Author: marina02

Development of a Vaccine Against Pasteurella atlantica: Addressing Emerging Health Challenges in Norwegian Salmon Aquaculture

Pasteurella atlantica is an emerging pathogen in Norwegian salmon aquaculture, with increasing outbreaks and severity since 2018. Fish health biologists and aquaculture farmers emphasize the urgent need for vaccination to control this disease, which manifests as blood-filled blisters in muscle and inner organs, inflammation, and the characteristic “bloody eye.” Our study shows two distinct variants of P. atlantica in Norway. The variant found in salmon is genotypically highly homogeneous and differs from the variant found in lumpsucker as well as the separate species P. skyensis. These differences are confirmed through ELISA and Western blotting. We have established a clinical disease model and demonstrated protection in fish vaccinated with inactivated P. atlantica. Efficacy studies reveal a dose-dependent protective effect in Atlantic salmon challenged with P. atlantica genomovar salmonicida*.

*The pre-clinical studies and the use of research animals were approved by the Norwegian Food Authorities and studies were performed in accordance with procedures approved by Zoetis Animal Ethics Committee.

1. Kamisinska, Agnieszka, Pharmaq AS, part of Zoetis, Presenter
2. Brudeseth, Bjørn, Pharmaq AS, part of Zoetis, Author
3. McAdam, Martin E., Pharmaq AS, part of Zoetis, Author
4. Heen, Vegar, Pharmaq AS, part of Zoetis, Author
5. Aeed, Paul, Pharmaq AS, part of Zoetis, Author
6. Gauthier, David, Pharmaq AS, part of Zoetis, Author
7. Sandlund, Liv, Pharmaq AS, part of Zoetis, Author
8. Alward, Larry, Pharmaq AS, part of Zoetis, Author
9. Klevan, Are, Pharmaq AS, part of Zoetis, Author

Atlantic cod gill poxvirus case in the gills of Atlantic cod suffering from cardiorespiratory disease

Atlantic cod (Gadus morhua), is an economically important fisheries in the North Atlantic. In Norway, attempt at Atlantic cod farming began few decades ago, with partially successful, but production collapsed around 2012 because of infectious disease problem. In the last four years, there has been a steady growth in the Atlantic cod industry in Norway, but diseases still cause problems with intestinal volvolus, spawning problems, circulatory disturbances and infectious diseases reported to be a great concerns. Increasing the knowledge of disease status in farmed Atlantic cod, represents a first and essential step for enhancing biosecurity and disease management to protect the Atlantic cod industry. Here, we characterized disease case in Atlantic cod from a Norwegian cod farm, which experienced increased mortality.

1. WELI, SIMON, NORWEGIAN VETERINARY INSTITUTE, AS, NORWAY, Presenter
2. Tengs, Torstein, 2Department of Breeding and Genetics, Nofima, Ås, Norway, Author
3. Nilsen, Hanne, NORWEGIAN VETERINARY INSTITUTE, AS, NORWAY, Author
4. Mohammad, Saima, NORWEGIAN VETERINARY INSTITUTE, AS, NORWAY, Author
5. Gjessing, Mona C, NORWEGIAN VETERINARY INSTITUTE, AS, NORWAY, Author

Establishment of SsB-1, a Solea senegalensis brain cell line – A versatile platform for fish virus research and transgene expression

Introduction
Cell lines serve as essential models in animal physiology and immunopathology, significantly minimizing the reliance on live animals in scientific research and supporting the 3Rs principle of reduction, refinement, and replacement. The Senegalese sole (Solea senegalensis) stands out as a highly promising flatfish species for Spanish aquaculture. Establishing cell lines from this species would greatly benefit the research community and contribute to reducing the need for laboratory animals.
Methodology:
Here, we established a new brain cell line from Senegalese sole, named SsB-1, target tissue for various neurotropic viruses. This cell line was characterised for its potential application in virology. We evaluated its susceptibility to a range of viruses relevant to aquaculture, as well as its immune response to sublethal infections. Additionally, the cell line’s ability to be transfected and express transgenes using various DNA plasmids was also addressed.
Results:
The SsB-1 cell line exhibits high susceptibility to the aquabirnavirus eel virus (EVE) and the alloherpesvirus Anguillid herpesvirus 1 (AnHV), while showing no susceptibility to the other viruses tested (EVEX, RGNNV, IPNV, SVCV). Noticeable, all viruses analysed triggered antiviral immune response except EVE. Also, the SsB-1 cell line can be transfected using transfection reagents and can express transgenes. Additionally, whilst functionalized polystyrene nanoparticles are able to enter into the cells, they were proven to be cytotoxic when used as transfection vehicles.
Conclusions:
The development of SsB-1 cell line from sole brain will serve as valuable tools for immunovirology research, especially regarding to neurotropic viruses, enhancing our understanding of viral susceptibility and immune responses while reducing the reliance on laboratory animals.
Funding: This research was carried out thanks to grant PID2022-139492NB-I00, funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU by the European Union, to A.C., as well as the grant IJC2020-042733-I from MICIU/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR, to Y.V.

