchestrates this through its encoded proteins, ORF093R and ORF102R. These proteins interact with the host proto-oncogene c-Myc, a master transcriptional regulator of metabolism. By binding c-Myc, ORF093R and ORF102R upregulate the expression of glutaminase 1 (GLS1), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (IDH2), all key enzymes in the glutaminolysis pathway [39]. This leads to increased glutamine uptake and metabolism, fueling the TCA cycle to provide energy and precursors for macromolecular synthesis.

Furthermore, ISKNV demonstrates a critical dependency on asparagine. Infection upregulates key enzymes of the malate-aspartate shuttle (MAS) pathway, including GOT1/2 and MDH1/2, as well as asparagine synthetase (ASNS) [41]. Inhibition of the MAS pathway or ASNS knockdown significantly reduces viral production. Strikingly, in the absence of glutamine, ISKNV replication can be fully restored by supplementing asparagine alone, indicating that the virus can bypass its reliance on glutamine if asparagine is available [41]. This positions asparagine as a critical, limiting metabolite for ISKNV protein synthesis and a potential target for therapeutic intervention.

Induction of Programmed Cell Death: Ferroptosis and Apoptosis

ISKNV has a complex relationship with host cell death pathways, inducing specific forms of programmed cell death at different stages of infection to facilitate its lifecycle. A landmark discovery is the demonstration that ISKNV triggers ferroptosis, a novel form of iron-dependent cell death, to enhance its replication [38]. ISKNV-infected CPB cells exhibit hallmark ferroptotic features, including mitochondrial shrinkage, increased membrane density, and reduced cristae. Biochemical assays confirm significant elevations in malondialdehyde (MDA, 1.7-fold), ROS (3.14-fold), and ferrous iron (Fe²⁺, 1.42-fold) [38]. Mechanistically, ISKNV downregulates glutathione peroxidase 4 (GPx4), the key enzyme that protects against lipid peroxidation, while upregulating acyl-CoA synthetase long-chain family member 4 (ACSL4), which enriches cellular membranes with oxidizable polyunsaturated fatty acids [38]. This shift creates a pro-ferroptotic environment. Critically, pharmacological induction of ferroptosis with erastin enhances viral replication, while inhibition with liproxstatin-1 suppresses viral yield, proving that ISKNV exploits ferroptosis as a mechanism to promote its progeny release [38].

ISKNV also manipulates the intrinsic, mitochondrial-mediated apoptotic pathway, but does so in a tightly regulated, dynamic manner. Infection leads to a loss of mitochondrial membrane potential (MMP) by up to 78% by day 5 post-infection [43]. This is driven by an imbalance in Bcl-2 family proteins: pro-apoptotic members Bax and Bak are significantly upregulated, while anti-apoptotic members Bcl-2 and Bcl-xL also increase but are unable to fully counteract the death signal [43]. The virus appears to fine-tune this balance to maintain mitochondrial function for ATP production during the early replication phase while eventually tipping the scales toward cell death via activation of caspase-9 and caspase-3 [43, 46]. The Nrf2-mediated oxidative stress response is intimately linked to this process, as ROS signals regulate the Bax/Bak-mediated death signaling, and antioxidant treatment blocks both the apoptotic cascade and viral replication [46]. This suggests that ISKNV uses the apoptotic pathway not for immediate destruction, but as a controlled exit strategy timed to optimize viral dissemination.

Host Transcriptomic Landscape and Innate Immune Evasion

Transcriptomic analyses of spleen tissues from ISKNV-infected Asian seabass provide a comprehensive view of the host's transcriptional response, revealing a complex battle between the host's antiviral defenses and the virus's immune evasion strategies. Over 69 key genes are implicated, including 41 hub genes within protein-protein interaction networks, spanning pathways related to immune regulation, endocytosis, cell cycle arrest, and programmed cell death [37]. The innate immune system is a primary battleground. ISKNV infection triggers the upregulation of stimulator of interferon genes (STING), a central component of the cytosolic DNA sensing pathway. The mandarin fish STING (scSTING) is localized in the endoplasmic reticulum and its overexpression increases expression of type I interferons (e.g., scIFN-h), interferon-stimulated genes (ISGs) like Mx, ISG15, PKR, and Viperin, and pro-inflammatory cytokines [47]. However, the virus has evolved countermeasures. The PI3K/AKT/p53 pathway, which is activated during infection, plays a dual role; it inhibits autophagy and promotes a more antiviral state, but its activation is not sufficient to fully clear the virus [45].

The virus also directly manipulates cell entry and signaling. ISKNV induces rapid activation of the Epidermal Growth Factor Receptor (EGFR), exploiting this receptor's signaling for entry. This activation triggers the downstream PI3K-Akt signaling pathway, which facilitates virus invasion by reorganizing the actin cytoskeleton [42, 48]. The virus's reliance on EGFR-mediated endocytosis is so profound that the EGFR inhibitor gefitinib can block ISKNV entry and infection both in vitro and in vivo [48]. The host's Wnt/β-catenin pathway is also subverted; mandarin fish tankyrase 1-like protein (TNKS1-like) is upregulated during infection, which in turn activates the Wnt/β-catenin pathway, a signal that is proviral and enhances ISKNV replication [40]. This multi-pronged attack on host signaling, activating proviral pathways (HIF, EGFR/PI3K/Akt, Wnt/β-catenin, c-Myc) while attempting to override or delay antiviral interferon responses, defines the molecular pathogenesis of ISKNV, turning the host cell into a dedicated viral factory before its controlled demolition via ferroptosis and apoptosis.

Epidemiology, Host Range, and Global Distribution

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus within the family Iridoviridae, represents one of the most significant emerging viral threats to global aquaculture and ornamental fish industries. The virus is classified as a notifiable pathogen by the World Organisation for Animal Health (WOAH), underscoring its transboundary importance and the critical need for robust epidemiological surveillance [1, 12, 18]. The epidemiology of ISKNV is characterized by a remarkably broad and expanding host range, a wide but increasingly interconnected global distribution, complex transmission dynamics involving both clinical and subclinical infections, and significant interactions with environmental stressors and co-infecting pathogens.

Host Range: An Expanding Pantheon of Susceptible Species

The host range of ISKNV is exceptionally broad, encompassing over 50 species of freshwater and marine fish, a figure that continues to grow with each new epidemiological investigation [8, 16]. This extensive host range is a defining feature of the virus and a primary driver of its global dissemination. The virus was first recognized as a significant pathogen in mandarin fish (Siniperca chuatsi) and red seabream (Pagrus major) in East Asia, but subsequent surveillance has revealed its capacity to infect a taxonomically diverse array of hosts across multiple continents.

Economically Important Food Fish Species: ISKNV causes devastating losses in several key aquaculture species. In tilapia (Oreochromis niloticus), ISKNV has emerged as a major pathogen, with outbreaks in Lake Volta, Ghana, in 2018 resulting in mortality rates between 60% and 90% and daily losses exceeding 10 tonnes of fish [13, 19]. This event marked the first detection of ISKNV in Africa and highlighted the virus's potential to cause catastrophic economic damage in new geographic regions. The virus has since been reported in tilapia in Brazil, Thailand, and other major producing nations [15, 21, 50]. Asian seabass (Lates calcarifer), also known as barramundi, is highly susceptible, with experimental challenges demonstrating dose-dependent mortality up to 77% [31]. In China, ISKNV genotype II (ISKNV-II) has been responsible for mass mortality events in farmed Asian seabass, with cumulative mortality reaching 85.89% in natural outbreaks [26]. Mandarin fish remain a primary host, with the virus causing significant economic losses to the Chinese aquaculture industry [9, 16]. Other economically important species affected include largemouth bass (Micropterus salmoides), pearl gentian grouper (Epinephelus fuscoguttatus × E. lanceolatus), and giant gourami (Osphronemus goramy) [3, 25, 52].

Ornamental Fish Species: A Reservoir for Global Dissemination: The ornamental fish trade serves as a critical pathway for the global spread of ISKNV. Numerous species, often carrying subclinical infections, act as reservoirs for the virus. The dwarf gourami (Trichogaster lalius) is a particularly well-documented carrier, and ISKNV has been detected in this species in India, Malaysia, and other regions [6, 24]. Other ornamental species confirmed as hosts include angelfish (Pterophyllum scalare), snakeskin gourami (Trichopodus pectoralis), three spot gourami (Trichopodus trichopterus), molly (Poecilia sphenops), and various cyprinids [4, 6, 23, 35]. A study in India detected ISKNV in 11.42% of ornamental fish samples (16 out of 140) representing 10 different species, with three of these species being reported as positive for the first time globally [23]. The presence of subclinical infections in these species is of paramount epidemiological significance, as apparently healthy fish can introduce the virus into new environments and naïve populations, bypassing standard quarantine measures [6, 32].

