Viral Hemorrhagic Septicemia Virus

Overview and Taxonomy of Viral Hemorrhagic Septicemia Virus

Viral hemorrhagic septicemia virus (VHSV) is the etiological agent of viral hemorrhagic septicemia (VHS), a highly contagious and economically devastating disease affecting a broad spectrum of marine and freshwater fish species worldwide. Recognized as a notifiable pathogen by the World Organisation for Animal Health (WOAH), VHSV represents one of the most significant viral threats to global aquaculture, causing mass mortality events in both wild and farmed fish populations [22, 29]. The virus is classified within the genus Novirhabdovirus, family Rhabdoviridae, a taxonomic grouping that distinguishes it from other rhabdoviruses by the presence of a unique non-virion (NV) gene [1, 12, 27]. This genomic hallmark is a defining feature of the genus and is central to the virus’s pathogenesis, as the NV protein plays a critical role in inhibiting host type I interferon responses and facilitating efficient viral replication [27, 37]. The family Rhabdoviridae encompasses a diverse array of enveloped, single-stranded negative-sense RNA viruses that infect a wide range of hosts, including plants, insects, and vertebrates. However, VHSV is distinguished by its remarkable host range, infecting over 140 fish species across marine, estuarine, and freshwater environments, and by its pronounced temperature-dependent pathogenicity [2, 3, 26].

Taxonomic Classification and Genomic Architecture

The taxonomic position of VHSV is firmly established within the order Mononegavirales, characterized by non-segmented, negative-sense RNA genomes. The virion exhibits the classic bullet-shaped morphology typical of rhabdoviruses, with a lipid envelope derived from the host cell membrane and a helical nucleocapsid core. The VHSV genome is approximately 11.1 to 11.2 kilobases in length and encodes six structural proteins in a conserved gene order: 3′-N-P-M-G-NV-L-5′ [10, 18, 27]. The nucleoprotein (N) encapsidates the viral RNA, forming the ribonucleoprotein complex essential for transcription and replication. The phosphoprotein (P) acts as a cofactor for the viral RNA-dependent RNA polymerase (L), while the matrix protein (M) orchestrates viral assembly and budding. The glycoprotein (G) forms trimeric spikes on the virion surface, mediating viral attachment to host cell receptors and membrane fusion, and is the primary target for neutralizing antibodies [1, 9, 24]. The non-virion (NV) protein, unique to novirhabdoviruses, is a multifunctional virulence factor that suppresses the host interferon system and modulates apoptosis [27, 37]. The large polymerase protein (L) contains the catalytic core for RNA synthesis, including a conserved GDNV motif that is a hallmark of the Novirhabdovirus genus, in contrast to the GDNQ motif found in most other mononegaviruses [16].

The genomic organization of VHSV is highly conserved across all genotypes, yet subtle variations in nucleotide sequence and gene length contribute to significant differences in virulence and host specificity. For instance, the complete genome of the Chinese isolate GY-2307 (genotype IVa) was determined to be 11,140 nucleotides in length, with 315 nucleotide substitutions relative to other VHSV sequences, 71 of which resulted in amino acid changes [10]. Such genetic variability, particularly within the G protein, is a major driver of antigenic diversity and poses a challenge for vaccine development, as cross-protection between genotypes can be limited [6, 26].

Phylogenetic Diversity and Genotypic Classification

Extensive phylogenetic analyses based on the complete G gene and, more recently, whole-genome sequences have delineated VHSV into four major genotypes (I–IV), each with distinct geographic distributions and host preferences [26, 33, 39]. Genotype I is predominantly found in European freshwater and marine environments and is further subdivided into several subgenotypes (Ia–Ie). Subgenotype Ia includes the highly virulent strains responsible for devastating outbreaks in farmed rainbow trout (Oncorhynchus mykiss) across continental Europe [30, 38]. Genotype II and III are primarily associated with marine fish in the Baltic Sea, Skagerrak, and the North Atlantic, and these isolates generally exhibit low or moderate virulence in rainbow trout [19, 30]. Genotype IV is divided into two distinct subgenogroups: IVa, which circulates in the North Pacific Ocean and along the Pacific coast of Asia, affecting species such as olive flounder (Paralichthys olivaceus) and Pacific herring (Clupea pallasii); and IVb, which emerged suddenly in the Laurentian Great Lakes of North America around 2003–2005, causing massive die-offs in over 30 freshwater fish species [7, 10, 33, 39].

The emergence of VHSV-IVb in the Great Lakes represents a landmark event in aquatic virology, as it demonstrated the capacity of a marine virus to jump into a freshwater ecosystem and adapt to a completely new suite of hosts. Population genetic analyses of VHSV-IVb have revealed significant spatial and temporal diversification, with the virus evolving into at least 36 G-gene haplotypes over less than two decades [39]. This rapid evolution is accompanied by a trend toward reduced virulence, which may facilitate long-term persistence in reservoir host populations [33, 39]. In contrast, genotype IVa isolates from Asia show a faster substitution rate (2.01 × 10⁻³ substitutions per site per year), indicating ongoing adaptive evolution in farmed environments [10, 33]. The genetic stratification of VHSV into these distinct subpopulations is driven by a combination of geographic isolation, host adaptation, and positive selection, particularly on the G protein, where differential selection signatures have been identified across codon sites [26].

Virulence Determinants and Molecular Markers

The molecular basis of VHSV virulence is complex and multifactorial, involving interactions between multiple viral proteins and host factors. Early studies using reverse genetics to generate chimeric viruses between a trout-virulent genotype Ia strain (DK-3592B) and a trout-avirulent genotype IVb strain (MI03) demonstrated that the N and P proteins together constitute the major determinants of host-specific virulence in rainbow trout [40]. This finding was surprising, as virulence in mammalian rhabdoviruses is typically governed by the M or P proteins. The N and P proteins are integral components of the viral polymerase complex, and their co-adaptation is hypothesized to enhance replication efficiency in the presence of a specific host factor(s) present in rainbow trout cells [40]. Further refinement of these virulence markers identified specific amino acid residues in the N protein (K46G and A241E) and the NV protein (R116S) that are critical for pathogenicity in rainbow trout [30]. Remarkably, the N protein variant K46G rendered the virus unable to establish infection in the fins, the primary portal of entry, thereby preventing systemic spread [30].

The NV protein, unique to novirhabdoviruses, is a key virulence factor that counteracts the host innate immune response. VHSV lacking a functional NV gene is severely attenuated in both cell culture and in vivo, exhibiting reduced replication and complete loss of pathogenicity in olive flounder [27]. Mechanistically, the NV protein modulates the PERK-eIF2α pathway to suppress host translation shutoff and interferon signaling, thereby creating a permissive environment for viral protein synthesis [37]. The N-terminal region of the NV protein is essential for this function, as truncation of this domain abolishes the protein’s ability to inhibit type I interferon responses and restore viral replication [27]. Additionally, the matrix protein (M) has been shown to undergo compensatory mutations that restore fitness when targeted mutations are introduced, highlighting the plasticity of the VHSV genome in maintaining virulence [28].

Host Range, Epidemiology, and Environmental Persistence

VHSV is renowned for its exceptionally broad host range, which now encompasses over 140 fish species from more than 50 families, including both teleosts and, as demonstrated recently, chondrosteans such as the endangered Pallid Sturgeon (Scaphirhynchus albus) [20, 26]. The virus is capable of infecting fish across a wide range of life stages, from fry to adults, and transmission occurs horizontally through waterborne exposure, with the gills and mucus layers serving as primary portals of entry [4, 23]. The minimum infective dose for olive flounder via immersion has been determined to be approximately 10³·⁴ TCID₅₀/mL, and viral shedding from infected individuals can be substantial, with a qPCR CT value of 23 (~10⁶ copies/mg) serving as a reliable indicator of high-risk infections [2, 23]. Importantly, convalescent fish can continue to shed virus at low levels for extended periods, acting as reservoirs that perpetuate the virus in the environment [36].

Environmental factors, particularly water temperature, exert a profound influence on VHSV epidemiology. The virus is classically associated with cold-water disease, with outbreaks typically occurring at temperatures below 15°C [3, 15]. At temperatures above 20°C, infection is generally not established in susceptible species such as olive flounder, although the virus can replicate at low levels in cell culture at 25°C [3]. This temperature dependence is mediated by both viral replication kinetics and host immune responses. At low temperatures (e.g., 13°C), VHSV transcription can exceed 5% of the host transcriptome, and the virus undergoes more frequent genetic changes, likely due to increased replication pressure [5]. Conversely, at higher temperatures, the host mounts a more robust interferon response, characterized by upregulation of ISG15 and Mx genes, which effectively suppresses viral replication [3, 25]. The critical temperature threshold for disease onset in olive flounder has been identified as 18.7°C, above which the risk of clinical VHS is significantly reduced [2].

The geographic distribution of VHSV is global, with endemic foci in Europe, North America, and Asia. In the Laurentian Great Lakes, VHSV-IVb has been detected in a wide range of wild fish species, including invasive Round Gobies (Neogobius melanostomus), which exhibit significantly higher infection prevalence and viral titers compared to native species, thereby amplifying reservoir competence [11]. In the St. Lawrence River, seropositivity to VHSV has been documented in 4.3% of Bluegill, 13.4% of Brown Trout, 19.3% of Northern Pike, and 18.3% of Walleye, indicating widespread exposure even in water bodies far from reported outbreaks [35]. In Asia, genotype IVa is enzootic in olive flounder farms along the coasts of Korea, Japan, and China, where it causes recurrent economic losses [2, 7, 10]. The virus has also been isolated from apparently healthy bastard halibut in China, suggesting the existence of subclinical carriers that may facilitate silent spread [7].

Immunological and Pathological Considerations

The pathogenesis of VHSV is characterized by a systemic hemorrhagic disease, with clinical signs including exophthalmia, abdominal distension due to ascites, petechial hemorrhages in the skin and musculature, and severe anemia [15, 22]. At the cellular level, VHSV has evolved sophisticated strategies to subvert host immune defenses. The N protein, for example, suppresses STAT1-mediated MHC class II transcription by promoting K48-linked ubiquitination and proteasomal degradation of STAT1, thereby impairing antigen presentation and adaptive immunity [21]. Similarly, the virus activates the integrated stress response (ISR) and induces stress granule formation via the PERK pathway, but paradoxically requires PERK activity for efficient viral replication and interferon production [17]. This intricate interplay between viral evasion and host defense underscores the complexity of VHSV pathogenesis.

Despite the lack of evidence for zoonotic potential, VHSV does not replicate in mammalian cells at 37°C and fails to cause disease in experimentally infected mice, the virus remains a pathogen of significant regulatory concern [32]. The WOAH lists VHSV as a notifiable disease, and international trade restrictions are often imposed on fish products from affected regions [22, 29]. The economic impact of VHSV on aquaculture is substantial, with losses attributed to direct mortality, reduced growth performance, and the costs of biosecurity measures and vaccination programs [8, 31]. Given the absence of effective antiviral therapies, current control strategies rely on strict biosecurity, early detection through molecular diagnostics such as RT-qPCR and recombinase polymerase amplification (RPA), and the development of effective vaccines, including inactivated, DNA, and live-attenuated platforms [1, 6, 13, 14, 34].

Molecular Pathogenesis and Glycoprotein Structure of VHSV

As a leading veterinary researcher specializing in aquatic viral pathogens, I present this exhaustive analysis of the molecular pathogenesis and glycoprotein structure of the Viral Hemorrhagic Septicemia Virus (VHSV). VHSV, a member of the genus Novirhabdovirus within the family Rhabdoviridae, represents one of the most significant viral threats to global aquaculture and wild fish populations, with the World Organisation for Animal Health (WOAH) listing it as a notifiable pathogen. The virus's single-stranded, negative-sense RNA genome, approximately 11,100–11,200 nucleotides in length, encodes six proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV), and RNA-dependent RNA polymerase (L), arranged in the canonical 3′-N-P-M-G-NV-L-5′ order [10, 33, 38]. Understanding the intricate molecular architecture of these proteins, particularly the glycoprotein, and their collective role in pathogenesis is paramount to developing effective countermeasures.

Molecular Architecture and Structural Integrity of the Glycoprotein (G)

The viral glycoprotein (G) is the primary antigenic determinant and the critical mediator of host cell entry, making it the central focus of vaccine development and neutralization strategies. The G protein forms homotrimeric spikes on the viral envelope, responsible for receptor binding and pH-dependent membrane fusion. Detailed genomic analyses of VHSV isolates from China, specifically the GY-2307 strain (genotype IVa), have revealed that the G gene is subject to significant evolutionary pressure, with specific nucleotide and amino acid variations observed when compared across 28 global isolates [10]. In the GY-2307 genome, 9 of the 23 nucleotide substitutions in the G gene resulted in non-synonymous amino acid changes, underscoring the protein's adaptive evolution within farm environments [10].

The structural integrity of the G protein, particularly its conserved disulfide bond-dependent conformation, is absolutely critical for immunogenicity. A landmark comparative study evaluating different vaccine inactivation methods demonstrated that the preservation of the G protein's monomeric structure is directly correlated with vaccine efficacy [1]. Both formalin- and β-propiolactone (BPL)-inactivated VHSV vaccines conserved the monomeric G protein structure, similar to non-inactivated virus, resulting in remarkably high survival rates of 80% and 90%, respectively, in olive flounder. In stark contrast, binary ethylenimine (BEI)- and heat-inactivated vaccines exhibited structural alterations in the monomeric G protein, leading to severely compromised protection, with survival rates plummeting to only 20% and 30% [1]. This direct correlation between G protein conformational integrity and protective immunity was further validated by monoclonal antibody binding assays, firmly establishing that the native, disulfide bond-stabilized structure of the G protein is a prerequisite for eliciting robust neutralizing antibody responses.