1. CUESTA, ALBERTO, UNIVERSIDAD MURCIA, Presenter
2. VALERO, YULEMA, UNIVERSIDAD MURCIA, Author

High-Throughput Screening of Antiviral Candidates Using VHSV as an RNA Virus Surrogate Model

RNA viruses, including those responsible for infectious diseases such as influenza, COVID-19, Ebola, and rabies, are highly prone to mutation. The high genetic variability and infectious nature of RNA viruses have led to increasing demand for the development of broad-spectrum antiviral agents. A surrogate virus serves as a safer alternative when biosafety level (BSL) regulations limit the handling of actual pathogenic viruses. Viral Hemorrhagic Septicemia Virus (VHSV), a single-stranded negative-sense RNA virus, is a marine virus with excellent experimental accessibility and no infectivity in humans, making it advantageous in terms of biosafety. In this study, we aimed to identify potential antiviral compounds suitable for drug development by using VHSV as a surrogate of RNA viruses. We conducted high-throughput screening of 17,265 chemical compounds and 2,100 natural extracts in Epithelioma papulosum cyprini (EPC) cells infected with a recombinant VHSV (rVHSV) expressing enhanced green fluorescent protein (eGFP). Screening was conducted based on cytopathic effect (CPE) observation and fluorescence-based analysis. The screening process identified three chemical compounds and one natural extract as candidate agents exhibiting antiviral activity against VHSV. Among the hits, ethyl 1-[(2-oxo-1,3,4,5-tetrahydro-1-benzazepin-7-yl)sulfonyl]piperidine-3-carboxylate, exhibited an infectivity rate of 0.0149% at 125μM, with an IC₅₀ value of 8.2μM. Cell viability at 800μM was 66.45%, and the CC₅₀ was estimated to exceed 800μM. These results suggest that this compound represents a promising candidate for further evaluation against a broad spectrum of RNA viruses affecting both humans and animals. Further studies will include additional in vitro validation of the compound’s efficacy and evaluation of its antiviral activity against pathogenic viruses beyond the surrogate model.

1. Kim, Su Yeon, PUKYONG NATIONAL UNIVERSITY, Presenter
2. Kim, Su Yeon, PUKYONG NATIONAL UNIVERSITY, Author
3. Shin, jiwoo, PUKYONG NATIONAL UNIVERSITY, Author
4. Jang, SuJin, PUKYONG NATIONAL UNIVERSITY, Author
5. Ahn, Heejin, PUKYONG NATIONAL UNIVERSITY, Author
6. Sung, Min-Kwan, PUKYONG NATIONAL UNIVERSITY, Author
7. Jo, So hyeon, PUKYONG NATIONAL UNIVERSITY, Author
8. Choi, Tae-Jin, PUKYONG NATIONAL UNIVERSITY, Author

Comparative Gene Expression and Viral Load Analysis in Distinct Carp Groups Infected with Koi Herpes Virus (KHV)

anju sebastian1*, mikolaj adamek2, ehdaa eltayeb eltigani abdelsalam1, anna martyrosyan1, ľubomír pojezdal3, kateřina matějíčková3, veronika piačková1

1university of south bohemia in české budějovice, faculty of fisheries and protection of waters, south bohemian research center of aquaculture and biodiversity of hydrocenoses, czech republic, sebastian@frov.jcu.cz

2university of veterinary medicine hannover, germany

3veterinary research institute, czech republic

Infectious disease prevention and treatment are continually evolving. One promising approach to reduce losses from infectious diseases in aquaculture is to use more resistant strains and hybrids. This experiment aimed to investigate the immune response (IgM) to Koi herpesvirus (KHV) across various carp species, strains, and hybrids: Cyprinus carpio (m2, m2 x al), C. rubrofuscus (as, koi), and Ctenopharyngodon idella (grass carp). Additionally, the study aimed to explore gene expression in these fish groups’ gills and head kidney.

Fish were infected through cohabitation with KHV-intraperitoneally-infected koi. The virus was detected in the gills of the fish as early as 7 days post-infection (dpi), with koi and all other fish groups showing similar viral loads. At 14 dpi, the viral load was particularly high in koi and the m2 strain, while the grass carp exhibited the lowest viral load. However, by 21 dpi, a noticeable decline in viral load was observed across all fish groups, and this level remained relatively stable until 35 dpi.