Wild Fish Populations and Native Species: The spillover of ISKNV from farmed or ornamental fish into wild populations represents a significant conservation concern. A notable example is the mass mortality of wild pearlspot (Etroplus suratensis) in Peechi Dam, located within the Western Ghats biodiversity hotspot in India, where ISKNV was confirmed as the aetiological agent [30]. This event underscores the potential for the virus to impact wild fish biodiversity. Furthermore, ISKNV has been detected in native South American cichlids, including peacock bass (Cichla ocellaris) and pearl cichlids (Geophagus brasiliensis) in Brazil, indicating that the virus is capable of establishing itself in endemic fish populations [11]. The experimental transmission of ISKNV from freshwater ornamental fish to the Australian marine species silver sweep (Scorpis lineolata) demonstrates a feasible pathway for the virus to bridge freshwater and marine environments, posing a threat to unique island ecosystems [36].

Global Distribution and Genomic Epidemiology

Initially considered an Asian pathogen, ISKNV has undergone a dramatic expansion in its known geographic range over the past two decades. The virus is now confirmed to be present in at least 15 countries across Asia, Africa, the Americas, and Oceania. The first detection in Africa occurred in 2018 in Ghana, where it caused a severe epidemic in farmed tilapia [19]. This was followed by the first report in South America in Brazil in 2020, where ISKNV was associated with a fatal outbreak in Nile tilapia [21]. Subsequent studies have confirmed its presence in multiple Brazilian states, affecting both farmed and native species [11]. In Asia, the virus is endemic in many countries, including China, Thailand, Indonesia, India, Malaysia, and Japan [12, 15, 23, 24, 26, 31]. Australia, which is considered free of megalocytiviruses, maintains strict import controls due to the high risk of introduction via the ornamental fish trade [32, 33, 36].

Genomic Epidemiology and Phylogenetic Structure: The species Infectious spleen and kidney necrosis virus is classified into three primary genotypes: ISKNV, red seabream iridovirus (RSIV), and turbot reddish body iridovirus (TRBIV) [12, 18]. However, genomic analyses have revealed a more nuanced structure, with ISKNV isolates further divided into clades. Whole-genome sequencing of 16 isolates from Southeast Asia using a hybridization capture enrichment method demonstrated that 14 of these belonged to ISKNV Clade 1, exhibiting greater than 98.81% nucleotide similarity [12]. This high degree of genetic conservation is typical of large dsDNA viruses but does not preclude the accumulation of epidemiologically informative single nucleotide polymorphisms (SNPs). A tiled-PCR sequencing approach applied to ISKNV outbreaks in Lake Volta, Ghana, identified 20 SNPs over a four-year sampling period (2018–2022), allowing for fine-scale tracking of viral evolution and transmission dynamics [13]. Furthermore, water samples from cages containing infected tilapia were successfully used for genomic surveillance, demonstrating that non-invasive sampling can be employed to track predominant viral variants in an outbreak, a technique with significant potential for real-time epidemiological monitoring [14].

The phylogenetic analyses consistently show that ISKNV isolates from diverse geographic locations and host species are closely related. For instance, ISKNV strains from gourami in Thailand were found to be closely related to strains from other fish hosts, suggesting frequent cross-species transmission events [4]. Similarly, the MCP gene sequences from Indian ornamental fish isolates showed 98.76% to 100% identity with reference strains from Japan, Australia, and Malaysia [23]. This genetic homogeneity across vast distances and host ranges points to the movement of infected fish, particularly through the ornamental trade, as the primary driver of global dissemination rather than independent viral evolution in isolated ecosystems.

Transmission Dynamics and Risk Factors

ISKNV transmission occurs through multiple routes, including horizontal transmission via waterborne exposure, direct contact, and ingestion of infected tissues. The virus can be shed in feces, urine, and skin mucus, contaminating the aquatic environment. Experimental evidence demonstrates that ISKNV can remain infectious in water without a fish host for at least 48 hours at 25°C, facilitating rapid spread within and between aquaculture facilities [33].

The Role of Frozen Fish Products: A critical and previously underappreciated transmission pathway is the trade in frozen fish products. A landmark study using an albino rainbow shark challenge model demonstrated that ISKNV remains infectious after storage at -20°C for seven days [5]. Inocula prepared from skin-on fillets, head-on eviscerated products, and peritoneal viscera all caused infection and disease in recipient fish via both intraperitoneal injection and bath immersion. The median infectious dose (ID50) was estimated to be just 42 ISKNV genome equivalents (95% CI: 19–98), highlighting the extreme infectivity of the virus [5]. This finding has profound implications for international trade and import risk assessments, as frozen seafood products are a major globally traded commodity.

Environmental and Host Factors Influencing Outbreaks: Several environmental and host factors modulate the epidemiology of ISKNV. Water temperature is a critical determinant of disease outcome. Experimental infections in juvenile Nile tilapia demonstrated that maintaining fish at temperatures above 30°C (32°C and 34°C) resulted in no mortality and a significant reduction in viral load, while fish kept at 26°C to 30°C developed severe disease and died [50]. This suggests that non-lethal hyperthermia could be used as a management strategy. Conversely, hypoxia, a common stressor in intensive aquaculture, directly triggers ISKNV outbreaks. The virus possesses hypoxia response elements (HREs) in its genome, and hypoxic conditions activate the hypoxia-inducible factor (HIF) pathway, which in turn upregulates viral gene expression and replication [8, 44]. This creates a positive feedback loop where environmental hypoxia promotes viral replication, leading to disease outbreaks.

Co-infections and Synergistic Pathogenesis: ISKNV frequently participates in polymicrobial infections, which often result in more severe disease than single-pathogen infections. Co-infection with the bacterium Aeromonas hydrophila is common in mandarin fish and has been shown to have a synergistic lethal effect, with mixed infections resulting in higher mortality than either pathogen alone [53]. Similarly, co-infections with Streptococcus agalactiae have been documented in tilapia and grouper, and these bacterial pathogens are thought to exacerbate the pathology caused by ISKNV [19, 21]. In Asian seabass, concurrent infection with piscine intestinal coccidia and ISKNV was associated with mass mortality and severe gastrointestinal pathology, suggesting that parasitic infections can also potentiate viral disease [51]. Co-infections with other viruses, such as tilapia lake virus (TiLV) in Thailand and nervous necrosis virus (NNV) in grouper, are also common, further complicating diagnosis and control [15, 54, 55]. The presence of these co-infections necessitates integrated health management strategies that address multiple pathogens simultaneously.

Surveillance, Detection, and Biosecurity Implications

The epidemiology of ISKNV is characterized by a high prevalence of subclinical infections, particularly in ornamental fish species. This poses a significant challenge for surveillance and control, as infected fish may appear healthy and pass visual inspection at borders. A study evaluating the impact of sample pooling on surveillance sensitivity found that individual testing is necessary to achieve adequate confidence of freedom from infection at low prevalence (e.g., 2%), while pooled testing can be effective when prevalence exceeds 10% [32]. The development of highly sensitive molecular tools, such as droplet digital PCR (ddPCR) with a detection limit of 1.5 copies/μL, has improved the ability to detect low-level infections in carrier fish [29]. Multiplex qPCR assays that can simultaneously detect ISKNV and other common pathogens like largemouth bass virus (LMBV) or NNV are also being developed to streamline diagnostic workflows [10, 55]. The WOAH listing of ISKNV as a notifiable disease mandates reporting of outbreaks, but the effectiveness of this system is contingent upon the diagnostic capacity and reporting infrastructure of individual nations. The demonstrated ability of ISKNV to survive freezing and its presence in a wide range of asymptomatic ornamental and food fish species underscores the need for stringent biosecurity protocols, including effective disinfection measures (e.g., heating to 65°C, pH extremes, 1% Virkon™, 1000 ppm sodium hypochlorite) and robust import risk assessments [5, 33].

Clinical Signs and Pathological Manifestations in Susceptible Species

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus within the family Iridoviridae, is a highly pathogenic double-stranded DNA virus responsible for devastating systemic disease across an alarmingly broad and expanding host range. The virus has been documented to infect over 50 species of both freshwater and marine fish, representing a significant threat to global aquaculture and wild fish populations [1, 8, 16]. The clinical presentation and pathological hallmarks of ISKNV infection are remarkably consistent across susceptible species, though the severity and specific manifestations can vary based on host species, age, environmental conditions, and the presence of co-infections. The World Organisation for Animal Health (WOAH) recognizes ISKNV as a notifiable pathogen, underscoring its economic and ecological significance [5, 18].