The G protein's functional domains extend beyond simple receptor binding. Molecular docking studies have identified that the pleckstrin homology domain of the VHSV glycoprotein is a potential target for antiviral compounds, such as umbelliferone, which may inhibit viral entry by binding this critical region [42]. Furthermore, the signal peptide (SP) of the G protein plays a crucial role in its targeting, secretion, and subsequent immunogenicity. Heterologous substitution of the native VHSV G SP with a piscidin-derived signal peptide (Psp) paradoxically enhanced secretory efficiency in a luciferase reporter assay but simultaneously attenuated viral replication in vivo [41]. Recombinant viruses bearing the Psp-fused glycoprotein (rVHSV-PspvG) exhibited slower replication kinetics, smaller plaque formation, and increased ER stress in host cells, leading to reduced viral loads in target tissues and complete attenuation of pathogenicity [41]. While the DNA vaccine encoding this modified G construct conferred only partial protection (60% survival), compared to complete protection from the native G, this demonstrates the exquisite sensitivity of viral pathogenesis to even subtle alterations in glycoprotein structure and processing.

Molecular Pathogenesis: The Orchestrated Subversion of Host Defense

The molecular pathogenesis of VHSV is a multi-phasic process involving sophisticated evasion of host innate immunity, subversion of antigen presentation, and manipulation of cellular stress responses. The initial stages of infection are mediated by the G protein binding to host cell receptors, with subsequent viral entry occurring via endocytosis and pH-dependent membrane fusion. Early-stage adhesion dynamics studies have elucidated that the gills and mucus layers serve as the primary adsorption sites, with viral RNA detectable within 1 hour post-infection, peaking between 1-3 hours at permissive temperatures [4]. This rapid initial attachment is followed by systemic dissemination, with the virus exploiting the host's own cellular machinery for replication.

A critical virulence determinant resides within the nucleoprotein (N) and phosphoprotein (P) genes. Through elegant reverse genetics and chimeric virus construction, it has been definitively established that the N and P proteins together constitute the major determinants of host-specific virulence in rainbow trout [40]. Reciprocal exchange of these two genes between a trout-virulent European strain (genotype Ia) and a trout-avirulent North American strain (genotype IVb) resulted in a complete gain-of-function in the avirulent background (82% mortality) and a complete loss-of-function in the virulent background (0% mortality) [40]. Furthermore, specific amino acid markers within the N protein (K46G and A241E) and the NV protein (R116S) have been identified as regulating virulence in rainbow trout, with the N K46G mutation rendering the virus incapable of establishing active infection at the fin portal of entry [30]. Paradoxically, while the N and P proteins are essential for virulence, the glycoprotein (G), NV protein, and polymerase (L) were not found to be major determinants of host-specific virulence in rainbow trout when tested in isolation [38], underscoring that the N-P complex is the key driver of pathogenesis in this species.

The NV protein, unique to novirhabdoviruses, orchestrates several critical strategies for immune evasion. It functions as a potent suppressor of the host type I interferon (IFN) response, a mechanism essential for efficient viral replication [27, 37]. Recombinant viruses lacking a functional NV gene (rVHSV-ΔNV) are completely attenuated in vivo and fail to replicate efficiently in cell culture, demonstrating the protein's non-redundant role in counteracting the host antiviral state [27]. The NV protein accomplishes this, in part, by modulating the PERK-eIF2α pathway, a branch of the integrated stress response (ISR). VHSV infection induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) via PERK, leading to a global host translation shutoff; however, viral protein synthesis paradoxically proceeds despite this hostile environment, with the NV protein being critical for maintaining efficient viral translation under these stress conditions [37]. The NV protein further contributes to pathogenesis by influencing viral genome length and replication efficiency, with longer genomes (e.g., those containing an additional NV gene) showing altered replication kinetics and reduced pathogenicity compared to wild-type viruses [27].

Subversion of Antigen Presentation and Manipulation of Cellular Proteostasis

VHSV has evolved sophisticated mechanisms to directly impair the host's ability to mount an adaptive immune response, targeting the antigen presentation machinery at multiple levels. The N protein of VHSV actively suppresses STAT1-mediated MHC class II transcription, thereby impairing antigen presentation by professional antigen-presenting cells [21]. This is achieved through a two-pronged attack: first, VHSV infection blocks interferon-gamma (IFN-γ)-induced expression of IRF1, CIITA, and MHC-II genes via STAT1 degradation. The N protein directly interacts with STAT1 and enhances its K48-linked ubiquitination, marking it for proteasomal degradation, which effectively neutralizes the IFN-γ signaling cascade required for CD4+ T cell activation [21].

Concurrently, the virus engages in a sophisticated feedback loop with the host's antiviral protein, viperin. Viperin exerts its antiviral activity by directly binding to both the N and P proteins of VHSV and targeting them for degradation through the autophagy pathway [43]. This represents an evolutionarily conserved antiviral mechanism. However, VHSV has evolved a counter-strategy: the N protein itself targets and degrades the transcription factors IRF1 and IRF9, key activators of viperin expression, through the ubiquitin-proteasome pathway, thereby creating a molecular tug-of-war between host defense and viral immune evasion [43].

The matrix (M) protein also plays a significant, albeit less characterized, role in pathogenesis. Compensatory mutations in the M protein are readily selected in response to engineered mutations at positions D62 and E181, suggesting that the M protein's function in viral assembly and budding is subject to strong selective pressure [28]. Furthermore, VHSV infection actively manipulates cellular stress responses through the formation of stress granules (SGs). The activation of the PERK pathway, but not PKR, is required for VHSV-induced SG formation, and this process is dependent on active viral replication, as individual VHSV proteins or inactive virus fail to induce SGs [17]. The SG scaffolding protein G3BP1 plays a critical role in regulating VHSV replication; cells lacking G3BP1 produce increased IFN and antiviral gene expression but paradoxically exhibit reduced viral protein synthesis and titers, highlighting a novel role for G3BP1 in the viral life cycle [17].

Temperature-Dependent Pathogenesis and Transcriptional Dynamics

VHSV pathogenesis is exquisitely sensitive to environmental temperature, which profoundly influences both viral replication kinetics and host immune responses. At permissive temperatures (below 15°C), VHSV establishes robust infection, while at elevated temperatures (above 20°C), infection is typically aborted. Detailed dual RNA-seq analyses have revealed that at lower temperatures (13°C), VHSV transcription can exceed 5% of the host transcriptome, and single nucleotide variants (SNVs) appear more frequently compared to infections at 20°C [5]. This suggests that cooler temperatures not only favor viral replication but also generate greater genetic diversity, potentially driving evolutionary adaptation.

Strand-specific RT-qPCR analyses have elucidated the dynamics of viral RNA species during replication. At 20°C, viral mRNA transcription proceeds at a higher speed, and the copy number of the complementary RNA (cRNA) replication intermediate is significantly higher (more than 10-fold at 12-36 hours post-infection) compared to 15°C, indicating a positive effect of higher temperature on the initial steps of viral RNA replication [44]. Crucially, the copy number of cRNA never exceeds that of vRNA at any time point, suggesting a regulatory bottleneck that limits the cRNA pool, potentially governing the switch between transcription and replication [44]. In primary olive flounder spleen cell cultures, viral mRNA levels on day 5 post-challenge differed more than 10-fold between temperatures, being highest at 15°C, followed by 20°C, and lowest at 25°C [3]. However, at 25°C, while viral replication was lower than at 20°C, the expression of interferon-related and pro-inflammatory cytokine genes was also relatively low, indicating a dual failure of both viral replication and host immune activation at the highest temperature [3]. Comparative transcriptomics between high and low pathogenic VHSV strains in rainbow trout revealed that the number of differentially expressed genes progressively increased over time, with a greater number observed in fish infected with the highly virulent strain, and functions related to inflammation were modulated during the first days of infection regardless of strain pathogenicity [19]. This integrated view of temperature-dependent pathogenesis underscores the complex interplay between viral replication efficiency, host immune competence, and environmental conditions in determining disease outcome.

Epidemiology and Risk Factors: Temperature and Host Susceptibility

The epidemiology of Viral Hemorrhagic Septicemia Virus (VHSV) is inextricably linked to a complex interplay of environmental determinants, most notably water temperature, and intrinsic host factors that govern susceptibility. As a pathogen listed by the World Organisation for Animal Health (WOAH) due to its devastating impact on global aquaculture and wild fish populations, understanding the nuanced relationship between thermal regime and host vulnerability is paramount for predictive modeling, biosecurity planning, and the development of effective intervention strategies. VHSV, a member of the genus Novirhabdovirus within the family Rhabdoviridae, exhibits a pronounced temperature-dependent pathogenesis that dictates its geographic distribution, seasonal outbreak patterns, and the severity of epizootics across a diverse range of over 140 susceptible fish species [22, 26].

The Thermal Threshold: Defining the Permissive Zone for VHSV Replication and Disease

The cardinal principle governing VHSV epidemiology is its strict association with cold-water environments. Clinical disease and mass mortality events are overwhelmingly observed at water temperatures below 15°C, with the most severe outbreaks typically occurring in the range of 8°C to 12°C [3, 15]. Conversely, temperatures exceeding 18-20°C are generally considered restrictive, and infection is rarely established at or above 25°C in susceptible species like the olive flounder (Paralichthys olivaceus) [3]. This thermal restriction is not merely a correlative observation but is rooted in fundamental virological and immunological mechanisms.

At the cellular level, temperature exerts a profound influence on VHSV replication kinetics. In vitro studies using primary olive flounder spleen cell cultures have demonstrated that viral mRNA levels at 5 days post-challenge are more than 10-fold higher at 15°C compared to 20°C, with replication at 25°C being minimal [3]. This pattern is corroborated by strand-specific RT-qPCR analyses in epithelioma papulosum cyprini (EPC) cells, which revealed that the copy number of the viral complementary RNA (cRNA), a critical replicative intermediate, is significantly higher (more than 10 times at 12-36 hours) at 20°C compared to 15°C [44]. While this suggests that higher temperatures within the permissive range can accelerate certain steps of the viral life cycle, the ultimate outcome in vivo is dictated by the host's capacity to mount a protective immune response. The paradox of faster in vitro replication at 20°C but higher in vivo mortality at 15°C highlights the dominant role of host immunity at the organismal level.

Crucially, the temperature-dependent restriction of VHSV is not absolute. While infection is not established at 25°C in olive flounder, the virus can persist and replicate at low levels within cells at this temperature without causing overt cytopathology [3]. This phenomenon has significant epidemiological implications, suggesting that sub-clinical or covert infections may occur at the upper thermal limits, potentially allowing the virus to persist in a population during warmer months and re-emerge when temperatures drop. This is supported by field surveillance data from olive flounder farms on Jeju Island, South Korea, which identified 18.7°C as a critical susceptibility threshold; detection rates were significantly higher below this temperature, but the virus was still detectable in fish and water samples at temperatures approaching this boundary [2]. The concept of a "thermal refuge" for VHSV is further reinforced by studies on Pacific herring (Clupea pallasii), where long-term shedding from convalesced individuals was linked to cooler or decreasing water temperatures, providing a mechanism for viral perpetuation during inter-epizootic periods [36].

Host Susceptibility: Age, Size, and Species as Modulating Factors

Superimposed upon the thermal landscape are intrinsic host factors that modulate susceptibility, with fish size and age being among the most critical. A comprehensive 8-month surveillance study across six olive flounder farms established 158 grams as a critical weight threshold for susceptibility; smaller fish exhibited significantly higher VHSV detection rates and viral loads [2]. This age-dependent susceptibility is a well-documented phenomenon in viral hemorrhagic septicemia, where juvenile and fingerling populations are disproportionately affected. The underlying mechanisms are multifactorial, involving the ontogeny of the immune system. In olive flounder, the expression of key immune components, such as the polymeric immunoglobulin receptor (pIgR), is developmentally regulated, with high expression observed in the late juvenile period as immune tissues mature [46]. Younger fish, with a less developed adaptive immune repertoire and potentially lower baseline levels of innate antiviral factors, are thus more vulnerable to rapid viral dissemination.

Species-specific susceptibility is another cornerstone of VHSV epidemiology, dictating the composition of host communities and the dynamics of viral reservoirs. The virus is notorious for its broad host range, infecting over 140 species, but susceptibility and reservoir competence vary dramatically. In the Laurentian Great Lakes, the introduction of VHSV genotype IVb led to massive die-offs in species such as round goby (Neogobius melanostomus), yellow perch (Perca flavescens), and several sunfish species (Lepomis spp.) [11]. Comparative surveillance in the St. Lawrence River revealed that invasive round gobies exhibited significantly higher infection prevalence and median viral titers compared to native species, identifying them as amplified reservoir hosts capable of sustaining viral transmission within the fish community [11]. This differential competence is not merely a function of exposure but reflects intrinsic differences in host-pathogen coevolution and antiviral capacity. For instance, the endangered pallid sturgeon (Scaphirhynchus albus) has been confirmed as a susceptible host for VHSV-IVb, with experimental infections demonstrating viral replication and histopathological lesions, although the degree of pathogenicity can be confounded by co-infections with other pathogens like Missouri River sturgeon iridovirus [20].