Regarding mortality, a high rate was observed in the hybrid m2 x al from 14 dpi to 35 dpi, and the lowest occurred for the as group, whereas no mortality occurred in the grass carp throughout the experiment. Specific antibody concentrations were analyzed from the blood serum using indirect ELISA and virus neutralization assay.

1. SEBASTIAN, ANJU, UNIVERSITY OF SOUTH BOHEMIA, Presenter

Molecular Cloning and Characterization of Peroxiredoxin-6 (Prx6) from seven-band grouper (Hyporthodus septemfasciatus)

Peroxiredoxin 6 (Prx6) is a member of the 1-Cys Prx family that primarily catalyzes the reduction of peroxides and peroxynitrites, and is involved in H₂O₂-mediated signal transduction, cell differentiation, proliferation, and gene expression. Nervous necrosis virus (NNV), a major etiological agent causing mass mortality in juvenile sevenband groupers (Hyporthodus septemfasciatus) during seed production, encodes the B2 protein from its subgenomic RNA3. The B2 protein induces mitochondria-mediated necrotic cell death and stimulates oxidative stress via hydrogen peroxide production, which in turn activates host antioxidant enzyme expression and modulates viral replication during early infection.

In this study, the full-length cDNA of HsPrx6 was identified from sevenband grouper. The ORF is 666 bp in length, encoding a 221-amino-acid protein with a predicted molecular mass of 24.27 kDa and an isoelectric point of 5.98. Multiple sequence alignment and homology analysis confirmed the conservation of the 1-Cys Prx signature motif (⁴⁴PVCTTE⁴⁹) and catalytic triad (T⁴³, C⁴⁶, R¹³⁰) across species. Phylogenetic analysis revealed that HsPrx6 is closely related to Prx6 homologs in other teleosts.

Expression analysis showed that HsPrx6 was significantly upregulated in the kidney following NNV infection. Functional assays demonstrated that recombinant HsPrx6 (rHsPrx6) protected covalently closed circular DNA from H₂O₂-induced oxidative damage in a dose-dependent manner. These findings suggest that HsPrx6 may act as an antioxidant in response to NNV-induced oxidative stress, contributing to cellular protection during viral infection.

1. JUNG, JAE-EUN, PUKYONG NATIONAL UNIVERSITY, Presenter
2. SEO, JUNG-MIN, PUKYONG NATIONAL UNIVERSITY, Author
3. LIM, CHAE-WON, PUKYONG NATIONAL UNIVERSITY, Author
4. JEON, SO-YEON, PUKYONG NATIONAL UNIVERSITY, Author
5. BYUN, JEONG-WON, PUKYONG NATIONAL UNIVERSITY, Author
6. Jeong, Ji-hyun, PUKYONG NATIONAL UNIVERSITY, Author
7. OH, MYUNGJOO, CHONNAM NATIONAL UNIVERSITY, Author
8. KIM, JONG-OH, PUKYONG NATIONAL UNIVERSITY, Author

Molecular Cloning and Characterization of Septin2 (Sept2) from seven band grouper (Hyporthodus septemfasciatus)

The sevenband grouper (Hyporthodus septemfasciatus) is a commercially important species in Asian aquaculture, particularly in South Korea. However, it is highly susceptible to nervous necrosis virus (NNV), a virulent pathogen that infects over 120 marine and freshwater fish species. NNV induces high mortality rates during the larval and juvenile stages, posing a significant threat to aquaculture productivity and causing substantial economic losses. Recent studies suggest that Septin2 (Sept2) plays a role in antiviral defense by interacting with immune-related genes such as heat shock proteins and interferons during viral infection. Septin2, a member of the SEPT2 subgroup, is presumed to contribute to cytoskeletal organization and innate immune responses. However, the functional roles of fish Septins, particularly in host-pathogen interactions involving NNV, remain largely unexplored. To investigate the molecular characteristics of Septin2 in teleosts, we cloned the full-length Septin2, gene from sevenband grouper and analyzed its expression. The cDNA was inserted into the pET-28a(+) expression vector using NdeI and XhoI restriction sites, and transformed into Escherichia coli BL21(DE3) cells. Optimal protein expression was achieved by induction with 1.0 mM IPTG at 37°C for 6 hours and confirmed via SDS-PAGE. The open reading frame of Septin2 is 1,083 bp in length and encodes a protein of 360 amino acids, with a predicted molecular mass of 41 kDa as determined using the ExPASy Compute pI/Mw tool. Septin2 was found to contain a canonical GTPase domain (pfam00735), including characteristic G1–G5 motifs and Switch regions, as identified using the NCBI Conserved Domain Database. These features are typical of the P-loop NTPase superfamily and support the protein’s role in cytoskeletal regulation. Furthermore, Phylogenetic analysis conducted using MEGA11 with the Maximum Likelihood method and 1,000 bootstrap replicates revealed that H. septemfasciatus Septin2 shares 100% sequence identity with Epinephelus lanceolatus (accession no. XP_033506927.1) and forms a distinct clade separate from mammalian homologs, indicating evolutionary divergence between teleosts and mammals. Collectively, these findings confirm the conserved nature of Septin2 in fish and provide a molecular basis for further investigation into its antiviral function against NNV.