Gross Clinical Signs in Susceptible Species

The clinical progression of ISKNV disease is typically acute to peracute, with mortality events often reaching 60% to over 90% in farmed populations [4, 13, 25]. The incubation period is generally short, with clinical signs appearing as early as three days post-infection under experimental conditions [50]. Affected fish exhibit a constellation of non-specific but characteristic behavioral and external signs. Lethargy and anorexia are among the earliest indicators, with diseased fish displaying slow, erratic swimming, congregating near the water surface or pond edges, and exhibiting a loss of equilibrium [19, 26, 56]. A pronounced darkening of the body surface (melanism) is a frequently reported external sign across numerous species, including Asian seabass (Lates calcarifer), grouper (Epinephelus spp.), and pearl gentian grouper hybrids [19, 26, 56]. In gourami species (Trichopodus spp.), clinical outbreaks are marked by skin hemorrhage and scale loss [4]. Abdominal distension due to ascites is another common finding, particularly noted in tilapia (Oreochromis niloticus) and barramundi during severe epizootics [19, 31]. Exophthalmia (pop-eye) and gill pallor or petechiae are also observed in some cases, reflecting the systemic nature of the infection [56]. In ornamental species like angelfish (Pterophyllum scalare), whirling behavior has been documented, indicating potential neurological involvement [6]. It is critical to note that subclinical infections are common, particularly in ornamental species such as dwarf gourami (Trichogaster lalius) and certain cyprinids, where apparently healthy fish can harbor and shed the virus, acting as silent vectors for transboundary spread [6, 12, 24, 35].

Gross Pathological Findings at Necropsy

Upon necropsy, the most striking and consistent gross lesions are observed in the target organs: the spleen and kidney. Splenomegaly (enlargement of the spleen) and renomegaly (enlargement of the kidney) are pathognomonic features of ISKNV infection, reported across a wide taxonomic range including mandarin fish (Siniperca chuatsi), Asian seabass, tilapia, grouper, and gourami [4, 6, 26, 56]. The affected organs are often friable, congested, and may appear mottled or discolored. The liver is also frequently involved, presenting with hepatomegaly, congestion, pallor, and a mottled or necrotic appearance [26, 27, 56]. In severe cases, the liver may be reduced in size and pale [27]. The gastrointestinal tract may be empty, and the visceral peritoneum can exhibit congestion or petechial hemorrhages. Ascitic fluid, when present, is typically clear or serosanguinous [19]. In pearl gentian grouper co-infected with Francisella sp., the absence of apparent white nodules in the spleen and kidney was noted, distinguishing the pathology from pure bacterial granulomatous diseases [56].

Histopathological Hallmarks: The Megalocytic Cell

The definitive histopathological lesion of ISKNV infection is the presence of abnormally enlarged, basophilic cells, termed megalocytes. These cells are the hallmark of megalocytiviral disease and are considered pathognomonic for ISKNV infection [4, 15, 21, 26]. Megalocytes are hypertrophic cells, often measuring 20–50 µm in diameter, with a large, pale nucleus and a cytoplasm that is distended and filled with abundant basophilic, finely granular material. This granular material represents viral inclusion bodies, specifically the viral assembly sites or "viral factories" where virion morphogenesis occurs. Transmission electron microscopy (TEM) confirms that these cells are packed with icosahedral virions approximately 140–160 nm in diameter, consistent with the Iridoviridae family [19, 21, 25, 26].

These megalocytic cells are distributed systemically but are most numerous in the hematopoietic tissues of the spleen and kidney, where they cause extensive necrosis and obliteration of normal tissue architecture [4, 26]. They are also consistently found in the liver, often within the sinusoids and parenchyma, displacing hepatocytes [26, 30]. Other organs where megalocytes are frequently observed include the heart, gills, gastrointestinal tract, pancreas, and brain, reflecting the pantropic nature of the virus [26, 51, 54]. In the spleen and kidney, the presence of megalocytes is accompanied by severe necrosis of hematopoietic elements, leading to the depletion of erythroid and lymphoid precursors. This hematopoietic necrosis is a primary driver of the anemia and immunosuppression observed in infected fish. Concurrently, an increase in melanomacrophage centers (MMCs) is often noted in the spleen and kidney, particularly in fish infected with attenuated or less virulent strains, suggesting an active but ultimately insufficient host scavenging response [20]. In tilapia, basophilic intracytoplasmic inclusion bodies within megalocytes are frequently associated with hemosiderin deposits in the anterior kidney and spleen, indicating a disruption of iron metabolism and erythrocyte turnover [15].

Organ-Specific and Cellular Pathological Manifestations

Spleen and Kidney: These are the primary target organs. Histological sections reveal a loss of normal lymphoid and hematopoietic architecture, replaced by sheets of megalocytes and necrotic cellular debris. The splenic ellipsoids and renal interstitium are heavily infiltrated. In severe cases, the parenchyma is almost entirely effaced by viral infection [4, 26].

Liver: Hepatic lesions range from mild congestion to severe multifocal necrosis. Megalocytes are observed within the sinusoids and hepatic cords. Hepatocytes may exhibit vacuolar degeneration, pyknosis, and karyorrhexis. In pearlspot (Etroplus suratensis), intracytoplasmic eosinophilic inclusions have been reported alongside the basophilic megalocytes, indicating a complex cytopathic effect [30].

Gastrointestinal Tract: In cases of co-infection, such as with piscine intestinal coccidia in juvenile barramundi, the gastrointestinal pathology is markedly intensified. This includes severe inflammation, epithelial desquamation, and necrosis of the lamina propria, with megalocytes present in the submucosa and muscularis [51]. This synergistic pathology suggests that ISKNV can exacerbate damage from other enteric pathogens.

Gills and Heart: Megalocytes can be found in the gill filaments and lamellae, contributing to respiratory distress. In the heart, they are observed in the epicardium and myocardium, potentially leading to cardiac dysfunction [26].

Cellular and Molecular Pathology: At the cellular level, ISKNV induces a complex interplay of cell death pathways. The virus triggers mitochondria-mediated apoptosis through an imbalance of pro-apoptotic (Bax/Bak) over anti-apoptotic (Bcl-2/Bcl-xL) proteins, leading to loss of mitochondrial membrane potential (ΔΨm) and activation of caspase-9 and caspase-3 [43]. Concurrently, ISKNV induces ferroptosis, an iron-dependent, non-apoptotic cell death pathway, characterized by mitochondrial shrinkage, increased membrane density, and elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS), and ferrous iron (Fe²⁺). This is mediated by the downregulation of glutathione peroxidase 4 (GPx4) and upregulation of acyl-CoA synthetase long-chain family member 4 (ACSL4) [38]. The virus also triggers an oxidative stress response via the Nrf2 pathway, which regulates the ROS-mediated Bax/Bak death signals [46]. These cell death mechanisms are not merely pathological bystanders; they are actively exploited by ISKNV to facilitate viral replication and dissemination [38].

Influence of Environmental Factors and Co-infections on Pathology

Temperature: Water temperature is a critical determinant of disease outcome. Experimental infections in Nile tilapia demonstrate that maintaining fish at temperatures above 30°C (specifically 32°C and 34°C) significantly reduces mortality and viral load, suggesting that hyperthermia can be a non-lethal therapeutic intervention [50]. Conversely, lower temperatures (22°C–28°C) favor viral replication and severe pathology. This temperature-dependency is linked to the virus's manipulation of the hypoxia-inducible factor (HIF) pathway. Hypoxia, common in warm, eutrophic aquaculture waters, triggers the HIF pathway, which in turn activates viral hypoxia response elements (HREs), dramatically increasing ISKNV replication and precipitating disease outbreaks [8, 44]. The viral protein VP077R directly interacts with and inhibits the host's HIF degradation machinery (VHL and FIH), creating a positive feedback loop that amplifies viral replication under hypoxic stress [8].