The genetic determinants of host-specific virulence have been a subject of intense investigation. Through the use of reverse genetics and chimeric viruses, it has been demonstrated that the nucleoprotein (N) and phosphoprotein (P) genes are the major determinants of VHSV virulence in rainbow trout (Oncorhynchus mykiss) [40]. This finding suggests a novel host-specific virulence mechanism involving the viral polymerase complex and its interaction with a host component, a mechanism distinct from that observed in mammalian rhabdoviruses. Further refinement of these virulence markers has identified specific amino acid residues in the N protein (K46G and A241E) and the NV protein (R116S) that drastically affect pathogenicity in rainbow trout [30]. The N protein variant K46G, for example, renders the virus unable to establish an active infection in the fins, the primary portal of entry, thereby preventing systemic spread [30]. These molecular insights explain why certain VHSV strains are highly virulent in one species but avirulent in another, a phenomenon critical for risk assessment and the development of targeted vaccines.

The Host-Pathogen Interface: Immune Modulation and Temperature-Dependent Signaling

The temperature-dependent outcome of VHSV infection is ultimately a reflection of the dynamic interplay between viral replication and host immune defense. At low, permissive temperatures (e.g., 13-15°C), VHSV not only replicates more efficiently but also exerts a more profound influence on the host transcriptome. Dual RNA-seq analyses have revealed that VHSV transcription can exceed 5% of the host transcriptome during infection, and single nucleotide variants (SNVs) appear more frequently in the virus at lower temperatures, suggesting that the selective pressure exerted by the host immune response is more intense under these conditions [5]. This is accompanied by a stronger, but often dysregulated, host immune response. At 13°C, there is a notable upregulation of genes associated with TNF signaling, necroptosis, and the NF-kappa B pathway, indicative of a robust inflammatory response [5]. However, this response may be insufficient or too slow to contain the rapidly replicating virus, leading to immunopathology and high mortality.

Conversely, at higher temperatures (e.g., 20-25°C), the host is capable of mounting a more effective and rapid antiviral state. The expression of interferon-related genes, such as ISG15 and Mx, is significantly upregulated in the spleen of olive flounder within 24 hours post-challenge at 25°C, a response that is markedly faster and more robust than at lower temperatures [3]. This early and potent interferon response is critical for establishing an antiviral state that restricts viral replication before it can reach pathogenic levels. The temperature-dependent efficacy of this response is further highlighted by studies using myeloperoxidase (MPO) inhibitors. Treatment of VHSV-infected olive flounder at 20°C with 4-aminobenzoic hydrazide (4-AH), an MPO inhibitor, increased cumulative mortality by 35%, demonstrating that the innate immune mechanisms effective at higher temperatures are actively required for protection [25]. The protective response at 20°C involves a complex signaling cascade that includes the proper translocation of GLUT4 to the cell surface to ensure adequate glucose supply for immune cell function; disruption of this pathway, as seen at lower temperatures or with MPO inhibition, leads to immune suppression and enhanced viral replication [25].

The role of specific immune cells and molecules in this temperature-dependent protection is becoming clearer. Non-specific cytotoxic cells (NCCs), the teleost equivalent of natural killer cells, play a crucial role. In olive flounder infected with VHSV at a suboptimal temperature of 17°C, there is a steady increase in the NCC population and CD8 gene expression [15]. Upon re-challenge at the optimal temperature of 10°C, a rapid and robust expansion of NCCs and increased expression of CD4 and CD8 genes are observed, demonstrating the establishment of immune memory by these innate-like cells [15]. This suggests that even at temperatures where clinical disease is suppressed, a protective immunological memory can be established, a concept that has profound implications for vaccination strategies. Furthermore, the development of a rapid neutralization assay using a chimeric rhabdovirus (rSHRV-Gvhsv-eGFP) has provided a high-throughput tool for assessing the functional antibody response, which is a key correlate of vaccine-induced protection [24].

Environmental and Anthropogenic Drivers of VHSV Emergence and Spread

Beyond the direct effects of temperature on the virus and host, broader environmental and anthropogenic factors act as powerful drivers of VHSV epidemiology. The introduction of VHSV genotype IVb into the Laurentian Great Lakes serves as a stark example of how human-mediated transport can lead to the emergence of a novel, highly virulent pathogen in a naive ecosystem. Phylogenetic analyses suggest that the virus was introduced from the northeastern Pacific Ocean (genotype IVa) and subsequently evolved into a distinct subgenogroup (IVb) that caused massive mortality in over 30 freshwater fish species [33, 39]. The evolutionary trajectory of VHSV-IVb in the Great Lakes has been characterized by continued genetic diversification, with a trend toward declining virulence over time, a pattern that may facilitate its long-term persistence in resident fish populations [33, 39]. This evolution is driven by both spatial and temporal factors, with virus populations in the Upper, Central, and Lower Great Lakes showing significant genetic divergence [39].

Environmental stressors, such as pollution, can further compromise host resistance and exacerbate VHSV outbreaks. Exposure to heavy oil (HO) in Japanese flounder (Paralichthys olivaceus) has been shown to suppress antiviral activities, leading to higher virus titers and increased mortality upon VHSV challenge [49]. Gene expression profiling revealed that HO exposure downregulates genes involved in interferon production and apoptosis induction, effectively crippling the host's ability to control viral replication [49]. This finding underscores the importance of considering environmental quality in disease risk assessments, particularly in coastal aquaculture zones that may be subject to industrial pollution. Similarly, the use of immunostimulants like polyinosinic-polycytidylic acid (poly I:C) and flagellins has been explored to enhance host resistance. Poly I:C-potentiated vaccination, for instance, has been shown to induce a robust CD4-2+ T cell response in olive flounder, providing outstanding protection against VHSV [48]. Flagellins from Marinobacter algicola can also reduce VHSV replication in trout cell lines, highlighting the potential for immunostimulatory feed additives to bolster antiviral defenses [47].

The role of asymptomatic carriers and environmental reservoirs is a critical, yet often underappreciated, aspect of VHSV epidemiology. Convalesced Pacific herring have been shown to continue shedding virus at a low rate for extended periods (at least 6 months after cessation of overt disease), and this shed virus can infect naïve sentinel fish [36]. This long-term shedding, which is linked to cooler temperatures, provides a mechanism for viral persistence in the marine environment during inter-epizootic periods. The development of environmental RNA (eRNA)-based surveillance methods offers a powerful tool for detecting these covert infections in aquaculture systems. Studies on Jeju Island have demonstrated that eRNA monitoring can detect VHSV in farm outlet water, with a qPCR CT value of 23 (~10⁶ copies/mg) serving as a reliable indicator for high-risk infections [2]. This non-invasive approach allows for the early detection of viral circulation before clinical signs appear, enabling proactive biosecurity interventions. The sensitivity of such methods can be enhanced by concentration techniques like iron flocculation, with oxalic acid buffer proving superior for preserving viral infectivity during the concentration process [45].

In conclusion, the epidemiology of VHSV is a complex, multi-factorial system where water temperature acts as the master regulator, defining the permissive window for disease outbreaks. Within this thermal framework, host susceptibility is modulated by age, size, species, and genetic background, while environmental stressors and anthropogenic activities can tip the balance toward more severe epizootics. The virus's ability to persist in covert infections and its ongoing evolutionary diversification, as seen in the Great Lakes, present ongoing challenges for disease control. A comprehensive understanding of these interacting risk factors is essential for developing predictive models, implementing targeted surveillance using advanced tools like eRNA, and designing effective vaccination and biosecurity strategies to mitigate the devastating economic and ecological impacts of VHSV.

Clinical Manifestations and Pathological Features of VHSV Infection

Viral hemorrhagic septicemia virus (VHSV) induces a complex, multi-systemic disease characterized by a spectrum of clinical manifestations that are profoundly influenced by host species, viral genotype, environmental temperature, and life stage. The clinical presentation of VHS is broadly categorized into acute, subacute, and chronic forms, though in many outbreaks, particularly those involving highly virulent strains and susceptible juvenile populations, an acute peracute course predominates. The hallmark of the disease is a severe hemorrhagic diathesis, reflecting widespread endothelial damage and a dysregulated host inflammatory response. The clinical signs, which are comprehensively documented across experimental and natural outbreaks, provide critical diagnostic clues but often require laboratory confirmation due to overlap with other septicemic conditions.

Gross Clinical Signs and Disease Progression

In susceptible species, the incubation period following exposure is temperature-dependent, typically ranging from 5 to 14 days at permissive temperatures below 15°C [3, 15, 23]. The initial clinical signs are often non-specific and include lethargy, anorexia, and a darkened body coloration, particularly along the dorsal surface and fins. As the disease progresses, the pathognomonic signs of VHS emerge. Exophthalmia (pop-eye), often bilateral, is a frequent finding, attributed to retro-orbital edema and hemorrhage. Abdominal distension due to ascites (fluid accumulation in the peritoneal cavity) is another common feature, reflecting vascular leakage and impaired osmoregulation. Petechial and ecchymotic hemorrhages are observed on the skin, particularly over the ventral abdomen, flanks, and around the bases of the fins, gill opercula, and the anus [8, 19]. The gills may appear pale or show petechiae. Anemia is a consistent finding, evidenced by pallor of the gills and internal organs, and is likely a consequence of hemorrhage and hemolysis. In severe cases, fish may exhibit a spiraling or corkscrew swimming behavior immediately prior to death, indicative of neurological involvement, although this is more commonly associated with other rhabdoviruses like infectious hematopoietic necrosis virus (IHNV). It is critical to note that the clinical picture can vary significantly; for instance, while acute mortality with pronounced hemorrhages is typical in rainbow trout (Oncorhynchus mykiss) and olive flounder (Paralichthys olivaceus), some host species, such as certain marine fish, may present with a more chronic, non-hemorrhagic syndrome characterized by lethargy and emaciation. The invasive round goby (Neogobius melanostomus) in the Laurentian Great Lakes has been identified as a key reservoir host, often carrying high viral loads without displaying overt clinical signs, which facilitates the virus's persistence and transmission to more susceptible native species like yellow perch (Perca flavescens) and sunfish species (Lepomis spp.) [11].

Gross and Histopathological Features

The pathological lesions of VHSV infection are systemic but exhibit particular predilection for the hematopoietic organs, the vascular endothelium, and the excretory and respiratory surfaces. On necropsy, the most striking findings are within the body cavity. The peritoneal cavity typically contains copious amounts of serosanguinous (blood-tinged) or clear ascitic fluid. The swim bladder may be hemorrhagic. The liver is often pale, friable, and mottled with areas of congestion and necrosis. The spleen and the anterior kidney (the primary hematopoietic and immune organs in teleosts) are frequently enlarged (splenomegaly and renomegaly), congested, and friable, with the spleen potentially showing a characteristic "nutmeg" appearance due to focal hemorrhagic necrosis [20, 25]. The intestinal tract may be hyperemic with a catarrhal exudate, and the kidney tissue can be liquefied in severe cases. Histopathological examination reveals the underlying cellular damage. The most consistent lesion is multifocal necrosis within the hematopoietic tissue of the spleen and kidney. In the kidney, necrosis of the interrenal tissue and hematopoietic cells is prominent, with a concurrent reduction in normal hematopoietic cells, directly correlating with the observed anemia and immunosuppression [20]. The liver exhibits focal to confluent coagulative necrosis of hepatocytes, often accompanied by hemorrhage. A key feature of the cytopathology is the presence of intracytoplasmic, eosinophilic inclusion bodies, particularly within hepatocytes and endothelial cells, though these are not always readily apparent and require skilled observation [10]. The gill epithelium may show hyperplasia, lamellar fusion, and necrosis, contributing to respiratory distress. In the brain, while not a primary target in all cases, perivascular cuffing, gliosis, and focal necrosis can be observed, correlating with neurological signs in some outbreaks. The extensive hemorrhages observed grossly are attributable to viral-induced damage to vascular endothelial cells, leading to increased vascular permeability and a coagulopathy, likely exacerbated by liver failure and consumption of clotting factors.

Molecular and Cellular Pathophysiology

At the molecular level, the clinical manifestations are the culmination of a complex interplay between viral replication, host innate immune activation, and immune evasion strategies employed by VHSV. The virus initially gains entry through mucosal surfaces, with the gills and skin mucus serving as primary sites of attachment and replication within 1-3 hours post-exposure at permissive temperatures (15-20°C) [4]. From these initial foci, the virus disseminates to internal organs via the bloodstream. The kinetics of viral replication are exquisitely temperature-sensitive; at 15°C, viral RNA levels in the spleen can increase more than 10-fold compared to 20°C, and at 25°C, viral replication is effectively abrogated in the host, though it can persist in cell culture [3, 5]. This temperature dependency is a cornerstone of VHSV epidemiology. The host's response is a double-edged sword. The activation of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), triggers a massive induction of type I interferons (IFN) and pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-8 [8, 15, 50]. This "cytokine storm" is a major driver of the systemic pathology. While the IFN response is crucial for limiting viral spread, the excessive and dysregulated inflammatory response contributes significantly to vascular leakage and tissue necrosis. The virus, however, has evolved sophisticated countermeasures. The non-virion (NV) protein is a key virulence factor that suppresses the host's type I IFN response and triggers host translational shut-off via the PERK-eIF2α pathway, allowing viral protein synthesis to proceed while dampening the production of antiviral effector proteins [27, 37]. Furthermore, the viral N protein directly targets the host's antigen presentation machinery by degrading STAT1, thereby inhibiting MHC class II expression and impairing the adaptive immune response [21]. This dual strategy of triggering a damaging inflammation while suppressing the specific antiviral immunity explains the intense necrosis and hemorrhagic pathology that characterizes acute VHS.

