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Given the lack of antiviral prophylactic measures authorized in aquaculture, vaccination is the most effective method to prevent and control diseases provoked by viral agents. Viral hemorrhagic septicemia virus (VHSV) is an important fish pathogen, known to induce high mortality rates and large economic losses worldwide in many aquaculture fish species, including rainbow trout (Oncorhynchus mykiss). DNA vaccination against VHSV has been proven a very effective method to induce a long-term protection against the virus, yet the mechanisms through which this vaccine induces protection are still unclear. To provide further insight on this matter, in the current study, we have studied the transcriptional response of leukocytes obtained from vaccinated fish when they were exposed in vitro to the virus. For this, fish were intramuscularly (i.m.) injected with the VHSV vaccine or mock-vaccinated with either the empty plasmid or saline solution and 30 days post-vaccination sacrificed to isolate kidney and spleen leukocytes. Isolated leukocytes were then exposed or not to inactivated VHSV and the transcription of a range of genes related to the antiviral response analyzed. Supernatants were also taken from these cultures and tested for antiviral activity. Our results revealed that leukocytes obtained from vaccinated fish, especially those derived from kidney, transcribed some specific antiviral genes and specially MDA5 at higher levels when exposed to the virus, correlating with an increased secretion of antiviral factors to the supernatants. The results obtained provide important insights on how leukocytes from fish effectively vaccinated respond to a posterior viral encounter, information of value to optimize this and other antiviral vaccines for use in aquaculture and establish correlates of protection. •Kidney leukocytes from VHSV DNA vaccinated fish, when exposed to VHSV, transcribe higher levels of ifn1, ifn2 and irf7.•Kidney leukocytes from vaccinated fish, when exposed to VHSV, transcribe higher levels of mda5 than those of mock-vaccinated fish.•The differential response of DNA vaccinated fish to VHSV is more pronounced in kidney than in spleen.•Supernatants from leukocytes obtained from DNA vaccinated fish have a basal antiviral activity.

•Two microRNAs (miR-462/-731) are upregulated in fish immunized with a DNA vaccine.•miR-462 and miR-731 were also induced by poly I:C and interferons.•Inhibiting miR-462 and miR-731 reduced the protective effect of poly I:C.•miR-462 and miR-731 directly contribute to interferon-mediated protection. Mechanisms that account for the high protective efficacy in teleost fish of a DNA vaccine expressing the glycoprotein (G) of Viral hemorrhagic septicemia virus (VHSV) are thought to involve early innate immune responses mediated by interferons (IFNs). Microribonucleic acids (miRNAs) are a diverse class of small (18–22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes and are emerging as critical regulators of cellular processes, including immune responses. We have recently reported that miR-462 and miR-731 were strongly induced in rainbow trout infected with VHSV. In this study, we analyzed the expression of these miRNAs in fish following administration of the DNA vaccine and their potential functions. Quantitative RT-PCR analysis revealed the increased levels of miR-462, and miR-731 in the skeletal muscle tissue at the site of vaccine administration and in the liver of vaccinated fish relative to empty plasmid backbone-injected controls. The increased expression of these miRNAs in the skeletal muscle correlated with the increased levels of the type I interferon (IFN)-inducible gene Mx, type I IFN and IFN-γ genes at the vaccination site. Intramuscular injection of fish with either type I IFN or IFN-γ plasmid construct resulted in the upregulation of miR-462 and miR-731 at the site of injection, suggesting that the induction of these miRNAs is elicited by IFNs. To analyze the function of miR-462 and miR-731, specific silencing of these miRNAs using anti-miRNA oligonucleotides was conducted in poly I:C-treated rainbow trout fingerlings. Following VHSV challenge, anti-miRNA-injected fish had faster development of disease and higher mortalities than control fish, indicating that miR-462/731 may be involved in IFN-mediated protection conferred by poly I:C.