Co-infections: ISKNV frequently participates in polymicrobial infections, which dramatically alter the clinical and pathological picture. Co-infection with Aeromonas hydrophila in Chinese perch results in a synergistic lethal effect, with higher mortality and more severe clinical signs than either pathogen alone [2, 53]. Similarly, co-infection with Streptococcus agalactiae in tilapia and Francisella sp. in grouper exacerbates pathology and mortality [19, 56]. In barramundi, concurrent infection with piscine intestinal coccidia leads to severely intensified gastrointestinal lesions, including extensive epithelial desquamation and necrosis [51]. The presence of Tilapia lake virus (TiLV) and ectoparasites alongside ISKNV in red hybrid tilapia further complicates the clinical presentation, highlighting the need for comprehensive diagnostic approaches [15]. These interactions underscore that ISKNV pathology in a field setting is rarely a pure viral disease but rather a complex, multi-factorial syndrome.

Advanced Molecular Diagnostics: LAMP and Other Detection Methods

The rapid, sensitive, and specific detection of Infectious Spleen and Kidney Necrosis Virus (ISKNV) is paramount for effective disease surveillance, outbreak response, and trade compliance, particularly given its status as a notifiable pathogen under the World Organisation for Animal Health (WOAH) [1]. The expanding host range of ISKNV, now documented across more than 50 fish species including economically critical aquaculture species such as tilapia (Oreochromis niloticus), mandarin fish (Siniperca chuatsi), Asian seabass (Lates calcarifer), and various ornamental gouramis, demands diagnostic tools that can be deployed both in centralized laboratories and in resource-limited field settings [4, 6, 12, 19]. While conventional polymerase chain reaction (PCR) and nested PCR (nPCR) remain widely used for initial screening and confirmatory sequencing of the major capsid protein (MCP) gene [11, 15, 24, 35], the inherent limitations of thermocycling-dependent methods, namely their requirement for expensive instrumentation, skilled personnel, and extended turnaround times, have driven the development of isothermal amplification strategies, digital quantification platforms, and CRISPR-based systems. This section provides a comprehensive, mechanistic examination of advanced molecular diagnostics for ISKNV, focusing on loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) integrated with CRISPR/Cas or lateral flow readouts, multiplex quantitative PCR (qPCR), droplet digital PCR (ddPCR), and tiled-PCR approaches for whole-genome sequencing.

Loop-Mediated Isothermal Amplification (LAMP) and Duplex Detection Platforms

Isothermal amplification methods circumvent the need for thermal cycling by leveraging enzyme-driven strand displacement and self-priming mechanisms at a constant temperature, typically 60–65 °C. For ISKNV, LAMP has been refined to enable simultaneous detection of co-infecting pathogens, a critical capability given the frequent polymicrobial disease presentations observed in aquaculture. He et al. (2025) developed a duplex LAMP assay incorporating hydroxynaphthol blue (HNB) as a colorimetric indicator, targeting the conserved MCP gene of ISKNV and the hlyA gene of Aeromonas hydrophila, a bacterial pathogen frequently implicated in co-infections with ISKNV in Chinese perch [2]. The assay’s design relies on four to six primer sets recognizing distinct regions of each target, generating characteristic ladder-like banding patterns on agarose gels. The HNB dye undergoes a color transition from violet to sky blue upon positive amplification due to chelation of magnesium ions by pyrophosphate, enabling visual readout without specialized equipment. This duplex LAMP achieved a limit of detection (LOD) of 100 femtograms (fg) for each individual pathogen and 1 picogram (pg) for mixed templates, with no cross-reactivity against a panel of other fish viruses or bacteria [2]. The restriction enzyme digestion profiles of the LAMP products, yielding fragments of 136, 117, and 96 base pairs for ISKNV, provided additional confirmation, enhancing specificity. The significance of this approach lies in its capacity to diagnose co-infections in a single reaction, thereby informing treatment decisions (e.g., antibiotic versus antiviral therapy) and reducing diagnostic turnaround from hours to under 60 minutes. The isothermal nature (63 °C for 60 min) makes it compatible with simple heating blocks or water baths, facilitating deployment in rural aquaculture settings.

Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a Integration

RPA represents a paradigm shift in field-deployable nucleic acid detection, operating at a low and constant temperature (37–42 °C) through the concerted action of recombinase, single-stranded DNA binding proteins, and strand-displacing polymerase. Unlike LAMP, RPA requires only a single pair of primers and can amplify target sequences in under 30 minutes. Li et al. (2020) first demonstrated RPA for ISKNV by targeting the MCP and ORF007 genes, achieving an LOD of 10² copies/µL, ten-fold more sensitive than conventional PCR, and integrating lateral flow dipsticks (RPA-LFD) for amplicon visualization within 30 minutes at 38 °C [28]. The RPA-LFD format relies on dual-labeled probes (FITC and biotin) that bind to anti-FITC antibodies and streptavidin on the dipstick, producing a visible test line. This method was validated on 24 field samples from mandarin fish farms, showing 100% concordance with PCR [28], underscoring its utility for on-farm rapid screening.

Building upon RPA, the integration of CRISPR/Cas12a systems has dramatically enhanced both sensitivity and specificity. Lu et al. (2024) developed an RPA-CRISPR/Cas12a assay for simultaneous detection of ISKNV and mandarin fish ranavirus (MRV) [7]. The Cas12a ribonucleoprotein complex, guided by a CRISPR RNA (crRNA) complementary to the ISKNV MCP amplicon, exhibits collateral cleavage activity upon target recognition, degrading a fluorophore-quencher reporter to generate a fluorescent signal. The assay operates at 37–39 °C, ideal for field conditions, and can detect as low as 0.1 copy/µL of ISKNV DNA and 1 copy/µL of MRV, representing one of the most sensitive molecular diagnostics reported for any megalocytivirus [7]. The total workflow requires only 45 minutes (30 min RPA + 15 min Cas detection), with optional extension to 60 minutes for low-concentration samples. Moreover, the RPA system can perform reverse transcription, enabling detection of viral mRNA transcripts, which may indicate active replication. The use of trans-cleavage probes that do not require excitation light for visual readout further enhances field applicability. This combination of RPA’s rapid amplification with CRISPR/Cas12a’s programmable specificity and high signal-to-noise ratio positions this platform as a leading candidate for point-of-care ISKNV diagnostics in aquaculture, especially in regions like Southeast Asia and West Africa where ISKNV outbreaks have caused catastrophic losses [13, 19].

Digital Droplet PCR (ddPCR) and TaqMan Quantitative PCR (qPCR)

For absolute quantification of viral load with unprecedented sensitivity, droplet digital PCR (ddPCR) has emerged as a transformative technology. Lin et al. (2020) established an EvaGreen-based ddPCR assay for ISKNV targeting a conserved 79-bp fragment of the MCP gene [29]. The principle involves partitioning the PCR mixture into thousands of nanoliter-sized droplets, each undergoing individual endpoint amplification. Poisson statistics are then applied to count the number of positive droplets, yielding an absolute quantification without reliance on a standard curve. The assay exhibited an analytical LOD of 1.5 copies/µL, approximately 20-fold more sensitive than TaqMan qPCR (LOD 34 copies/µL) [29]. In a comparative study of 23 mandarin fish fry samples, ddPCR detected ISKNV in 65.22% (15/23) compared to only 30.43% (7/23) by qPCR, demonstrating its superiority for detecting low-level or subclinical infections [29]. This capability is critical for identifying carrier fish, a major concern for the ornamental fish trade, where subclinically infected individuals have been shown to harbor viral loads below the detection threshold of conventional qPCR [32]. ddPCR is also less susceptible to inhibitors commonly present in fish tissue homogenates and water samples, making it robust for environmental surveillance.

Complementary to ddPCR, a partially validated TaqMan qPCR assay, the ISKNV104R assay, was developed by Koda et al. (2023) to detect all three genotypes of ISKNV (ISKNV, RSIV, and TRBIV) [18]. Targeting a conserved region of a hypothetical protein, the assay demonstrated a mean efficiency of 99.97%, a correlation coefficient of 1.000, and an analytical LOD of ≤10 copies per reaction [18]. Diagnostic sensitivity and specificity were 91.99% and 89.80%, respectively, against a panel of 397 samples from 21 source populations. This assay is particularly relevant for WOAH reporting requirements, as it can detect the RSIV genotype responsible for notifiable red seabream iridoviral disease [18]. However, pooled sample testing, while cost-effective for surveillance, can reduce diagnostic sensitivity; Johnson et al. (2019) showed that pooling more than five fish per test dramatically decreased detection rates for low-prevalence infections, necessitating larger sample sizes to maintain surveillance sensitivity [32]. Thus, for high-consequence applications such as import certification, individual testing by ddPCR or highly sensitive TaqMan qPCR remains the gold standard.