Differential Susceptibility and Host-Specific Virulence

The clinical manifestation is not a uniform entity; it is heavily modulated by viral genotype and host species. The genetic determinants of virulence have been localized to the N and P proteins, which form the viral polymerase complex [40]. Reciprocal exchange of these genes between a trout-virulent (genotype Ia) and a trout-avirulent (genotype IVb) strain can completely switch the virulence phenotype; the N gene provides an essential determinant that is strongly enhanced by the P gene [38, 40]. This suggests a host-specific adaptation mechanism where the viral polymerase complex is more efficient in interacting with a host factor(s) in the permissive species. For example, genotype Ia strains are devastating to rainbow trout, causing high mortality with classic hemorrhagic signs, whereas genotype IVb strains from the Great Lakes are essentially avirulent in trout but cause high mortality in multiple wild freshwater species like yellow perch and gobies [33, 38]. The clinical signs in these latter species can be similar (hemorrhages, exophthalmia, ascites) but often appear with a lower within-species prevalence and more variable severity. The invasive round goby, in particular, acts as a robust amplifier host, showing high infection prevalence (significantly higher than native species) and high viral titers while often remaining clinically normal, thus serving as a silent vector for the virus [11]. The specific amino acid substitutions in the N (e.g., K46G and A241E) and NV (e.g., R116S) proteins are critical molecular markers that can predict virulence in rainbow trout, with the N K46G mutation causing the virus to fail in establishing infection at the fin, its primary portal of entry [30]. This highlights the fine-tuned molecular interaction that dictates whether a host will suffer a lethal, hemorrhagic disease, or a covert, persistent infection.

Co-factors and Environmental Modulation

The clinical outcome is further compounded by environmental stressors. Exposure to sublethal concentrations of pollutants, such as heavy fuel oil, can severely suppress the host's antiviral responses. Oil-exposed fish show reduced expression of IFN and apoptosis-related genes, leading to significantly higher viral titers in target organs (e.g., heart) and increased mortality compared to virus-only controls, despite not altering the fundamental pathological lesions [49]. Similarly, underlying infections with other pathogens, such as the Missouri River sturgeon iridovirus (MRSIV) in endangered pallid sturgeon (Scaphirhynchus albus), can confound experimental outcomes and likely exacerbate VHSV pathology in natural settings, although histopathological assessments confirmed that VHSV-specific lesions (e.g., hemorrhagic gastrointestinal organs and reduced hematopoietic cells) were present only in VHSV-exposed fish [20]. Nutritional state, stress from handling or high-density stocking, and the developmental stage of the fish also influence susceptibility. Notably, smaller fish (under 158g) are significantly more susceptible to infection and clinical disease [2, 23]. In survivors of an acute outbreak, the virus can persist, and these convalescent fish can shed low levels of infectious virus for extended periods, especially associated with the gills, and this shedding is potentiated by decreasing water temperatures [2, 36]. This carrier state allows the virus to persist in a population, creating a reservoir for future epizootics. The WHO and WOAH recognize VHSV as a notifiable pathogen due to its devastating economic impact on global aquaculture and its ability to cause mass mortality in wild fish populations, disrupting aquatic ecosystems.

Diagnostic Methods: From Conventional Assays to Environmental RNA Surveillance

The accurate and timely detection of Viral Hemorrhagic Septicemia Virus (VHSV) is paramount for implementing effective control measures, preventing international spread, and managing outbreaks in both aquaculture and wild fish populations. As a notifiable pathogen to the World Organisation for Animal Health (WOAH), VHSV diagnosis must adhere to rigorous standards that balance sensitivity, specificity, speed, and practical applicability across diverse settings. The diagnostic landscape for VHSV has evolved dramatically, progressing from foundational virological techniques through advanced molecular assays to, most recently, pioneering environmental surveillance methods that promise non-invasive, population-level monitoring. This section provides a comprehensive examination of these diagnostic modalities, analyzing their underlying principles, performance characteristics, limitations, and roles within a holistic disease management framework.

Conventional Virological and Immunological Assays

The historical cornerstone of VHSV diagnosis has been virus isolation in cell culture, followed by serological or molecular confirmation. The standard protocol involves inoculating susceptible cell lines, most commonly epithelioma papulosum cyprini (EPC) cells, but also fathead minnow (FHM) cells or rainbow trout gonad (RTG-2) cells, with homogenized tissue samples from target organs such as kidney, spleen, heart, and brain [7, 22]. The identification of characteristic cytopathic effects (CPE), typically observed within 3–7 days post-inoculation, constitutes a presumptive positive result. This approach offers the distinct advantage of recovering live virus, which is essential for subsequent characterization, virulence assessment, and epidemiological tracing. However, cell culture is labor-intensive, requires specialized facilities and trained personnel, and can be relatively slow, often delaying critical management decisions during outbreaks. Furthermore, the sensitivity of isolation can be compromised by sample toxicity, inadequate preservation during transport, or the presence of low viral loads, a particular concern in carrier fish or samples from convalescent populations where long-term shedding occurs at low levels [36].

Following virus isolation, definitive identification has traditionally relied on serological methods, particularly the use of specific antisera or monoclonal antibodies in neutralization tests or immunofluorescence assays. The development of monoclonal antibodies targeting the VHSV glycoprotein (G) has been instrumental, enabling the detection of specific viral antigens with high specificity. For instance, a panel of seven anti-VHSV monoclonal antibodies was used to develop a lateral flow immunochromatographic assay (LFIA) for the rapid detection of VHSV genotype IVa in olive flounder [53]. This LFIA demonstrated a detection limit in the range of 10^5.05 to 10^5.55 TCID50 per 100 μL, with 100% specificity and 93.9% sensitivity when validated using field samples from infected fish hearts [53]. Such immunochromatographic strip tests represent a significant advancement for point-of-care diagnostics, offering results within minutes without the need for sophisticated laboratory equipment. However, their sensitivity is typically lower than that of molecular methods, making them more suitable for screening during acute outbreaks than for detecting subclinical infections.

Complementary serological approaches have focused on detecting host antibodies as a marker of past or current exposure. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect anti-VHSV antibodies, particularly non-neutralizing antibodies against the nucleocapsid (N) protein. A blocking ELISA, for example, was applied to survey wild fish populations in Wisconsin, revealing widespread seropositivity in species such as northern pike and walleye, even in water bodies geographically distant from known outbreak events [35]. This underscores the value of serology for understanding viral distribution and dynamics, but its utility is tempered by the fact that antibody levels can wane over time and are not a direct indicator of active infection. More specific antibody detection has been achieved using recombinant VHSV G protein (rgG) in an indirect ELISA format. This assay demonstrated high sensitivity, detecting antibodies in rainbow trout sera at dilutions up to 1:15,625, outperforming traditional virus neutralization tests [55]. The neutralization assay itself has been modernized through the development of recombinant reporter viruses. The chimeric snakehead rhabdovirus rSHRV-Gvhsv-eGFP, which expresses the VHSV G protein and enhanced green fluorescent protein (eGFP), allows for a rapid, high-throughput neutralization assay that reduces the time for titer determination from several days to just 24 hours [24]. This innovative approach mitigates operator bias and eliminates the need for fresh complement sources, streamlining the evaluation of protective immune responses following vaccination or natural infection.

Molecular Detection: PCR-Based Platforms and Their Refinements

The advent of polymerase chain reaction (PCR) technology revolutionized VHSV diagnostics, providing unparalleled sensitivity, specificity, and speed compared to conventional virology. The detection of viral RNA requires a reverse transcription step, yielding complementary DNA (cDNA) for subsequent amplification. Both conventional reverse transcription PCR (RT-PCR) and real-time quantitative RT-PCR (RT-qPCR) are widely employed. A direct comparison of these two methods for detecting VHSV in marine ornamental fish found that both exhibited identical sensitivity (detecting 100 copies of viral RNA) and specificity, with complete concordance in sample testing [52]. While RT-PCR is simpler and less expensive, RT-qPCR offers the critical advantages of real-time quantification, reduced risk of contamination due to a closed-tube format, and a shorter turnaround time.

The target gene for these assays is typically the nucleoprotein (N) gene, which is highly conserved and abundantly transcribed during replication. Numerous validated RT-qPCR protocols exist, including a binary multiplex RT-qPCR system (bmRT-qPCR) that has been adapted for simultaneous detection, typing, and quantification of VHSV strains from all known genotypes on a macroarray platform [54]. This system demonstrated high analytical sensitivity (5–50 TCID50/mL), specificity, and reliability, with the added advantage that pre-prepared 96-well plates could be stored at -25°C for up to one year without significant loss of efficiency [54]. Such innovations facilitate high-throughput screening and batch processing, which is essential for large-scale surveillance programs.

A critical challenge in molecular diagnostics is the risk of false-negative results due to technical failures during RNA extraction or reverse transcription, or the presence of PCR inhibitors in tissue samples. To address this, researchers have evaluated the use of heat-inactivated snakehead rhabdovirus (SHRV) as an internal positive control (IPC) for VHSV RT-qPCR [51]. SHRV was selected for its structural and genomic similarity to VHSV, and heat inactivation preserved RNA integrity without affecting assay sensitivity. The inclusion of this IPC allows for the reliable identification of false negatives, thereby enhancing the diagnostic confidence of the assay [51].

Beyond RNA detection, understanding viral replication dynamics is crucial for pathogenesis studies and evaluating antiviral interventions. Strand-specific RT-qPCR (ssRT-qPCR) has been optimized to discriminate between the three forms of VHSV RNA: genomic viral RNA (vRNA), antigenomic complementary RNA (cRNA), and messenger RNA (mRNA). By employing tagged primers, researchers successfully quantified these strands in EPC cells, revealing that the copy number of cRNA never exceeded that of vRNA, suggesting a regulatory mechanism that limits cRNA synthesis [44]. This technique has been applied to study the effects of temperature and host immune status on replication, demonstrating that higher temperatures (20°C) promote faster mRNA transcription and cRNA synthesis compared to lower temperatures (15°C) [44]. Such nuanced insights into viral replication strategies are invaluable for developing targeted control measures.

Isothermal Amplification and Point-of-Care Innovations

While PCR-based methods remain the gold standard for laboratory-based diagnosis, they require expensive thermal cyclers and skilled personnel, limiting their deployment in field settings. Isothermal amplification technologies, particularly recombinase polymerase amplification (RPA), have emerged as powerful alternatives that overcome these logistical barriers. RPA operates at a constant low temperature (typically 37–42°C) and can amplify nucleic acids to detectable levels within 15–30 minutes, making it ideal for on-site testing.

A highly sensitive RPA assay targeting the VHSV N gene was established and optimized. The assay demonstrated a detection limit of 8.3 copies/μL, outperforming conventional RT-PCR, and showed no cross-reactivity with other fish viruses [34]. When validated against 1,924 field samples, the RPA assay yielded results fully consistent with traditional RT-PCR, confirming its robustness and suitability for clinical and quarantine applications [34]. Building upon this platform, a duplex lateral flow RT-RPA (lfRT-RPA) diagnostic kit was developed for the simultaneous detection of VHSV and infectious pancreatic necrosis virus (IPNV), two major viral threats to trout farming [13]. This kit integrates RPA amplification with a lateral flow strip for visual readout, providing a truly integrated sample-to-answer system. The lfRT-RPA was nearly as sensitive as RT-qPCR for detecting VHSV, although it was approximately 10-fold less sensitive for IPNV [13]. Despite this limitation, the ability to conduct duplex testing in the field within 30 minutes represents a transformative tool for rapid outbreak response, enabling farmers and veterinarians to make informed decisions about quarantine and treatment without the delays inherent to laboratory submission.

Biosensor Technologies and Alternative Detection Platforms

The search for ever more rapid, sensitive, and user-friendly diagnostic methods has spurred the development of biosensor-based platforms. These devices convert a biological recognition event (e.g., nucleic acid hybridization, antigen-antibody binding) into a measurable physical signal, such as an electrical current or optical change. An electrochemical genosensor was fabricated for VHSV detection by immobilizing a thiolated DNA probe specific to the G gene on a reduced graphene oxide/gold nanocomposite-modified pencil graphite electrode [29]. Target hybridization was detected by changes in current and charge transfer resistance using cyclic voltammetry and differential pulse voltammetry. This genosensor achieved a limit of detection of 125 pM of DNA target within a broad linear range (10^-4 to 10^-10 M), with acceptable selectivity, stability, and reproducibility [29]. While still in the developmental stage, such biosensors hold promise for integration into portable, low-cost devices for real-time environmental or clinical monitoring.

Another innovative approach leverages synthetic binding proteins derived from the variable lymphocyte receptor B (VLRB) of hagfish, termed ccombodies. Two candidates, V4B and V4H, were identified that specifically bind to the VHSV G protein in its native conformation [9]. These ccombodies demonstrated utility in ELISA, Western blotting, and immunofluorescence assays, and the V4B candidate showed neutralizing activity by attenuating CPE in VHSV-infected cell cultures [9]. As recombinant proteins produced in bacteria, ccombodies offer a renewable and scalable alternative to monoclonal antibodies for diagnostic and potentially therapeutic applications.

Environmental RNA (eRNA) Surveillance: A Paradigm Shift in Disease Monitoring

The most significant recent advancement in VHSV diagnostics is the application of environmental RNA (eRNA)-based surveillance. This non-invasive approach detects viral RNA shed by infected fish into the surrounding water, offering the potential to monitor entire populations without the need to capture, handle, and sacrifice individual animals. This paradigm is particularly attractive for aquaculture and wild fisheries, where traditional sampling can be logistically challenging, stressful to the fish, and may underestimate true prevalence due to spatial and temporal heterogeneity in viral distribution.