Viral hemorrhagic septicemia virus (VHSV) is a major pathogen in aquaculture worldwide, causing significant outbreaks through waterborne transmission. This study aimed to evaluate the effectiveness of environmental RNA (eRNA)‐based surveillance for detecting VHSV in aquaculture systems, focusing on the relationship between infection dynamics, water temperature, and fish weight. Over an 8‐month period, VHSV prevalence was assessed in fish and outlet water samples from six olive flounder farms in Jeju Island, South Korea. Higher detection rates were observed at lower temperatures (< 18°C) and in smaller fish, with 18.7°C and 158 g identified as critical susceptibility thresholds. Controlled laboratory experiments further confirmed a strong correlation between VHSV shedding and infection intensity, with a qPCR CT value of 23 (~106 copies mg−1) serving as a reliable indicator for high‐risk infections. The eRNA method successfully detected VHSV in farms with severe infections, demonstrating its potential for noninvasive disease surveillance, although sensitivity was limited at low infection rates. These findings provide actionable insights for early detection, biosecurity enhancement, and sustainable aquaculture management, particularly in temperature‐sensitive regions like Jeju Island. This study supports the integration of eRNA‐based surveillance into routine disease monitoring, contributing to both aquaculture sustainability and environmental health.

Red sea bream iridovirus (RSIV) and Viral hemorrhagic septicemia virus (VHSV ) are the most important viral marine pathogens in South Korea because RSIV and VHSV infect and cause high mortality rates in major fish species such as Paralichthys olivaceus and Sebastes schlegelii . These viruses can be transmitted both vertically and horizontally, and early diagnosis is imperative. In this research, RSIV and VHSV viral genomes are detected by PCR-lateral flow assay (LFA). PCR-LFA is sensitive, capable of detecting a viral genome at a concentration of 2–200 fg/µL. Development of this detection method is very meaningful because LFA is simple, requiring a minimum of personnel training to perform. Additionally, LFA requires less time than other detection methods and can be an immediate detection tool that is indispensable in preventing rapid viral spread.

Even though DNA vaccination has proven as one of the most effective methods in controlling fish rhabdoviruses, the immune mechanisms responsible for protection are still unknown. Many studies have focused on studying which cytokines and immune genes are triggered in response to the vaccine at different times post-vaccination. However, to elucidate the mechanism(s) responsible for protection, to our understanding it is also of great relevance to study the immune response to the virus in fish that have been previously vaccinated and compare it to the effects that the virus might have on non-vaccinated fish. This type of study has never been performed to date in fish. Thus, in the current work, we vaccinated rainbow trout ( Oncorhynchus mykiss) with a DNA vaccine against viral hemorrhagic septicemia virus (VHSV), and 30 days post-vaccination we challenged the fish with a virulent VHSV. It was then, that we studied the immune response to the virus at very early times post-infection in fish, in order to compare the effects of VHSV on vaccinated or non-vaccinated trout. We studied the levels of expression of interleukin 1β (IL-1β), major histocompatibility complex (MHC) class Iα and IIα genes, immunoglobulin M (IgM), CD8α, type I interferon (IFN), Mx, IFN-γ and natural killer enhancing factor (NKEF) in head kidney, spleen and blood. When we compared the effect that VHSV had on vaccinated fish to the effect that the virus produced in fish vaccinated with the empty plasmid, the genes that were significantly up-regulated were IL-1β and MHC IIα in the spleen at day 1 post-infection, MHC Iα in all organs at day 1 post-infection, and IFN and Mx in the spleen and blood at days 1 and 3 post-infection, respectively. Genes that correlate with an increased specific immune response were not significantly increased in response to VHSV in these vaccinated animals. The results suggest that DNA vaccination induces a memory state in fish that, on the contrary to what would occur in mammals, primes the non-specific immune responses upon a later encounter with the virus.

Viral hemorrhagic septicemia virus (VHSV) is a problematic pathogen in olive flounder (Paralichthys olivaceus) aquaculture farms in Korea. Thus, it is necessary to develop a rapid and accurate diagnostic method to detect this virus. We developed a quantitative RT-PCR (qRT-PCR) method based on the nucleocapsid (N) gene sequence of Korean VHSV isolate (Genogroup IVa). The slope and R2 values of the primer set developed in this study were −0.2928 (96% efficiency) and 0.9979, respectively. Its comparison with viral infectivity calculated by traditional quantifying method (TCID50) showed a similar pattern of kinetic changes in vitro and in vivo. The qRT-PCR method reduced detection time compared to that of TCID50, making it a very useful tool for VHSV diagnosis.