Multiplex qPCR and Conventional PCR Variants

Simultaneous detection of ISKNV alongside other viral or bacterial pathogens is essential for accurate etiological diagnosis, as co-infections with TiLV, NNV, Francisella spp., and Streptococcus agalactiae are frequently documented [15, 51, 54, 56]. Cao et al. (2024) developed a multiplex qPCR that simultaneously amplifies ISKNV and largemouth bass virus (LMBV), another iridovirus, with LODs of 7 copies/µL and 4 copies/µL, respectively [10]. The assay demonstrated excellent linearity (R² > 0.99) and intra-/inter-assay coefficients of variation below 3%, and it was validated on 229 field samples, outperforming conventional PCR in sensitivity [10]. Similarly, Kumalasari et al. (2022) employed a multiplex PCR targeting the MCP gene of ISKNV and the CP gene of nervous necrosis virus (NNV) for simultaneous screening of marine fish, identifying co-infections in 18 grouper samples [55]. These multiplex formats reduce reagent costs and labor while providing a comprehensive disease profile, which is particularly valuable for outbreak investigations involving mass mortality events [27].

Conventional PCR and nested PCR (nPCR) remain essential for initial detection, especially in laboratories with limited real-time PCR capacity. The WOAH-recommended MCP-targeting primers amplify a 563-bp fragment and have been used to confirm ISKNV in numerous host species, including dwarf gourami, angelfish, and pearl cichlids [4, 6, 11]. Nested PCR offers enhanced sensitivity for low-copy-number samples, as demonstrated by Pereira et al. (2024) in the first detection of ISKNV in South American cichlids [11]. However, nested PCR is prone to carryover contamination, and its semi-quantitative nature limits its utility for viral load monitoring.

Tiled-PCR and Field-Based Whole-Genome Sequencing

Understanding viral evolution and transmission dynamics requires whole-genome sequences. The large dsDNA genome of ISKNV (>110 kbp) poses challenges for standard shotgun sequencing approaches, particularly from clinical specimens with low viral DNA yields. Alathari et al. (2023) pioneered a multiplexed, tiled-PCR method that amplifies overlapping amplicons covering the entire ISKNV genome using 55 primer pairs [13, 57]. Coupled with long-read sequencing (e.g., Oxford Nanopore), this approach enables real-time genomic surveillance directly from field samples. The tiled-PCR can recover 50% of the genome from as little as 275 femtograms of template (~2,410 viral copies per 5 µL reaction) [13]. Applied to outbreaks in Lake Volta, Ghana, over a four-year period, the method detected 20 single nucleotide polymorphisms (SNPs) despite the low mutation rate typical of dsDNA viruses, revealing subtle population-level changes [13].

Remarkably, Alathari et al. (2024) extended this approach to water samples, demonstrating that viral fractions concentrated from cage-side water yielded genome sequences matching those from infected fish [14]. This “lab in a suitcase” concept, combining tiled-PCR, portable MinION sequencers, and rapid DNA extraction, allows non-destructive sampling and real-time genomic epidemiology in resource-limited settings. Such capabilities are transformative for early warning systems and for tracking introduction pathways, such as the documented risk of ISKNV spread via frozen fish fillets [5]. The stability of ISKNV at −20 °C for at least seven days [5] and its resistance to drying [33] underscore the need for robust, field-validated genomic tools.

Comparative Sensitivity and Field Applicability

A synthesis of the detection limits reported across platforms highlights the trade-offs between sensitivity, throughput, and field-readiness. ddPCR offers the highest analytical sensitivity (1.5 copies/µL) but requires a partition-generating instrument [29]. RPA-CRISPR/Cas12a achieves comparable sensitivity (0.1 copies/µL) with isothermal amplification and no need for thermal cycling, although the CRISPR step adds reagent cost and complexity [7]. LAMP with HNB dye is slightly less sensitive (100 fg, equivalent to ~10²–10³ copies) but is the simplest to implement in a low-resource context [2]. TaqMan qPCR remains the workhorse for high-throughput laboratory diagnostics, with robust performance across diverse matrices [18]. For epidemiological studies requiring genome resolution, tiled-PCR coupled with portable sequencing is unrivaled, albeit with higher per-sample cost [13, 14]. The choice of method must thus be guided by the specific diagnostic objective: rapid on-site screening (LAMP or RPA-LFD), absolute quantification (ddPCR), multiplex surveillance (multiplex qPCR), or evolutionary tracking (tiled-PCR sequencing). Given WOAH’s emphasis on notification of ISKNV genotypes causing RSIVD, adoption of validated assays like ISKNV104R qPCR is recommended for border inspection and outbreak confirmation [18]. Meanwhile, the integration of CRISPR-based diagnostics into national surveillance programs offers a promising avenue for democratizing access to high-sensitivity testing, particularly in the Global South where ISKNV has emerged as a major threat to food security and rural livelihoods [19].

Immune Evasion Mechanisms and Host Immune Response

The pathogenesis of Infectious Spleen and Kidney Necrosis Virus (ISKNV) is a masterclass in viral subversion, characterized by a sophisticated, multi-layered strategy to dismantle host defenses while simultaneously hijacking cellular machinery for replication. As a member of the genus Megalocytivirus within the family Iridoviridae, this dsDNA virus has evolved a remarkable arsenal to evade, suppress, and redirect the host immune response, ensuring systemic infection and often, high mortality. The virus does not merely exist within the host; it actively manipulates fundamental signaling pathways, metabolic networks, and cell death programs to create a permissive environment for its propagation. Understanding these complex host-virus interactions is critical for developing effective vaccines and therapeutics.

Subversion of Innate Immune Signaling Pathways

A cornerstone of ISKNV's pathogenic success lies in its ability to directly target and incapacitate key innate immune signaling cascades. The virus encodes proteins that act as molecular mimics and decoys to disrupt the critical pathways that would normally trigger an antiviral state.

The Hypoxia-Inducible Factor (HIF) Pathway Hijack: One of the most elegantly characterized mechanisms is the exploitation of the HIF pathway. ISKNV encodes the protein VP077R, which acts as a master manipulator of the cellular oxygen-sensing machinery. Under normoxic conditions, the transcription factor HIF-1α is targeted for degradation by the von Hippel-Lindau (VHL) protein and its activity is further inhibited by factor-inhibiting HIF (FIH). VP077R directly binds to both VHL and FIH, effectively releasing the "brakes" on the HIF pathway [8]. By competitively inhibiting the interaction between HIF-1α and VHL, VP077R prevents HIF degradation. Concurrently, VP077R recruits FIH and Skp1 to form a complex that promotes FIH degradation via ubiquitination [8]. This dual-action strategy results in a stable, active HIF pathway, which then binds to viral hypoxia response elements (HREs) within the ISKNV genome, most notably within the promoter of orf077r itself, creating a powerful positive feedback loop [8, 44]. This activation leads to a dramatic upregulation of viral replication, particularly in aquatic environments where hypoxic stress is common [44]. This represents a direct environmental connection to viral pathogenesis, where the virus has evolved to thrive under the very conditions that stress its host.

Interference with Interferon and JAK-STAT Signaling: The type I interferon (IFN) response is a frontline antiviral defense, and ISKNV has demonstrated a capacity to blunt its efficacy. Transcriptomic analyses of ISKNV-infected Asian seabass spleen tissue reveal a complex dysregulation of immune pathways, indicating that the virus actively shapes the transcriptional landscape to its advantage [37]. While the expression of interferon-inducible genes is observed, the virus's ability to replicate efficiently suggests it either suppresses the magnitude of this response or acts downstream of IFN signaling. The development of successful vaccines often relies on counteracting this suppression; for instance, chitosan-selenium nanoparticles (CTS-SeNPs) have been shown to upregulate type I interferons (IFN φ2 and IFN φ3), thereby enhancing viral suppression [58]. This implies that the virally-imposed block on IFN induction is a critical weakness to be targeted therapeutically. Furthermore, the stimulator of interferon genes (STING) pathway, a central component of the cytosolic DNA sensing machinery, is actively involved in the host response. Mandarin fish STING (scSTING) is upregulated upon ISKNV infection and its overexpression leads to the induction of IFN-h, Mx, ISG15, and other antiviral effectors, suppressing ISKNV replication [47]. The fact that ISKNV can still establish a productive infection suggests it possesses mechanisms to antagonize STING or its downstream signaling, a likely focal point for future research.

Manipulation of Programmed Cell Death: A Balancing Act

ISKNV exerts exquisite control over host cell fate, delaying apoptosis to facilitate viral replication while ultimately inducing cell death to release progeny. This is a dynamic and tightly regulated process.