The foundational premise of eRNA surveillance for VHSV rests on the understanding of viral shedding kinetics. Controlled laboratory experiments have demonstrated a strong correlation between VHSV infection intensity and viral shedding into the water. Specifically, a qPCR cycle threshold (Ct) value of 23, corresponding to approximately 10^6 copies per mg of tissue, was identified as a reliable indicator of high-risk infections that are likely to result in substantial environmental contamination [2]. The dynamics of early-stage adhesion and release are equally critical. Studies using Centricon ultrafiltration (30 kDa) to concentrate virus from seawater revealed that VHSV could be detected in the water within 1 hour post-infection of fish, with initial viral loads in tanks containing both fish and virus being initially lower than in virus-only controls before eventually equalizing [4]. This supports a model where viruses adsorb to fish surfaces (gills, mucus) and are subsequently re-released, establishing a dynamic equilibrium between the host and its aqueous environment [4]. The mucus layer, in particular, appears to play a dual role, initially trapping virus but later facilitating re-release, a process that complicates interpretation of eRNA signals but underscores the method's ability to capture active transmission.

The practical application of eRNA surveillance was rigorously evaluated in an 8-month field study conducted across six olive flounder farms on Jeju Island, South Korea [2]. Water samples were collected from farm outlet pipes and analyzed alongside traditional fish tissue samples. The results confirmed the utility of eRNA for detecting VHSV, with higher detection rates observed at lower water temperatures (<18°C) and in smaller fish (threshold weight of 158 g), both of which are recognized risk factors for VHS outbreaks. The method successfully identified farms experiencing severe infections, demonstrating its potential as an early warning system. However, sensitivity was notably limited at low infection prevalence or when viral loads in the water were low [2]. This limitation necessitates further optimization of concentration methods and sampling strategies to improve the detection of subclinical or pre-outbreak conditions.

The efficiency of viral recovery from water samples is a critical determinant of eRNA surveillance sensitivity. Iron flocculation has emerged as a robust method for concentrating VHSV from large volumes of seawater. This technique involves forming an iron hydroxide-virus floc, which is then collected and eluted. A comparative study of elution buffers found that oxalic acid was superior to ascorbic acid for preserving viral infectivity, with mean infective recovery yields of 23.8% and 4.4%, respectively [45]. While oxalic acid maintained infectivity at concentrations above 10^5 PFU/mL, recovery was insufficient at lower viral concentrations (10^2 PFU/mL, <10%), highlighting a key challenge for detecting nascent infections [45]. Nevertheless, for genomic detection via qRT-PCR, both buffers performed similarly, with mean genome recovery yields of 71.2% (oxalic acid) and 81.4% (ascorbic acid) [45].

The integration of eRNA data with risk assessment models is the ultimate goal. By establishing critical thresholds for water temperature and fish size, and correlating water Ct values with tissue viral loads, eRNA surveillance can move beyond simple detection to provide actionable risk stratification [2]. This aligns with the principles of precision aquaculture, where biosecurity measures can be dynamically adjusted based on real-time risk assessments. The WOAH-recognized need for non-lethal, population-level monitoring tools positions eRNA surveillance as a transformative technology, one that, with continued refinement, promises to become a cornerstone of VHSV management in the 21st century.

Immune Response and Vaccine Development Against VHSV

The host immune response to Viral Hemorrhagic Septicemia Virus (VHSV) is a complex, multi-layered process that is profoundly influenced by environmental temperature, viral genotype, and the specific host species. As a notifiable pathogen to the World Organisation for Animal Health (WOAH) and the cause of significant economic losses in global aquaculture, understanding these immunological mechanisms is paramount for the development of effective prophylactic and therapeutic strategies. The interplay between the virus’s sophisticated immune evasion tactics and the host’s innate and adaptive defenses dictates the outcome of infection, ranging from rapid mortality to sterile immunity or persistent, covert infection. This section provides an exhaustive analysis of the host immune landscape during VHSV infection and critically evaluates the diverse array of vaccine platforms being developed to combat this devastating pathogen.

Innate Immune Response: The First Line of Defense

The innate immune system serves as the critical frontline defense against VHSV, with its efficacy being tightly linked to water temperature. The virus is highly pathogenic at temperatures below 15°C, while infection is poorly established above 20°C, a phenomenon recapitulated at the cellular level where viral mRNA replication at 15°C is over 10-fold higher than at 25°C [3, 5]. This temperature-dependent restriction is not merely a reflection of viral polymerase kinetics but is fundamentally driven by the host’s ability to mount a robust interferon (IFN) response.

The Interferon and Interferon-Stimulated Gene (ISG) Axis

Upon VHSV entry, pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) detect viral RNA, initiating a signaling cascade that converges on the activation of interferon regulatory factors (IRFs). The critical role of IRF3 in this pathway has been definitively demonstrated using CRISPR/Cas9-mediated knockout zebrafish, where irf3-KO animals exhibit dose-dependent, early mortality upon VHSV challenge due to a severely blunted expression of type I IFNs (ifnphi1 and ifnphi2) and downstream antiviral effectors like ISG15 and viperin [60]. This deficiency in IRF3 leads to a delayed immune response, resulting in unchecked viral replication and exacerbated inflammation [60]. Interferon regulatory factor 7 (IRF7) appears to play a compensatory role later in infection, potentially driving ifnphi3 expression independently of IRF3 [60].

The antiviral state is subsequently executed by a suite of ISGs. Mx proteins and ISG15 are consistently and strongly upregulated in surviving fish and in vitro models, correlating with viral control [1, 3, 14, 61, 63, 65]. For instance, formalin- and β-propiolactone (BPL)-inactivated vaccines that confer high protection (80-90% survival) induce significant upregulation of Mx and ISG15 in the spleen [1]. Furthermore, microRNA-155 (miR-155) has been identified as a potent positive regulator of this axis; transfection of miR-155 mimics into Epithelioma papulosum cyprini (EPC) cells confers complete resistance to VHSV-induced cytopathic effect by inducing Mx1 and ISG15 expression, effectively blocking viral replication [63]. This mechanism has been successfully translated in vivo using MS2 bacteriophage virus-like particles (MS2-VLPs) to deliver miR-155, resulting in significantly lower cumulative mortality (26%) compared to controls (80%) in olive flounder [58].

Viperin represents another archetypal ISG with direct anti-VHSV activity. Viperin from sea perch (Ljviperin) is induced by both IFN-dependent and independent pathways. Its C-terminal domain is critical for exerting its antiviral function by binding to the viral nucleoprotein (N) and phosphoprotein (P), targeting them for degradation via the host’s autophagy pathway [43]. This reveals an evolutionarily conserved mechanism where the host co-opts a cellular degradation system to eliminate viral components.

The Integrated Stress Response and Stress Granules

VHSV infection activates the integrated stress response (ISR), a cellular pathway that modulates translation under stress conditions. The virus induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) primarily through PKR-like endoplasmic reticulum kinase (PERK) [17, 37]. Paradoxically, while host translation is shut off, VHSV protein synthesis proceeds efficiently despite high levels of phosphorylated eIF2α, indicating a viral strategy to hijack the translation machinery [37]. This PERK activation is also required for optimal IFN production and stress granule (SG) formation [17]. SGs, which are cytoplasmic aggregates of stalled mRNA and RNA-binding proteins, form in response to VHSV infection in a PERK-dependent manner. The SG scaffolding protein G3BP1 plays a nuanced role; its absence leads to increased IFN and antiviral gene expression but paradoxically reduces viral protein synthesis and titers, suggesting G3BP1 is co-opted by the virus for efficient replication [17]. The non-virion (NV) protein is a key mediator of this process, with NV-null VHSV mutants inducing less eIF2α phosphorylation and exhibiting reduced viral protein synthesis, underscoring the NV protein’s role in manipulating the ISR for viral benefit [37].

Adaptive Immune Response and Immune Memory

While the innate response is critical for early control, long-term protection against VHSV requires the coordinated action of the adaptive immune system, encompassing both humoral (B cell) and cell-mediated (T cell) arms.

Cell-Mediated Immunity and T Cell Dynamics

Cytotoxic T lymphocytes (CTLs) are essential for clearing infected cells. A hallmark of effective vaccination is the upregulation of CD8α, a marker of cytotoxic T cells [1, 15]. In olive flounder, VHSV infection at a suboptimal temperature (17°C) leads to a steady increase in non-specific cytotoxic cells (NCCs) and CD8 gene expression [15]. Crucially, upon re-challenge at the optimal temperature (10°C), a rapid and robust expansion of NCCs and heightened expression of both CD4 and CD8 genes occurs, demonstrating the establishment of immunological memory [15]. This memory response is also a target for vaccine enhancement. Poly (I:C)-potentiated vaccination, which involves co-administration of the TLR3 agonist with live VHSV, dramatically increases the frequency of CD4-2+ T cells, which play a dominant role in the antiviral response and drive the outstanding protective efficacy observed [48].

Humoral Immunity and the Role of B Cells

VHSV-specific immunoglobulin M (IgM) is a key correlate of protection for many vaccine platforms. Fish immunized with formalin- or BPL-inactivated vaccines show significantly elevated serum VHSV-specific IgM titers, which directly correlate with reduced viral loads and high survival [1]. The neutralizing capacity of these antibodies is directed against the viral glycoprotein (G), the major target for neutralization [24, 55]. As an example, recombinantly expressed G protein (rgG) can serve as a sensitive antigen in ELISA-based serosurveillance, capable of detecting antibodies at high dilutions [55]. This humoral response is not only protective upon vaccination but is also a marker of past exposure in wild populations, as demonstrated by widespread seropositivity against VHSV nucleoprotein in sport fishes in Wisconsin, indicating enzootic transmission at sub-outbreak levels [35]. The polymeric immunoglobulin receptor (pIgR), which mediates transport of IgM to mucosal surfaces, is also upregulated in response to VHSV, highlighting the importance of mucosal immunity [46].

Viral Immune Evasion Mechanisms

VHSV, as a highly successful pathogen, has evolved sophisticated mechanisms to subvert host immunity, primarily through its N, P, and NV proteins, ensuring its replication and spread.

Suppression of Antigen Presentation

The N protein is a potent suppressor of adaptive immunity, specifically targeting the MHC class II antigen presentation pathway. In sea perch, the N protein of VHSV directly interacts with STAT1 and enhances its K48-linked ubiquitination, leading to its degradation via the ubiquitin-proteasome pathway [21]. This degrades STAT1, a crucial transcription factor for the IFN-γ signaling cascade, thereby blocking the expression of IRF1, CIITA, and ultimately MHC-II-α and -β [21]. By crippling the ability of antigen-presenting cells to display viral peptides to CD4+ helper T cells, VHSV effectively blunts the adaptive immune response, a strategy mirroring that of many mammalian viruses.

Disruption of Interferon Signaling

The NV protein is a multifunctional virulence factor central to VHSV’s pathogenicity. Beyond its role in the ISR, the NV protein is essential for inhibiting the host type I IFN response. Recombinant VHSV lacking the NV gene (rVHSV-ΔNV) is completely attenuated in vivo, unable to block interferon production, and fails to induce disease [27]. The N-terminal region of the NV protein is critical for this function, and the dynamics of the NV gene and viral genome length are tightly linked to the suppression of the antiviral state [27]. Furthermore, the N protein itself can inhibit host defense by targeting transcription factors (IRF1 and IRF9) that upregulate viperin, creating a negative feedback loop that favors viral replication [43].

Vaccine Development: From Inactivated to Next-Generation Platforms

The quest for an effective VHSV vaccine has been a major focus of research, driven by the severe economic impact of the disease. A diverse range of platforms have been explored, each with distinct advantages and limitations.

Inactivated Virus Vaccines

Inactivated vaccines represent a traditional and safe approach. A critical factor influencing their efficacy is the method of inactivation. Comparative studies have demonstrated that formalin and β-propiolactone (BPL) are superior to binary ethylenimine (BEI) or heat treatment [1]. This difference is rooted in the structural integrity of the glycoprotein (G). Formalin and BPL preserve the disulfide bond-dependent monomeric conformation of the G protein, which is essential for inducing protective immune responses. Conversely, BEI and heat denature this structure, resulting in poorly immunogenic vaccines that yield survival rates of only 20-30% [1]. A formalin-inactivated vaccine delivered via intraperitoneal injection has shown remarkable efficacy in largemouth bass, achieving a 99% relative percent survival (RPS) and inducing a robust innate immune response characterized by upregulation of genes involved in antigen processing and presentation, necroptosis, and type I IFN regulation [14]. The practical application of inactivated vaccines is further enhanced by formulation. A chitosan-PLGA-encapsulated trivalent immersion vaccine containing formalin-killed VHSV has demonstrated 66.63% RPS against VHS, offering a non-stressful, scalable delivery route [31].

Live-Attenuated and Single-Cycle Vaccines

Reverse genetics has revolutionized VHSV vaccinology, enabling the rational design of rationally attenuated and single-cycle vaccines. A key breakthrough was the identification of virulence markers in the N (K46G, A241E) and NV (R116S) proteins; mutations in these residues drastically attenuate virulence in rainbow trout [30]. Similarly, substituting the G protein’s native signal peptide with a piscidin signal peptide (Psp) results in a recombinant virus (rVHSV-PspvG) with slower replication, enhanced ER stress, and attenuated pathogenicity in olive flounder [41].