Olive flounder (Paralichthys olivaceus) is one of the most economically important aquaculture fish. However, its production is often affected by various diseases, especially viral hemorrhagic septicemia virus (VHSV) that cause serious economic losses. In this study, we sequenced the whole transcriptome of the P. olivaceus using Illumina RNA-sEq. De novo assembly of control and virus-infected cDNA libraries of head kidney at 13 and 20 °C was accomplished with 2,007,532,438 raw reads, resulting in 244,578 unigenes with an average length of 533 bp and found 65,535 candidate coding unigenes with homology to other species by BLAST analysis. DEG analysis among control and virus-infected head kidney samples of 13 and 20 °C revealed that 1290 up-regulated and 162 down-regulated genes (p ≤ 0.01), linked to metabolism, virulence factors, adhesion and immune-response. We constructed an expressed gene catalog for the P. olivaceus to serve as a resource for marine environmental genomic and immuno-genetic/genomic studies focused on uncovering the molecular mechanisms underlying the responses of P. olivaceus to VHSV under different temperature.

The protection conferred by antiviral DNA vaccines in fish is known to rely greatly on innate immune responses. Since oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG motifs) have been shown to induce potential innate immune responses, we have introduced several copies (either two or four) of a fragment containing multiple CpG sequences of known immunostimulatory effects into a DNA vaccine against viral hemorrhagic septicemia virus (VHSV). We have determined the effects of this introduction on the vaccine immunogenicity, measured as immune gene induction, serum neutralizing activity and antigen-dependent proliferation. When comparing the effects of the vaccine containing 2 copies of this CpG fragment (pVHSV-2CpG) or that containing 4 copies of the fragment (pVHSV-4CpG) with the original VHSV DNA vaccine (pVHSV), we found that the levels of expression of type I interferon (IFN) were significantly up-regulated in muscle and spleen when the CpG fragments were introduced. An up-regulation in the levels of MHC-I expression in spleen were also observed in response to the modified vaccines, whereas, the levels of transcription of interleukin 1β (IL-1β) were strongly reduced in comparison to the original vaccine. Important but very variable differences were also observed concerning the vaccine induction of IFN-γ. Moreover, the serum neutralizing capacity was strongly increased as fish were vaccinated with plasmids containing more CpG fragments. Taken together, all these results demonstrate a strongly increased immunogenicity of the VHSV DNA vaccine, through the introduction of this multicopy CpG fragment.

Researchers have only begun to study the role of shipping in the spread of invasive species in the Laurentian Great Lakes despite a well-documented history of introductions in these lakes due to ballast water release. Here, we determine whether ballast water discharge was a likely vector of spread of the fish disease, viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb), throughout the Great Lakes and St. Lawrence Seaway. Three models were developed to assess whether the spread of VHSV was due to (1) chance (random model), or (2) ballast water discharge (location model), and whether (3) increased propagule pressure, as measured by the number of visitations by ships carrying ballast water from VHSV infected areas, increased the likelihood of a discharge location becoming infected with VHSV (propagule pressure model). The third model was also used to assess the probable point of initial introduction of VHSV. Presence and absence accuracies and weighted Cohen’s kappa were calculated to determine which models best predicted observed presences and absences of VHSV. Location models explain the patterns of VHSV detections better than random models, and inclusion of “propagule pressure” often improved model fit; however, the relationship is weak likely because of a long lag time between introduction and detection, a high rate of false negatives in reporting, and the possible contribution of other vectors of spread. Montreal was also identified as the more likely introduction site of VHSV, rather than Lake St. Clair, the site where the virus was first detected.

Outbreaks of infectious diseases among freshwater fish damage the aquaculture industry. A technique for detecting pathogens in environmental water samples has recently been developed. In the present work, the monitoring of pathogenic viruses in Lake Erhai and Lake Dianchi (Yunnan, China) was performed. Yunnan possesses a rich diversity of cyprinid fish, and the aquaculture of freshwater fish is a highly prosperous industry in this region. Thus, Cyprinid herpesvirus 1, 2, and 3 as well as three rhabdoviruses, Infectious hematopoietic necrosis virus, Viral hemorrhagic septicemia virus, and the Spring viremia of carp virus, all of which have severe impacts on aquaculture, were targeted. Viruses in lake surface water were concentrated and then quantified/detected by real-time PCR/real-time reverse transcription PCR. Five of the six tested viruses were detected in lake water. Although there has been no reported viral outbreak in the survey lakes, our results indicate a potential risk for these viral diseases.