Delaying Apoptosis for Viral Factories: Early in infection, ISKNV actively inhibits apoptosis to preserve the cellular machinery required for its own genome replication and virion assembly. The virus manipulates the Bcl-2 family of proteins, a critical checkpoint for mitochondrial-mediated apoptosis. ISKNV infection in GF-1 cells induces a rapid upregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, which counterbalance the pro-apoptotic signals from Bax and Bak, thereby maintaining mitochondrial membrane potential (MMP or ΔΨm) in the early to middle stages of infection [43]. This delicate balance allows the virus to complete its replication cycle within a functional cellular environment.

Exploiting Ferroptosis for Viral Egress: More recently, ISKNV has been demonstrated to induce a distinct, iron-dependent form of programmed cell death known as ferroptosis to enhance its replication [38]. In CPB cells, ISKNV infection triggers the hallmarks of ferroptosis, including mitochondrial shrinkage, increased membrane density, a significant 3.14-fold increase in reactive oxygen species (ROS), and a 1.7-fold increase in malondialdehyde (MDA) [38]. Mechanistically, the virus orchestrates this process by downregulating the key ferroptosis gatekeeper, glutathione peroxidase 4 (GPx4), while simultaneously upregulating the pro-ferroptotic enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) [38]. The functional significance of this is clear: induction of ferroptosis with erastin enhances viral yield, while its inhibition with liproxstatin-1 significantly suppresses viral production. This strategy contrasts with classical apoptosis, suggesting that ISKNV may trigger ferroptosis to release virions in a manner that is less immunogenic or more efficient for its spread.

Triggering Oxidative Stress and Apoptosis in Later Stages: As infection progresses, the mitochondrial balance tips. The virus induces significant reactive oxidative species (ROS) production, triggering an Nrf2-mediated oxidative stress response [46]. This stress signal ultimately activates the pro-apoptotic members Bax and Bak, leading to a loss of MMP, activation of caspase-9 and -3, and host cell death [43, 46]. Interestingly, treatment with antioxidants like GSH and NAC not only blocks this apoptotic cascade but also significantly reduces viral protein (MCP) expression and virus titers, demonstrating that the virus relies on this specific cell death pathway for efficient completion of its life cycle [46].

Metabolic Reprogramming: Feeding the Viral Machine

ISKNV is a master of metabolic manipulation, redirecting host metabolic pathways to satisfy its immense demands for energy, nucleotides, and amino acids. This metabolic hijacking is a critical immune evasion strategy, as it creates a favorable intracellular milieu.

Glutamine and Asparagine Dependency: ISKNV forces a reprogramming of glutamine metabolism to support viral proliferation. The viral proteins ORF093R and ORF102R interact directly with the host transcription factor c-Myc, which then upregulates key enzymes of glutaminolysis, including glutaminase 1 (GLS1), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (IDH2) [39]. This ensures a steady supply of metabolic intermediates. Critically, ISKNV has an absolute requirement for asparagine. Infection upregulates the malate-aspartate shuttle (MAS) and asparagine synthetase (ASNS), and when asparagine is limiting, viral replication is severely curtailed. Remarkably, the addition of asparagine alone to a glutamine-free medium can rescue ISKNV replication to 92% of normal levels, highlighting its role as a critical limiting metabolite [41].

Metabolic Shift to Aerobic Glycolysis: ISKNV also induces a profound shift in glucose metabolism. The virus enhances the pentose phosphate pathway (PPP) early in infection, likely to supply ribose-5-phosphate for nucleotide biosynthesis. As infection progresses, it drives a switch to aerobic glycolysis (the Warburg effect), where glucose is fermented to lactate even in the presence of oxygen [49]. This provides a rapid supply of ATP and biosynthetic precursors, demonstrating how the virus co-opts fundamental cellular bioenergetics to fuel its own replication.

Modulating the Host Inflammatory and Adaptive Immune Response

Beyond subverting innate pathways, ISKNV actively modulates the broader immune landscape, including systemic inflammation and the development of adaptive immunity.

Cytokine and Chemokine Dysregulation: The systemic impact of ISKNV infection is characterized by a complex and often dysregulated cytokine response. In vivo studies show a significant upregulation of pro-inflammatory cytokines like TNF-α, IL-1β, and IRF1, particularly in co-infection scenarios with Aeromonas hydrophila [53]. This suggests that the virus contributes to a pathological inflammatory state that exacerbates tissue damage. Transcriptomic data from the spleen, a primary target organ, reveals profound alterations in pathways related to immune regulation, cell communication, and programmed cell death, indicating a holistic disruption of organ-level immune function [37].

Viral Mimicry of Host Immune Proteins: ISKNV has evolved to produce direct homologs of host immunomodulatory molecules. A prime example is ORF069L, which encodes a protein homologous to the human B7-CD28 family protein HHLA2, a type 1 transmembrane molecule involved in T-cell co-stimulation and regulation [34]. This viral mimicry likely allows ISKNV to directly short-circuit T-cell activation or promote an immunosuppressive environment. Deletion of this gene dramatically reduces viral virulence by 40% and leads to a near 100% protection rate in immunized fish, strongly suggesting that ORF069L is a key virulence factor that dampens the adaptive immune response [34].

Humoral and Cell-Mediated Response: Despite these evasion tactics, a robust adaptive immune response can be elicited. The major capsid protein (MCP) is a potent immunogen. Successful vaccination strategies, including subunit, DNA, and recombinant fusion protein vaccines, consistently demonstrate the induction of high-titer specific neutralizing antibodies [3, 22, 59]. This humoral response is a critical correlate of protection. Furthermore, effective immunity requires a strong cell-mediated component. Vaccinated fish show significant upregulation of CD4 and CD8α T-cell markers, MHC class I and II molecules, and type I interferons [9, 47, 52]. A multi-epitope vaccine designed from the capsid protein was shown to simultaneously upregulate both cellular (CD4, CD8α, MHC-Iα) and humoral (IgM, MHC-IIα) markers, achieving an 85.33% survival rate [52]. The development of gene-deleted live attenuated vaccines (e.g., Δorf103r/tk, Δorf037l) has proven exceptionally effective, providing over 95% protection by mimicking a natural infection that primes both arms of the immune system without causing severe disease [9, 16].

The Complex Role of Co-infections and Environmental Stress

In a natural setting, the host immune response is never fighting a single adversary. ISKNV frequently participates in polymicrobial interactions that dramatically alter disease outcomes.

Synergistic Pathology with Co-infections: Co-infection with Aeromonas hydrophila is common and devastating, resulting in significantly higher mortality than either pathogen alone due to a synergistic lethal effect [53]. The primary viral infection is thought to suppress the host's ability to control the bacterial infection, a phenomenon known as viral-bacterial synergism. Similarly, co-infections with Streptococcus agalactiae [19, 21], Francisella sp. [56], piscine intestinal coccidia [51], and Nervous Necrosis Virus (NNV) [15, 54, 55] have been documented. The presence of multiple pathogens likely overwhelms a host immune system already compromised by ISKNV's immunosuppressive mechanisms. The detection of megalocytes and hemosiderin deposits in co-infected fish underscores the severe pathological outcomes of these interactions [15].

Temperature as a Determinant of Immune Efficacy: The host immune response to ISKNV is profoundly influenced by environmental temperature. Lower temperatures (22–26°C) favor severe disease and high mortality, while higher temperatures (32–34°C) can completely abrogate mortality and significantly reduce viral loads [17, 50]. This has critical implications for vaccine development. While gene-deleted vaccines like Δorf074r retain residual virulence at permissive temperatures (28°C), a low-temperature (22°C) immersion strategy can further attenuate the virus, allowing for safe immunization [17]. This temperature-dependent susceptibility highlights a key mechanism: at lower temperatures, the poikilothermic fish host's immune system is less robust, providing a wider window for viral replication and immune evasion.

Prevention, Control Strategies, and Biosecurity Measures

The prevention and control of Infectious Spleen and Kidney Necrosis Virus (ISKNV) necessitates a multifaceted, integrated approach that combines vaccination strategies, antiviral therapeutics, rigorous biosecurity protocols, environmental management, and advanced surveillance systems. As a WOAH-notifiable pathogen responsible for devastating economic losses across global aquaculture, with mortality rates frequently exceeding 60% in susceptible populations [4, 13], ISKNV demands a comprehensive disease management framework that addresses both clinical outbreaks and subclinical carrier states. The virus’s expanding host range, now documented in over 50 freshwater and marine species across Asia, Africa, and South America [11, 19, 37], coupled with its demonstrated ability to persist in frozen fish products [5] and survive in aquatic environments for extended periods [33], underscores the critical importance of robust prevention and control programs.