A particularly promising strategy is the single-cycle vaccine, where the G gene is deleted (rVHSV-ΔG). This vaccine can only undergo a single round of infection and cannot spread, providing an inherent safety advantage. Remarkably, a genotype IVa-based rVHSV-ΔG vaccine demonstrated superior protection against a heterologous genotype Ia challenge in rainbow trout compared to a formalin-killed vaccine [6]. This cross-protection was associated with elevated expression of pro-inflammatory cytokines (TNF-α, IL-1β) and the chemokine CXCL8, as well as histone modifications (H3K4me3, H3K27ac) at the promoters of these genes, suggesting the induction of trained immunity (innate immune memory) [6]. This same single-cycle platform also provided cross-protection against infectious hematopoietic necrosis virus (IHNV), a related novirhabdovirus, which was independent of humoral adaptive immunity and further supports the role of trained immunity in broad-spectrum protection [62].

Other live-attenuated approaches include modifying the L protein polymerase motif. Replacing the GDNV motif with GDNQ (the motif found in most other mononegavirales) accelerates viral replication in vitro but paradoxically results in lower overall mortality in vivo [16]. Recombinant VHSVs with rearranged genomes, engineered to express heterologous antigens like the capsid protein of nervous necrosis virus (NNV), are being developed as bivalent vaccines. One such candidate (rVHSV N2G1C4) is protective against both VHSV and NNV in trout and sole, demonstrating the versatility of the VHSV genome as a delivery vector [18].

DNA Vaccines

The VHSV G protein-expressing DNA vaccine is one of the most potent preparations ever developed for fish. A vaccine encoding the native G protein can provide near-complete protection against homologous challenge. However, a DNA vaccine encoding the PspvG construct, while immunogenic, only provided partial protection (60% survival) compared to the full protection afforded by the native vG vaccine [41]. This highlights that even single amino acid changes or modifications to signal peptides can significantly impact immunogenicity. While highly effective against homologous strains, DNA vaccines have shown limited cross-protective efficacy against other novirhabdoviruses like IHNV [62].

Immunostimulants, Antivirals, and Novel Delivery Platforms

Beyond traditional vaccines, several compounds and biologics have demonstrated potent anti-VHSV activity, offering alternative pathways for disease control.

• Umbelliferone: A coumarin derivative, acts as a potent immersion therapeutic. It exhibits direct virucidal activity by potentially binding to the G protein and also modulates the host immune response by downregulating IFN-α and upregulating IFN-γ and ISG15 in the kidneys [42, 59]. A 2-hour immersion in 10 mg/L for three days improved survival by up to 90% [42].
• Ribavirin and Ivermectin: These compounds suppress VHSV replication in vitro and in vivo. Treatment with ivermectin (0.25 mg/kg) before or after infection significantly improves survival and reduces viral shedding, partly through an immunostimulatory effect involving sustained expression of Mx and TNF-α [64].
• Probiotics: The probiotic Bacillus subtilis and its secreted surfactant, surfactin, can prevent VHSV infection in intestinal epithelial cells (IECs), blocking the virus at the portal of entry and preventing systemic spread [66].
• Nanoparticle Delivery: The MS2-VLP platform provides a stable and effective method for delivering functional miRNAs (like miR-155) in vivo, representing a novel RNA-based vaccine approach [58].
• Exosome-Based Immunomodulation: Plasma-derived exosomes from VHSV-infected olive flounder (VHSV-Exo) exhibit superior immunomodulatory effects and are non-toxic, presenting a novel candidate for prophylactic applications [56].
• Flagellin Adjuvants: Recombinant flagellins, even those with deleted hypervariable regions, can reduce VHSV replication in cell lines [47].

Correlates of Protection and Pre-Existing Immunity

Identifying reliable correlates of protection is crucial for vaccine development. Viral load and shedding dynamics are key indicators. In olive flounder, a qPCR CT value of 23 (~10⁶ copies/mg) serves as a reliable threshold for identifying high-risk, actively shedding infections [2]. Vaccine efficacy is therefore measured not only by survival but by its ability to suppress viral replication below this shedding threshold.

The potential for vaccines to protect against a virus that has established long-term reservoirs in convalescent hosts is a significant challenge. Studies on Pacific herring have shown that fully recovered fish can continue to shed VHSV at low levels for at least six months, acting as reservoirs [36]. This is further complicated by the fact that pre-existing immunity does not guarantee resistance to reinfection, and the virus can persist in association with gills of recovered individuals, with shedding relapses triggered by temperature fluctuations [36]. This underscores the need for vaccines that can induce not just systemic immunity but also robust mucosal immunity to prevent establishment of persistent infection at portals of entry [4, 36]. The difficulties in applying in vitro findings to whole animal models, as highlighted in zebrafish studies, further emphasize the complexity of the in vivo immune landscape and the necessity for continued research using diverse models [57].

Control Strategies and Biosecurity Measures in Aquaculture

The management of Viral Hemorrhagic Septicemia Virus (VHSV) in aquaculture represents one of the most formidable challenges facing the global finfish industry, given the pathogen's broad host range encompassing over 140 species, its capacity for waterborne transmission, and its marked temperature-dependent pathogenesis [22, 26]. The World Organisation for Animal Health (WOAH) classifies VHSV as a notifiable pathogen, underscoring its economic and epizootic significance. Control strategies must therefore be multifaceted, integrating vaccination protocols, antiviral therapeutics, environmental surveillance, and rigorous biosecurity measures tailored to the virus’s unique ecological and molecular characteristics. The absence of any licensed commercial vaccine in most jurisdictions compounds the urgency of developing integrated management frameworks [67]. Recent advances in reverse genetics, immunomodulatory therapeutics, and environmental RNA (eRNA) surveillance have, however, dramatically expanded the toolkit available for VHSV mitigation.

Vaccine Development and Immunoprophylaxis

The cornerstone of long-term VHSV control lies in effective vaccination, yet the path to licensure has been obstructed by genotype diversity, variable immune responses across host species, and the challenge of delivery in high-density aquaculture settings. Inactivated vaccines have been the subject of intensive investigation, with the method of viral inactivation emerging as a critical determinant of immunogenicity. A comparative analysis of formalin-, binary ethylenimine (BEI)-, β-propiolactone (BPL)-, and heat-inactivation methods in olive flounder (Paralichthys olivaceus) revealed that both formalin and BPL treatment preserved the disulfide bond-dependent conformational integrity of the viral glycoprotein (G protein), which correlated with survival rates of 80% and 90%, respectively, upon homologous challenge [1]. In stark contrast, BEI- and heat-inactivated vaccines yielded only 20–30% survival, coinciding with detectable alterations in G protein monomer structure [1]. Mechanistically, BPL-inactivated vaccines elicited significant upregulation of Mx, ISG15, IL-10, IFNγ, and CD8α in the spleen, with subsequent challenge triggering robust IL-2, CD8α, and IgM responses alongside downregulation of Mx and IL-10, indicating a balanced engagement of both innate and adaptive arms [1]. These findings underscore that the structural preservation of surface epitopes is non-negotiable for inactivated VHSV vaccine efficacy.

Formalin-inactivated vaccines have also been evaluated in largemouth bass (Micropterus salmoides), where treatment with 0.05% formalin at 16°C for 48 h produced a remarkable 99% relative survival rate, accompanied by elevated serum neutralizing antibodies and transcriptional upregulation of annexin A1a, coxsackievirus and adenovirus receptor homolog, and heat shock protein 90 alpha [14]. RNA sequencing of splenic tissue revealed enrichment of differentially expressed genes in antigen processing and presentation pathways, necroptosis, and type I interferon regulation, with IRF3 and HSP90AA1.2 identified as hub genes coordinating the vaccine-induced immune response [14]. These data collectively validate the WOAH-recommended principle that vaccine formulation must prioritize antigenic fidelity.

A paradigm shift has emerged with the development of reverse genetics-based live vaccines, particularly single-cycle and gene-deleted constructs. A single-cycle VHSV genotype IVa vaccine (rVHSV-ΔG) lacking the glycoprotein gene demonstrated superior cross-protective efficacy against heterologous VHSV genotype Ia challenge in rainbow trout (Oncorhynchus mykiss) when compared to a formalin-killed counterpart [6]. Despite comparable cross-reactive antibody titers and type I interferon responses, the rVHSV-ΔG group exhibited significantly elevated expression of pro-inflammatory cytokines (tnf-α, il-1β, il-6) and the chemokine cxcl8 [6]. Crucially, elevated histone modifications (H3K4me3, H3K27ac) at the promoter regions of these cytokine genes suggested that live vaccines may induce trained immunity, a form of innate immune memory that provides broad-spectrum protection against diverse VHSV variants [6]. This mechanism was further supported by evidence that the single-cycle vaccine conferred cross-protection against infectious hematopoietic necrosis virus (IHNV), a closely related novirhabdovirus, whereas DNA vaccines encoding the VHSV G protein alone failed to do so [62]. The implication is profound: live attenuated platforms can reprogram the innate immune system to recognize conserved pathogen-associated molecular patterns, bypassing the limitations of strain-specific humoral immunity.

Reverse genetics has also enabled strategic engineering of the NV gene, a unique novirhabdovirus virulence factor. Recombinant VHSVs lacking NV or expressing N-terminally truncated NV proteins were completely attenuated in olive flounder, yet they failed to induce cytopathic effects or robust replication in vitro [27]. This attenuation correlated with derepression of Mx gene expression, confirming the NV protein’s role in suppressing type I interferon responses [27]. Conversely, insertion of an additional NV gene (rVHSV-dNV) paradoxically reduced replication efficiency due to increased genome length, though virulence remained comparable to wild-type, suggesting that genome length itself modulates interferon sensitivity [27]. These findings provide a rational basis for designing safe, replication-competent vaccines with defined attenuation markers.

The application of virus-like particles (VLPs) and nucleic acid-based vaccines represents another frontier. MS2 bacteriophage-based VLPs encapsulating precursor miR-155, a microRNA known to enhance antiviral immunity, conferred significant protection in olive flounder, with cumulative mortality of only 26% compared to 80% in PBS controls following VHSV challenge at 10⁶ PFU/fish [58]. Mature miR-155 was detected in kidney tissue three days post-immunization, confirming in vivo delivery and processing [58]. This platform exploits the intrinsic immunomodulatory capacity of miR-155 to upregulate Mx1 and ISG15 expression, thereby inhibiting viral replication at the transcriptional level [63]. Such approaches circumvent the antigenic variability of the G protein while leveraging host innate mechanisms.

Antiviral Therapeutics and Immunomodulatory Agents

In the absence of universally effective vaccines, antiviral compounds offer a critical adjunct for outbreak control, particularly during the peracute phase of epizootics when vaccination is impractical. The plant-derived coumarin umbelliferone (7-hydroxycoumarin) has emerged as a particularly promising immersion-based therapeutic. In vitro studies demonstrated that umbelliferone achieved 97% plaque reduction through direct virucidal activity, with molecular docking predicting binding to the pleckstrin homology domain of the VHSV glycoprotein [59]. Immersion treatment of olive flounder at 10 mg/L for 2 h × 3 days yielded a 90% improvement in survival, suppressed viral gene expression, and modulated innate immune responses, including downregulation of ifn-α and upregulation of ifn-γ and isg15 in the kidney [42]. Pharmacokinetic analysis revealed rapid distribution and elimination, with a Cmax lower than the in vitro IC50 (13.86 μg/mL), suggesting that immunomodulation, rather than direct virucidal activity, is the primary mechanism in vivo [42]. The compound’s half-life of 8.1 days at 15°C in seawater indicates rapid environmental degradation, a favorable property for minimizing ecotoxicological risk, though environmental safety assessments remain warranted [42].

Ribavirin and ivermectin have also demonstrated antiviral efficacy in vivo. In olive flounder, ribavirin (8.33 mg/kg) and ivermectin (0.25 mg/kg) administered prior to VHSV infection significantly improved survival rates, reduced viral shedding, and downregulated viral gene expression [64]. The persistent expression of Mx and upregulation of tumor necrosis factor-α in treated fish suggested an immunostimulatory component to their activity [64]. Similarly, α-lipoic acid (LA), a potent antioxidant, upregulated IRF7, viperin, and ISG15 expression while reducing VHSV-induced reactive oxygen species production, achieving a 38% survival rate in largemouth bass when administered post-infection [65]. The dual mechanism of LA, direct antiviral gene induction coupled with mitigation of oxidative stress, highlights the therapeutic potential of repurposed nutraceuticals.

Probiotic-based strategies represent a nascent but compelling approach to preemptive control. Bacillus subtilis and its secreted lipopeptide surfactin inhibited VHSV infection in olive flounder intestinal epithelial cells (IECs), preventing viral dissemination to internal organs [66]. Fish pretreated with B. subtilis via oral exposure showed no cytopathic effects in head kidney or spleen homogenates at 96 h post-challenge, whereas untreated fish exhibited systemic infection [66]. This protection was attributed to the physical barrier enhancement of the intestinal epithelium and the immunomodulatory properties of surfactin, which may interfere with viral attachment or entry at mucosal surfaces [66]. Given that the gills and mucus are the primary portals of VHSV entry, with viral RNA detectable within 1 h post-infection in these tissues [4], probiotic fortification of mucosal barriers could serve as a first line of defense in high-risk facilities.