Vaccination Strategies: From Attenuated to Subunit Platforms

The development of effective vaccines against ISKNV has progressed substantially, with multiple platforms demonstrating significant protective efficacy. Live attenuated vaccines represent one of the most promising approaches, capitalizing on their ability to mimic natural infection and stimulate robust, long-lasting immunity. The double-gene-deleted live attenuated immersion vaccine Δorf103r/tk, created through homologous recombination knockout of the orf103r and thymidine kinase (tk) genes, exemplifies this strategy’s potential. This vaccine strain demonstrated severe attenuation in mandarin fish (Siniperca chuatsi), inducing only mild histological lesions with a mortality rate of just 3%, and was completely eliminated within 21 days post-administration. Critically, a single immersion dose provided long-lasting protection exceeding 95% against lethal ISKNV challenge, accompanied by robust upregulation of interferon expression and production of specific neutralizing antibodies [16]. This work provides proof-of-principle evidence that gene-deletion strategies can yield safe, efficacious immersion vaccines suitable for large-scale aquaculture applications.

Similarly, deletion of the ORF71L gene resulted in virulence attenuation in mandarin fish, with infected fish showing lower cumulative mortality and reduced viral genome copy numbers in spleen tissues compared to wild-type ISKNV infection. Notably, ISKNV-Δ71-infected fish exhibited increased numbers of melanomacrophage centers (MMCs) in the spleen, suggesting enhanced antigen processing and immune activation [20]. The deletion of ORF069L, which encodes a viral HHLA2 homolog involved in immune evasion, reduced lethality by 40% relative to wild-type virus while achieving nearly 100% protection in immunized fish [34]. These findings collectively indicate that multiple viral genes contribute to ISKNV pathogenicity and can be systematically targeted for vaccine development.

Temperature optimization has emerged as a critical variable in vaccine safety, particularly for attenuated strains that retain residual virulence. The Δorf074r deletion strain, while attenuated at standard aquaculture temperatures (28°C), still induced 46.7% mortality in mandarin fish, rendering it unsuitable for direct application. However, low-temperature immersion immunization at 22°C further reduced residual virulence, resulting in 100% fish survival post-immunization. When these immunized fish were subsequently maintained at 28°C, 80% were protected against ISKNV challenge, demonstrating that temperature manipulation can serve as a safety switch for live attenuated vaccines [17]. This principle was further validated with the Δorf037l vaccine strain, where low-temperature (22°C) immersion immunization elevated survival rates to 90% and achieved a relative percentage survival (RPS) of 92.6%, while effectively triggering expression of key immune-related genes including IFN-h, IL-1, IκB, Mx, TNF-α, and Viperin, as well as inducing specific neutralizing antibodies [9].

Subunit vaccine development has focused on the major capsid protein (MCP), the primary structural component and key antigen mediating viral entry into host cells. Refolded recombinant MCP produced in Escherichia coli demonstrated superior immunogenicity compared to insoluble protein formulations, eliciting significantly higher serum antibody titers in Nile tilapia and upregulation of MHC-I, MHC-II, IL-1β, and IL-4 genes, with peak responses observed at 28 days post-immunization [22]. However, the protective efficacy of MCP-based subunit vaccines has historically been suboptimal for field applications. Addressing this limitation, a bioinformatics-driven approach identified two truncated proteins (tMCP and t051L) with rich antigenic epitope content, which were linked via a rigid linker peptide to create the fusion protein t051L-tMCP. This fusion construct demonstrated dramatically improved immunogenicity, with survival rates reaching 95.50% in largemouth bass, significantly superior to the 85.01% and 79.01% observed with tMCP and t051L alone, respectively. Furthermore, viral load detection at 7 days post-challenge revealed substantially reduced viral replication in head kidney, liver, and spleen tissues of vaccinated fish, with clear dose-dependent relationships in immunoassay results [3].

Multi-epitope vaccines represent an economical, rapid, and safe alternative. Using immunoinformatics to analyze capsid protein epitopes, researchers identified three T lymphocyte and five B lymphocyte epitopes exhibiting antigenicity, non-toxicity, and non-allergenicity. Fusion of these epitopes into a multi-epitope vaccine (MEV) significantly upregulated mRNA expression of IL-1β, TNF-α, CD4, CD8α, MHC-Iα, and MHC-IIα, while markedly increasing total IgM levels, indicating induction of both cellular and humoral immunity. Crucially, MEV vaccination conferred 85.33% survival against ISKNV challenge with significantly reduced viral loads in the spleen and head kidney, outperforming whole capsid protein immunization [52].

Oral vaccine platforms have been developed using surface display technologies in Saccharomyces cerevisiae and Bacillus subtilis to express ISKNV MCP. Oral immunization of largemouth bass with these recombinant strains significantly elevated expression of IFN-γ, TNF-α, IL-1, CD8α, MHC-I, IgM, and IgT in spleen and head kidney tissues, along with serum-specific antibody levels. Following challenge, survival rates of 53.3% and 50.0% were achieved with S. cerevisiae and B. subtilis platforms respectively, with histopathological examination revealing no obvious lesions in liver, spleen, or head kidney of immunized fish compared to severe pathology in controls [62]. While these RPS values of 30.0% and 25.0% are modest, the practicality of oral delivery for mass vaccination in aquaculture settings makes this approach worthy of further optimization.

Nanotechnology-based delivery systems have revolutionized immersion vaccination by overcoming biological barriers that traditionally limited vaccine efficacy. Single-walled carbon nanotubes (SWCNTs) functionalized with mannose-modified MCP (SWCNTs-M-MCP) target antigen-presenting cells (APCs), inducing robust immune responses through enhanced APC uptake and presentation. Bath immunization with this system achieved an RPS of 81.3% compared to only 41.5% with SWCNTs-MCP without mannose targeting [60]. Optimization studies identified optimal immune parameters: 8 hours of immune duration, 20 mg/L vaccine dose, and 8 fish per liter density, with immune duration exerting the greatest influence on immune response magnitude [66]. SWCNTs have similarly enhanced DNA vaccine delivery, with SWCNTs-pcDNA-MCP achieving 82.4% RPS compared to 54.2% with naked pcDNA-MCP at equivalent doses [59]. Recombinant baculovirus vector vaccines (BacMCP) delivering the MCP gene under a CMV promoter have also shown promise, achieving 100% vaccine efficacy in small largemouth bass and 85.7% in larger fish via injection, with immersion-vaccinated small bass achieving 77.3% efficacy [63].

Antiviral Therapeutics and Host-Directed Interventions

The metabolic reprogramming that occurs during ISKNV infection has revealed multiple therapeutic targets. ISKNV replication is critically dependent on glutamine metabolism, with viral proteins ORF093R and ORF102R interacting with c-Myc to upregulate glutaminase 1 (GLS1), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (IDH2), thereby reconstructing glutamine metabolism to satisfy the energy and macromolecular requirements for viral proliferation [39]. Furthermore, asparagine availability functions as a critical limiting factor for ISKNV protein synthesis. Inhibition of the malate-aspartate shuttle (MAS) pathway or reduction of asparagine synthetase (ASNS) expression decreased viral production by 1.3-fold and 0.6-fold respectively, while asparagine supplementation to glutamine-depleted medium restored ISKNV copy numbers to 92% of complete medium levels [41]. Similarly, accelerated aerobic glycolysis and pentose phosphate pathway activity are required for efficient ISKNV replication, providing additional metabolic intervention points [49].

The discovery that ISKNV triggers ferroptosis, an iron-dependent programmed cell death pathway, to enhance its replication has opened new therapeutic avenues. ISKNV infection induces hallmark morphological alterations including mitochondrial shrinkage, increased membrane density, and cristae reduction, accompanied by significant elevations in malondialdehyde (1.7-fold), reactive oxygen species (3.14-fold), and ferrous iron (1.42-fold). Mechanistically, the virus downregulates glutathione peroxidase 4 (GPx4) while upregulating acyl-CoA synthetase long-chain family member 4 (ACSL4). Pharmacological blockade of ferroptosis with liproxstatin-1 significantly suppressed viral yield, while induction with erastin enhanced replication, establishing ferroptosis inhibition as a promising intervention strategy [38].