RNA interference (RNAi) technologies have progressed from proof-of-concept to practical application. Short hairpin RNAs (shRNAs) targeting the NV gene of VHSV, delivered via lentiviral vectors, reduced viral titers by 99.99% in cell culture [12]. The specificity of NV targeting is strategically advantageous, as the NV protein is essential for interferon suppression; its knockdown thus restores host antiviral signaling [12]. While delivery challenges remain for in vivo application, the development of stable RNA delivery systems, such as MS2-VLPs [58], suggests that RNAi-based therapeutics may eventually be deployed as feed additives or immersion treatments.

Environmental Surveillance and Biosecurity Infrastructure

Effective biosecurity depends on early detection, and VHSV surveillance has been revolutionized by environmental RNA (eRNA) methodologies. An 8-month study across six olive flounder farms on Jeju Island, South Korea, demonstrated that eRNA-based monitoring could detect VHSV in outlet water samples, with detection rates inversely correlated with temperature [2]. A critical threshold of 18.7°C and fish weight of 158 g were identified as susceptibility inflection points; below these, the risk of infection increased markedly [2]. Quantitative PCR cycle threshold values of 23 (~10⁶ copies/mg) served as reliable indicators of high-risk infections, providing actionable cutoffs for preemptive harvesting or quarantine [2]. The eRNA approach, while less sensitive at low prevalence, offers a non-invasive, cost-effective alternative to lethal sampling and can be integrated into routine farm management.

The kinetics of viral entry and shedding inform both surveillance timing and biosecurity protocols. Immediately upon exposure, VHSV adsorbs to the gill epithelium and mucus within 1 h, with peak viral loads in these tissues occurring between 1–3 h at 15–20°C [4]. Ultrafiltration recovery of virus from seawater confirmed that adsorbed virus can be re-released into the environment, establishing an environmental reservoir that sustains horizontal transmission [4]. The minimum infective dose for horizontal transmission in juvenile olive flounder via immersion is 10³·⁴ TCID₅₀/mL at 15°C, with infection prevalence increasing linearly with viral concentration [23]. These quantitative parameters enable risk-based modeling of biosecurity interventions, for instance, reducing stocking density below 158 g per fish or raising water temperature above 18°C during high-risk periods could disrupt transmission dynamics [2, 23].

Temperature manipulation as a biosecurity measure is grounded in the well-characterized temperature-dependent replication kinetics of VHSV. At 25°C, VHSV replication is virtually abrogated in vivo, with ISG15 and Mx expression in the spleen significantly elevated as early as 24 h post-challenge [3]. Conversely, at 15°C, viral mRNA levels exceed those at 20°C by more than tenfold, and immune gene expression is delayed [3]. Dual RNA-seq analyses have revealed that at low temperatures (13°C), VHSV transcription can constitute over 5% of the host transcriptome, with single nucleotide variants accumulating more frequently, indicating active viral evolution under thermal stress [5]. The practical implication is that thermal therapy, raising water temperature to 20–25°C during an outbreak, could reduce viral replication sufficiently to allow immune clearance, provided the cultured species can tolerate such temperatures.

The identification of reservoir species is critical for biosecurity in open systems. In the St. Lawrence River, invasive round gobies (Neogobius melanostomus) exhibited significantly higher VHSV prevalence and viral titers compared to native yellow perch (Perca flavescens), sunfishes, rock bass, and brown bullhead [11]. This amplified reservoir competence means that round gobies can maintain and shed virus even at low enzootic levels, facilitating spillover into commercially valuable species [11]. Similarly, Pacific herring (Clupea pallasii) that survive VHS continue to shed low levels of virus for at least six months post-convalescence, with shedding linked to the gill-associated persistence that is reactivated by decreasing water temperatures [36]. These data argue for the inclusion of wildlife surveillance, particularly of invasive or highly susceptible forage fish, in farm-level biosecurity plans, and for the implementation of physical barriers or multi-site fallowing to break the transmission chain.

Diagnostic advances have further strengthened biosecurity. Recombinase polymerase amplification (RPA) assays targeting the N gene can detect VHSV at 8.3 copies/μL within 15 min at 37°C, surpassing traditional RT-PCR in speed while maintaining specificity [34]. Duplex lateral flow formats that simultaneously detect VHSV and infectious pancreatic necrosis virus (IPNV) have been developed, with sensitivity nearly equivalent to quantitative RT-PCR for VHSV detection [13]. These point-of-care tools enable on-site decision-making, allowing farmers to quarantine suspect stocks before laboratory confirmation. For laboratory-based surveillance, electrochemical genosensors using glycoprotein gene probes on reduced graphene oxide/gold nanocomposite electrodes achieve a limit of detection of 125 pM, with excellent reproducibility and stability [29]. The integration of such devices into automated water monitoring systems could provide real-time alerts of viral incursion.

Integrated Biosecurity Frameworks and Future Directions

No single intervention is sufficient to control VHSV in intensive aquaculture; a layered, risk-based approach is essential. This begins with the procurement of specific pathogen-free (SPF) seedstock and extends to the management of water sources, effluent treatment, and movement restrictions. The demonstration that heat-inactivated snakehead rhabdovirus (SHRV) can serve as an internal positive control for RT-qPCR diagnosis exemplifies the importance of quality assurance in surveillance programs [51]. False negatives due to RNA extraction failures or PCR inhibition can be detected through the use of such controls, reducing the risk of undetected introductions.

The development of trivalent immersion vaccines incorporating formalin-killed VHSV alongside bacterial and parasitic antigens, encapsulated in chitosan-poly(lactide-co-glycolide) nanoparticles, offers a practical solution for multi-pathogen protection in species such as olive flounder [31]. Prime-boost immersion vaccination achieved relative percent survival of 66.6% against VHSV, with significant upregulation of Ig genes in mucosal and systemic tissues [31]. The immersion route is particularly advantageous for mass vaccination of small fingerlings, circumventing the handling stress associated with injection.

From a One Health perspective, the risk of VHSV transmission to mammals, including humans, is negligible. VHSV is inactivated within 8 days at 37°C, and no cytopathic effects or replication occur in mammalian cell lines at physiological temperatures [32]. Mice intravenously injected with high doses (2.37 × 10⁵ TCID₅₀) showed no clinical signs, mortality, or infection, confirming the virus’s strict host tropism [32]. This zoonotic safety profile facilitates the use of VHSV-based platforms for heterologous antigen delivery, including the development of bivalent vaccines against fish nodaviruses [18] and even mammalian pathogens [67].

The principal challenge moving forward is the translation of these experimental advances into commercial products and field-implementable protocols. The genetic diversification of VHSV, particularly the emergence of genotype IVb in the Laurentian Great Lakes and the evolution of genotype IVa in Asian aquaculture, necessitates continuous antigenic surveillance and vaccine updating [10, 39]. The identification of the N and P proteins, rather than the G protein, as primary determinants of host-specific virulence in rainbow trout [40] suggests that live attenuated vaccines targeting polymerase complex functionality may offer broader genotype coverage than G protein-based subunit vaccines. Coupled with the trained immunity induced by single-cycle vaccines [6], the future of VHSV control lies in harnessing the host’s innate memory to transcend the constraints of antigenic variation.

References

[1] Kim J, Kim W, Shin S, Kim T, Jung S. Comparative analysis of immunogenicity for viral hemorrhagic septicemia virus (VHSV) vaccines inactivated by different methods.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.110217

[2] Kang H, Lee Y, Park J, Lee J, Heo Y, Kim N, et al.. Environmental RNA‐Based Surveillance of Viral Hemorrhagic Septicemia Virus (VHSV) in Olive Flounder (Paralichthys olivaceus) Aquaculture: Detection Dynamics and Risk Assessment. Environmental DNA. 2025. DOI: https://doi.org/10.1002/edn3.70083

[3] Jang Y, Yoon S, Krishnan R, Oh M. Temperature-dependent viral hemorrhagic septicemia virus (VHSV) infection kinetics and immune response in primary olive flounder spleen cell culture and the host.. Virology. 2025. DOI: https://doi.org/10.1016/j.virol.2025.110390

[4] Yoon S, Jang Y, Kim S, Oh M. Early‐Stage Adhesion Dynamics of Viral Hemorrhagic Septicemia Virus in Olive Flounder, Paralichthys olivaceus. Journal of Fish Diseases. 2025. DOI: https://doi.org/10.1111/jfd.70005

[5] Kang G, Roh H. Dual RNA-seq Analyses of Viral Hemorrhagic Septicemia Virus and Olive Flounder (Paralichthys olivaceus) Interactions at Low and High Water Temperatures.. Developmental and Comparative Immunology. 2025. DOI: https://doi.org/10.1016/j.dci.2025.105335

[6] Lee KM, Kim SY, Kim KH. Superior protective efficacy of genotype IVa-based single-cycle viral hemorrhagic septicemia virus (VHSV) vaccine compared to formalin-killed VHSV vaccine against VHSV genotype Ia in rainbow trout (Oncorhynchus mykiss).. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.110409

[7] Xu Y, Tang T, Wang J, Dou W, Wang N, Zhou Y, et al.. Isolation and characterization of viral hemorrhagic septicemia virus from farmed bastard halibut.. Diseases of Aquatic Organisms. 2025. DOI: https://doi.org/10.3354/dao03840

[8] Ghaderzadeh M, rahimi-Mianji G, Nejati-Javaremi A, Shahbazian N. Transcriptomic and biometric parameters analysis in rainbow trout (Oncorhynchus mykiss) challenged with viral hemorrhagic septicemia virus (VHSV). BMC Genomics. 2025. DOI: https://doi.org/10.1186/s12864-025-11300-x

[9] Rodelas-Angelia AJD, Angelia MRN, Jeong S, Kim YR, Choi MG, Kim H, et al.. Development of a neutralization Ccombody, synthetic hagfish antibody, for viral hemorrhagic septicemia virus (VHSV) targeting glycoprotein G.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.110994

[10] Xu Y, Tang T, Wang N, Ding C, Zhou Y, Wang F, et al.. Genome analysis of viral hemorrhagic septicemia virus isolated from Paralichthys olivaceus in China. Scientific Reports. 2025. DOI: https://doi.org/10.1038/s41598-025-17471-7

[11] Haws AL, Getchell R, Farrell JM. Distribution of viral hemorrhagic septicemia virus in the nearshore fish community of the St. Lawrence River.. Journal of Aquatic Animal Health. 2025. DOI: https://doi.org/10.1093/jahafs/vsaf011

[12] Dehkordi NP, Saffar B, Mokhtari A, Samani LA, Amini A. Utilizing shRNA-expressing lentivectors for viral hemorrhagic septicemia virus suppression via NV gene targeting. Frontiers in Veterinary Science. 2025. DOI: https://doi.org/10.3389/fvets.2025.1508470

[13] Tamer C, Kuruçay H, Gözel S, Müftoglu B, Kökluoğlu EN, Kadı H, et al.. Implementation of a Recombinase Polymerase Amplification-Driven Duplex Lateral Flow Diagnostic Kit for Concurrent Detecting Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus: A Comparative Analysis of RPA and PCR Methods.. Journal of Fish Diseases. 2025. DOI: https://doi.org/10.1111/jfd.70029

[14] Huang H, Lu X, Guo J, Chen Y, Yi M, Jia K. Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa.. Fish and Shellfish Immunology. 2024. DOI: https://doi.org/10.1016/j.fsi.2024.109691

[15] Jang Y, Yoon S, Krishnan R, Oh M. Kinetics of viral hemorrhagic septicemia virus (VHSV) and immune response in olive flounder (Paralichthys olivaceus) at altered temperature.. Fish and Shellfish Immunology. 2024. DOI: https://doi.org/10.1016/j.fsi.2024.110004

[16] Kwak JS, Kim JY, Kim KH. Effect of mutations in GDNV motif of viral hemorrhagic septicemia virus (VHSV) L protein on polymerase activity, viral growth, and in vivo virulence.. Virology. 2024. DOI: https://doi.org/10.1016/j.virol.2024.110257

[17] Ramnani B, Powell S, Shetty AG, Manivannan P, Hibbard BR, Leaman D, et al.. Viral Hemorrhagic Septicemia Virus Activates Integrated Stress Response Pathway and Induces Stress Granules to Regulate Virus Replication. Viruses. 2023. DOI: https://doi.org/10.3390/v15020466

[18] Souto S, Mérour E, Coupanec AL, Lamoureux A, Bernard J, Brémont M, et al.. Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection. Frontiers in Immunology. 2023. DOI: https://doi.org/10.3389/fimmu.2023.1138961

[19] Biasini L, Zamperin G, Pascoli F, Abbadi M, Buratin A, Marsella A, et al.. Transcriptome Profiling of Oncorhynchus mykiss Infected with Low or Highly Pathogenic Viral Hemorrhagic Septicemia Virus (VHSV). Microorganisms. 2023. DOI: https://doi.org/10.3390/microorganisms12010057

[20] Hopper LR, Glenn J, MacConnell E, Winton J, Emmenegger E. Susceptibility of Pallid Sturgeon to viral hemorrhagic septicemia virus genotype IVb.. Journal of Aquatic Animal Health. 2023. DOI: https://doi.org/10.1002/aah.10181

[21] Lu X, Li W, Guo J, Jia P, Zhang W, Yi M, et al.. N Protein of Viral Hemorrhagic Septicemia Virus Suppresses STAT1-Mediated MHC Class II Transcription to Impair Antigen Presentation in Sea Perch, Lateolabrax japonicus. Journal of Immunology. 2022. DOI: https://doi.org/10.4049/jimmunol.2100939

[22] Remington J. viral hemorrhagic septicemia virus. CABI Compendium. 2022. DOI: https://doi.org/10.1079/cabicompendium.66348