The hypoxia-inducible factor (HIF) pathway represents another critical host-virus interface. ISKNV encodes VP077R, which interacts with the Von Hippel-Lindau (VHL) protein at the HIF-binding region, competitively inhibiting HIF-1α degradation. Additionally, VP077R recruits factor-inhibiting HIF (FIH) and S-phase kinase-associated protein 1 (Skp1) to form an FIH-VP077R-Skp1 complex that promotes FIH degradation via ubiquitination. This dual mechanism releases two "brakes" on the HIF pathway, activating a positive feedback loop that dramatically enhances viral replication, particularly under hypoxic conditions common in aquaculture environments [8]. Fifteen viral hypoxia response elements (HREs) have been identified in the ISKNV genome, with the hre-orf077r promoter showing remarkable responsiveness to HIF pathway activation, providing potential biomarkers for assessing outbreak risk under hypoxic stress [44].

Repurposing of existing pharmaceuticals has yielded several candidates with anti-ISKNV activity. Gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, blocks ISKNV entry by suppressing EGFR/PI3K phosphorylation and inhibiting microfilament gathering, thereby preventing virus-mediated endocytosis. In vitro, gefitinib inhibited viral protein synthesis and decreased viral titers in a dose-dependent manner, while in vivo administration reduced pathogenicity and ISKNV infection [48]. The EGFR-PI3K-Akt signaling axis is essential for ISKNV invasion, and its pharmacological inhibition represents a viable therapeutic strategy [42]. The PI3K/AKT/p53 pathway, when activated, inhibits ISKNV replication by regulating autophagy and immune responses, with agonists or overexpression of pathway factors decreasing autophagy levels and elevating immune-related gene expression [45].

Nanocarrier-based drug delivery systems have dramatically improved the therapeutic index of antiviral compounds. Single-walled carbon nanotubes loaded with moroxydine hydrochloride (Mor-SWCNTs) achieved 182.35 μg/g drug concentration in target tissues compared to 103.48 μg/g with free Mor at equivalent doses, while extending drug detention time by over 48 hours. This resulted in cumulative mortality of only 11.51% and infection rate of 3.81%, compared to 43.34% mortality and 22.67% infection rate with free Mor [65]. Similarly, ganciclovir-loaded SWCNTs (G-SWCNTs) at half the dose (20 mg/L) achieved 14.75% cumulative mortality compared to 32.50% with free ganciclovir at 40 mg/L, with both substantially outperforming the 88.75% mortality in untreated controls [67].

Chitosan-selenium nanoparticles (CTS-SeNPs) represent a novel immunomodulatory antiviral approach. These nanoparticles, with an average size of 51.73 nm and moderate stability in freshwater environments for 24 hours, demonstrated 94.02% and 91.82% inhibition rates at 48 and 72 hours post-infection in dwarf gourami fin (DGF) cells, primarily affecting the attachment and replication stages of the ISKNV replication cycle. In zebrafish models, intraperitoneal injection and immersion administration of CTS-SeNPs at safe concentrations improved survival rates to 53.33% and 50.00% respectively, with survivors showing lower viral loads. Mechanistically, CTS-SeNPs upregulated interferon-inducible genes and, following ISKNV challenge, induced type I interferon expression (IFN φ2 and IFN φ3), suggesting immune modulation as the primary antiviral mechanism [58].

The plant-derived alkaloid anisodamine, when used as an adjuvant with inactivated ISKNV vaccine, significantly improved immune protective rates and inhibited viral replication, while chitosan similarly enhanced vaccine efficacy with stronger protection of liver and spleen tissues as demonstrated by pathological examination [64]. High-throughput virtual screening of marine algae metabolites identified four compounds (BC012, BC014, BS032, and RC009) with binding affinities of -9.2 to -9.9 kcal/mol against the MCP protein, exhibiting favorable ADMET characteristics and drug-likeness, though these require in vitro and in vivo validation [61].

Biosecurity Protocols and Environmental Control Measures

The demonstrated ability of ISKNV to survive and remain infectious under various environmental conditions necessitates rigorous biosecurity protocols. Comprehensive disinfection studies have established that ISKNV is effectively inactivated by heating to 65°C for 20 minutes, exposure to pH 3 or pH 11, treatment with 1% Virkon™, 1000 ppm sodium hypochlorite, and benzalkonium chloride at recommended concentrations and contact times. Critically, ISKNV can remain infectious in aquaria without fish for at least 48 hours at 25°C, highlighting the importance of thorough disinfection of equipment, tanks, and water systems between production cycles [33].

Temperature manipulation represents a non-pharmacological intervention with significant practical applications. Juvenile Nile tilapia maintained at water temperatures above 30°C (specifically 32°C and 34°C) showed no mortality following ISKNV challenge, with qPCR analysis revealing significant reduction in viral loads in kidney and spleen tissues. In contrast, fish maintained at 26°C, 28°C, and 30°C developed clinical disease with mortality, indicating enhanced viral activity at lower temperatures [50]. This non-lethal hyperthermia approach, consistent with observations in other aquatic viral diseases such as white spot syndrome virus in shrimp and koi herpesvirus in carp, provides a practical control strategy for tilapia aquaculture operations in regions where temperature manipulation is feasible. However, the paradoxical relationship between hypoxia and ISKNV pathogenesis complicates environmental management, as hypoxic conditions common in high-density aquaculture activate the HIF pathway and trigger ISKNV outbreaks through viral HREs, potentially creating conditions where even subclinical infections escalate to devastating disease [44].

The trade in frozen fish products represents a significant pathway for ISKNV introduction to new geographic regions. Experimental challenge models using albino rainbow sharks demonstrated that ISKNV remains infectious after storage at -20°C for 7 days, with all tissue pools, including skin-on fillet only products, causing infection and disease via intraperitoneal injection or immersion, whether used fresh or frozen. The estimated median infectious dose (ID50) was 42 ISKNV genome equivalents (95% CI: 19–98), providing critical data for import risk assessments [5]. This finding has profound implications for international trade regulations, as frozen fish products are frequently traded commodities with limited virological screening.

In the event of an outbreak, rapid containment is essential. Pooled testing strategies offer a cost-effective approach for surveillance, though diagnostic sensitivity is substantially reduced when testing tissue pools compared to individual testing. Surveillance sensitivity can be maximized by increasing sample size, with pooled testing being highly effective when prevalence exceeds 10%. For populations with high viral loads, surveillance sensitivity can be achieved using 260 fish in pools of 10 (26 tests total) or 200 fish in pools of 5 (40 tests total) [32]. The development of rapid, field-deployable diagnostic tools has enhanced outbreak response capabilities. The recombinase polymerase amplification (RPA)-CRISPR/Cas12a system can detect as low as 0.1 copy/μL of ISKNV at a constant temperature of 37–39°C, requiring only 30 minutes of RPA amplification followed by 15 minutes of CRISPR/Cas reaction, eliminating the need for complex laboratory equipment [7]. RPA combined with lateral flow dipsticks (RPA-LFD) achieves detection within 30 minutes at 38°C with 10² copies/μL sensitivity, 10-fold more sensitive than conventional PCR [28].

Integrated Health Management and Surveillance

The high prevalence of co-infections in ISKNV outbreaks necessitates integrated health management approaches that address polymicrobial interactions. Co-infection of ISKNV with Aeromonas hydrophila in Chinese perch results in synergistic lethal effects, with mixed-infection groups showing higher mortality than single-pathogen or secondary infection groups. ISKNV-primary infection increases mortality from secondary bacterial infections, while co-infected fish exhibit high expression of IRF1, Mx, Viperin, Hepcidin, TNFα, and IL-1β, indicating that concurrent infections activate host inflammatory responses that may exacerbate pathology [53]. Similarly, co-infection of ISKNV with Francisella sp. in pearl gentian grouper resulted in 52% cumulative mortality over 56 days [56], while concurrent infection with piscine intestinal coccidia in juvenile barramundi caused severe gastrointestinal lesions markedly intensified in co-infected tissues [51]. These findings emphasize that disease management programs must consider the entire pathogen landscape rather than focusing solely on ISKNV.

The role of ornamental fish in ISKNV dissemination deserves particular attention. Subclinical infections are common in ornamental species, with apparently healthy fish serving as asymptomatic carriers. In India, 29.4% of apparently healthy molly and angelfish screened positive for ISKNV [35], while 11.42% of ornamental fish from retailers representing 10 different species were positive, with three species reported positive for the first time globally [23]. The experimental transmission of ISKNV from freshwater ornamental fish (dwarf gourami) to marine silver sweep (Scorpis lineolata) via direct inoculation and cohabitation demonstrates a feasible pathway for pathogen exchange between freshwater and marine environments, with further transmission from infected marine silver sweep

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