[23] Yoon S, Jang Y, Kim S, Krishnan R, Oh M. Determination of the minimum infective dose of viral hemorrhagic septicemia virus (VHSV) in juvenile olive flounder, Paralichthys olivaceus using an immersion challenge model. Virus Research. 2023. DOI: https://doi.org/10.1016/j.virusres.2023.199305

[24] Lee KM, Kim DH, Kim KH. Development of rapid neutralization assay of viral hemorrhagic septicemia virus (VHSV) based on chimeric rhabdovirus expressing heterologous glycoprotein.. Journal of Virological Methods. 2022. DOI: https://doi.org/10.1016/j.jviromet.2022.114639

[25] Cho S, Kim S, Vaidya B, Kwon J, Kim D. Identification of rearing temperature-dependent host defense signaling against viral hemorrhagic septicemia virus infection.. Fish and Shellfish Immunology. 2022. DOI: https://doi.org/10.1016/j.fsi.2022.03.003

[26] Nigar K, Kakakhel S, Khan A, Khan H, Zaib K, Wen S. Population genetic analyses unveiled genetic stratification and differential natural selection signatures across the G-gene of viral hemorrhagic septicemia virus. Frontiers in Genetics. 2022. DOI: https://doi.org/10.3389/fgene.2022.982527

[27] Abdellaoui N, Kim SY, Kim KH, Kim MS. Effects of Non-Virion Gene Expression Level and Viral Genome Length on the Replication and Pathogenicity of Viral Hemorrhagic Septicemia Virus. Viruses. 2022. DOI: https://doi.org/10.3390/v14091886

[28] Kim SY, Kwak JS, Jung W, Kim MS, Kim KH. Compensatory mutations in the matrix protein of viral hemorrhagic septicemia virus (VHSV) genotype IVa in response to artificial mutation of two amino acids (D62A E181A). Virus Research. 2023. DOI: https://doi.org/10.1016/j.virusres.2023.199067

[29] Moattari G, Izadi Z, Shakhsi-Niaei M. Development of an electrochemical genosensor for detection of viral hemorrhagic septicemia virus (VHSV) using glycoprotein (G) gene probe. Aquaculture. 2021. DOI: https://doi.org/10.1016/J.AQUACULTURE.2021.736451

[30] Baillon L, Mérour E, Cabon J, Louboutin L, Vigouroux E, Alencar ALF, et al.. The Viral Hemorrhagic Septicemia Virus (VHSV) Markers of Virulence in Rainbow Trout (Oncorhynchus mykiss). Frontiers in Microbiology. 2020. DOI: https://doi.org/10.3389/fmicb.2020.574231

[31] Kole S, Dar S, Shin S, Jeong H, Jung S. Potential Efficacy of Chitosan-Poly (Lactide-Co-Glycolide)-Encapsulated Trivalent Immersion Vaccine in Olive Flounder (Paralichthys olivaceus) Against Viral Hemorrhagic Septicemia Virus, Streptococcus parauberis Serotype I, and Miamiensis avidus (Scuticociliate). Frontiers in Immunology. 2021. DOI: https://doi.org/10.3389/fimmu.2021.761130

[32] Ho DT, Kim N, Yun D, Kim K, Kim J, Jang GI, et al.. Potential harmful effects of viral hemorrhagic septicemia virus in mammals. Fisheries and aquatic sciences. 2022. DOI: https://doi.org/10.47853/fas.2022.e29

[33] Niner MD, Stepien CA, Gorgoglione B, Leaman D. Genomic and immunogenic changes of Piscine novirhabdovirus (Viral Hemorrhagic Septicemia Virus) over its evolutionary history in the Laurentian Great Lakes. PLoS ONE. 2021. DOI: https://doi.org/10.1371/journal.pone.0232923

[34] Weili Y, Wei H, Liu X, Ni Y, Bai Y, Qing S, et al.. Establishment of recombinase polymerase amplification technology for detecting Viral Hemorrhagic Septicemia Virus (VHSV) in cultured aquatic animals. Aquaculture. 2021. DOI: https://doi.org/10.1016/J.AQUACULTURE.2020.736338

[35] Thiel W, Toohey-Kurth K, Giehtbrock D, Baker BB, Finley MA, Goldberg T. Widespread Seropositivity to Viral Hemorrhagic Septicemia Virus (VHSV) in Four Species of Inland Sport Fishes in Wisconsin.. Journal of Aquatic Animal Health. 2021. DOI: https://doi.org/10.1002/aah.10120

[36] Hershberger P, Mackenzie A, Gregg J, Wilmot M, Powers RL, Purcell M. Long-term shedding from fully convalesced individuals indicates that Pacific herring are a reservoir for viral hemorrhagic septicemia virus.. Diseases of Aquatic Organisms. 2021. DOI: https://doi.org/10.3354/DAO03595

[37] Kesterson SP, Ringiesn JL, Vakharia V, Shepherd B, Leaman D, Malathi K. Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway. Viruses. 2020. DOI: https://doi.org/10.3390/v12050499

[38] Yusuff S, Kurath G, Kim MS, Tesfaye TM, Li J, McKenney DG, et al.. The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout. Virology Journal. 2019. DOI: https://doi.org/10.1186/s12985-019-1139-3

[39] Stepien CA, Niner MD. Evolutionary trajectory of fish Piscine novirhabdovirus (=Viral Hemorrhagic Septicemia Virus) across its Laurentian Great Lakes history: Spatial and temporal diversification. Ecology and Evolution. 2020. DOI: https://doi.org/10.1002/ece3.6611

[40] Vakharia V, Li J, McKenney DG, Kurath G. The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout. Journal of Virology. 2019. DOI: https://doi.org/10.1128/JVI.00382-19

[41] Bessaid M, Kim KH. Effect of exchange of viral hemorrhagic septicemia virus (VHSV) G protein's signal peptide with piscidin signal peptide on virus replication and immunogenicity.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.110455

[42] Mendis WRH, Kirindage KGIS, Ahn G, Jung S, Kim T, Kang S. Umbelliferone immersion therapy controls viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus) via direct virucidal and immunomodulatory effects.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.111015

[43] Lu X, Yi M, Hu Z, Yang T, Zhang W, Marsh E, et al.. Feedback loop regulation between viperin and viral hemorrhagic septicemia virus through competing protein degradation pathways. bioRxiv. 2024. DOI: https://doi.org/10.1101/2024.01.09.574905

[44] Lee EG, Kim KH. Effect of temperature and IRF-9 gene-knockout on dynamics of vRNA, cRNA, and mRNA of viral hemorrhagic septicemia virus (VHSV).. Fish and Shellfish Immunology. 2023. DOI: https://doi.org/10.1016/j.fsi.2023.108617

[45] Ryu N, Baek E, Kim M, Kim K. In Vitro Viral Recovery Yields under Different Re-Suspension Buffers in Iron Flocculation to Concentrate Viral Hemorrhagic Septicemia Virus Genotype IVa in Seawater. Animals. 2023. DOI: https://doi.org/10.3390/ani13050943

[46] Kim K, Park S, Park J, Jeong M, Kim J, Kim H, et al.. Analysis of Polymeric Immunoglobulin Receptor Expression in Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus. Development & Reproduction. 2023. DOI: https://doi.org/10.12717/DR.2023.27.2.67

[47] Nuñez-Ortiz N, Díaz‐Rosales P, García JA, Serra C, Enes P, Tafalla C, et al.. Immunostimulant properties of full-length and truncated Marinobacter algicola flagellins, and their effects against viral hemorrhagic septicemia virus (VHSV) in trout.. Fish and Shellfish Immunology. 2022. DOI: https://doi.org/10.1016/j.fsi.2022.08.018

[48] Chun JH, Jung J, Kim YR, Lazarte J, Kim SW, Kim J, et al.. Poly (I:C)-Potentiated Vaccination Enhances T Cell Response in Olive Flounder (Paralichthys olivaceus) Providing Protection against Viral Hemorrhagic Septicemia Virus (VHSV). Vaccines. 2021. DOI: https://doi.org/10.3390/vaccines9050482

[49] Song J, Kitamura S, Oh M, Nakayama K. Heavy oil exposure suppresses antiviral activities in Japanese flounder Paralichthys olivaceus infected with viral hemorrhagic septicemia virus (VHSV).. Fish and Shellfish Immunology. 2022. DOI: https://doi.org/10.1016/j.fsi.2022.03.046

[50] Kim K, Kim HC, Park C, Park J, Lee YM, Kim W. Interleukin-8 (IL-8) Expression in the Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus (VHSV) Challenge. Development & Reproduction. 2019. DOI: https://doi.org/10.12717/DR.2019.23.3.231

[51] Choi MG, Kim D, Kim H, Kim KH. Evaluation of Inactivated Snakehead Rhabdovirus as an Internal Positive Control for RT-qPCR Diagnosis of Viral Hemorrhagic Septicemia Virus in Fish.. Journal of Virological Methods. 2025. DOI: https://doi.org/10.1016/j.jviromet.2025.115128

[52] Abdulgani N, Sudaryatma PE, Berliana NPS. Comparison of RT-PCR and rRT-PCR Methods in Detection of Viral Hemorrhagic Septicemia Virus (VHSV) in Marine Ornamental Fish. BIO Web of Conferences. 2024. DOI: https://doi.org/10.1051/bioconf/20248902007

[53] Kong K, Jeong H, Shyam K, Oh M, Kim C, Kim H, et al.. Development and validation of a lateral flow immunochromatographic assay for specific detection of viral hemorrhagic septicemia virus (VHSV, genotype IVa) in olive flounder (Paralichthys olivaceus). Aquaculture. 2021. DOI: https://doi.org/10.1016/J.AQUACULTURE.2021.736491

[54] López-Vázquez C, Bandín I, Dopazo C. Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV). Animals. 2021. DOI: https://doi.org/10.3390/ani11030841

[55] Tamer C, Gumusova S, Albayrak H. Detections of Rainbow Trout Antibodies to Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus via a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay. Aquaculture Studies. 2021. DOI: https://doi.org/10.4194/aquast669

[56] Nikapitiya C, Jayathilaka E, Edirisinghe S, Oh C, Zoysa MD. Characterization, MicroRNA Profiling, and Immunomodulatory Role of Plasma-Derived Exosomes from Olive Flounder (Paralichthys olivaceus) in Response to Viral Hemorrhagic Septicemia Virus.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.110316

[57] Shanaka K, Madushani K, Kim M, Jung S, Lee J. Virus Infection Models: Zebrafish as an Infection Model to Study Immune Landscape During Viral Hemorrhagic Septicemia Virus (VHSV) Infection. Reviews in Aquaculture. 2025. DOI: https://doi.org/10.1111/raq.70011

[58] Kim SY, Kim Y, Kim MS. Protective efficacy of miR-155 encapsulated using MS2 bacteriophage-based virus-like particle (MS2-VLP) against viral hemorrhagic septicemia virus (VHSV) in olive flounder.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.111000

[59] Mendis WRH, Lim J, Jung S, Kang S. Antiviral effects of umbelliferone against viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus).. Fish and Shellfish Immunology. 2024. DOI: https://doi.org/10.1016/j.fsi.2024.109767

[60] Sellaththurai S, Jung S, Nadarajapillai K, Kim M, Lee J. Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model.. Developmental and Comparative Immunology. 2024. DOI: https://doi.org/10.1016/j.dci.2024.105208

[61] Choi H, Choi D, Park J, Jung H, Kim B, Hong S, et al.. Protective Effects of Long Double-Stranded RNA with Different CpG Motifs against Miamiensis avidus and Viral Hemorrhagic Septicemia Virus (VHSV) Infections in Olive Flounder (Paralichthys olivaceus). Fishes. 2024. DOI: https://doi.org/10.3390/fishes9060227

[62] Kim SY, Lee KM, Kim KH. Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).. Vaccine. 2023. DOI: https://doi.org/10.1016/j.vaccine.2023.07.058

[63] Abdellaoui N, Kim D, Jeon Y, Kim MS. Antiviral effect of miR-155 in Epithelioma papulosum cyprini (EPC) cells against viral hemorrhagic septicemia virus (VHSV) infection.. Fish and Shellfish Immunology. 2023. DOI: https://doi.org/10.1016/j.fsi.2023.108937

[64] Baek E, Kim MJ, Kim K. In vitro and in vivo evaluation of the antiviral activity of arctigenin, ribavirin, and ivermectin against viral hemorrhagic septicemia virus infection.. Fish and Shellfish Immunology. 2022. DOI: https://doi.org/10.1016/j.fsi.2022.108456

[65] Zhang W, Chen X, Yu F, Li F, Li W, Yi M, et al.. α-Lipoic Acid Exerts Its Antiviral Effect against Viral Hemorrhagic Septicemia Virus (VHSV) by Promoting Upregulation of Antiviral Genes and Suppressing VHSV-Induced Oxidative Stress. Virologica Sinica. 2021. DOI: https://doi.org/10.1007/s12250-021-00440-5

[66] Han S, Munang'andu H, Yeo I, Kim S. Bacillus subtilis Inhibits Viral Hemorrhagic Septicemia Virus Infection in Olive Flounder (Paralichthys olivaceus) Intestinal Epithelial Cells. Viruses. 2020. DOI: https://doi.org/10.3390/v13010028

[67] Kim MS, Kim KH. Genetically engineered viral hemorrhagic septicemia virus (VHSV) vaccines.. Fish and Shellfish Immunology. 2019. DOI: https://doi.org/10.1016/j.fsi.2019.10.031