1. JEON, SO YEON, PUKYONG NATIONAL UNIVERSITY, Presenter
2. JUNG, JAE EUN, PUKYONG NATIONAL UNIVERSITY, Author
3. SEO, JEONG MIN, PUKYONG NATIONAL UNIVERSITY, Author
4. PARK, JI HWAN, PUKYONG NATIONAL UNIVERSITY, Author
5. LIM, CHAE WON, PUKYONG NATIONAL UNIVERSITY, Author
6. LEE, SONGYI, PUKYONG NATIONAL UNIVERSITY, Author
7. OH, MYUNG JOO, CHONNAM NATIONAL UNIVERSITY, Author
8. KIM, JONG OH, PUKYONG NATIONAL UNIVERSITY, Author

The infection dynamics and immune response of the causative poxvirus in the course of the development of atypical cellular gill disease in ayu Plecoglossus altivelis

Atypical cellular gill disease (ACGD) in ayu caused by Plecoglossus altivelis poxvirus (PaPV) has led to significant economic losses in aquaculture. This disease manifests the appearance of atypical cells and fusion of secondary gill lamella, but the process of symptom development, virus infection dynamics, and host response are still unknown. In this study, we attempted to reveal the virus propagation and immune response in the course of disease development in experimentally infected ayu.

A PaPV core protein gene was inserted into a plasmid DNA vector to produce recombinant protein in E. coli, and a polyclonal antibody was obtained from a rabbit immunized with the protein. Ayu (4.5g) were challenged by immersion in rearing water added with the virus solution derived from homogenate ACGD dead fish gills for the infected group or MEM for the control group, and then the fish were kept at 18℃ in an aquarium with running water for 3 weeks. Virus DNA copy number in the rearing water and the organs in fish periodically sampled were measured in quantitative PCR, and virus antigen was detected on the gill sections using the polyclonal antibody. Immune-related gene expression in the gills and spleen were analyzed. In an additional experiment, mRNA for early and late PaPV-expressed proteins in each organ were detected by RT-PCR.

The cumulative mortality rate reached 80%, and acute mortality occurred. Histopathological examination of the gills revealed the appearance of many atypical cells and gill clubbing just before mortality occurred. Virus antigens were also detected in atypical cells at the site of hyperplasia. The amount of virus DNA in the rearing water increased after the middle stage of infection and peaked before mortality occurred. A similar increasing tendency in virus DNA was detected in the gills, heart, spleen, and fins. Virus mRNA was detected only in the gills and fins. Gene expression analysis showed upregulation of IL-1β, Granzyme A, and IFN-γ in the gills or spleen. These results indicate that PaPV propagates only in the gills and fins, and typical symptoms of ACGD develop during the significant virus increase, as well as strong immune responses including cellular proliferation in the gills.

1. Baba, Shuntaro, Tokyo University of Marine Science and Technology, Tokyo, Japan, Presenter
2. Mori, Tatsuya, Tochigi Prefectural Fisheries Experimental Station, Tochigi, Japan, Author
3. Takagi, Yuya, Tochigi Prefectural Fisheries Experimental Station, Tochigi, Japan, Author
4. Wada, Shinpei, Nippon Veterinary and Life Science University, Tokyo, Japan, Author
5. Kondo, Hidehiro, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
6. Matsumoto, Megumi, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
7. Kato, Goshi, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
8. Sano, Motohiko, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author

A Chlorella Virus Surrogate System for High-Throughput Screening of Antiviral Agents Against DNA Viruses

Emerging fish DNA viruses such as Red Sea Bream Iridovirus (RSIV) have significant social, cultural and economic impacts. Due to the lack of effective vaccines or treatments, outbreaks are currently managed by culling infected animals to prevent transmission, resulting in substantial economic losses. Therefore, there is an urgent need to find effective substances for the control of these and related viruses. However, the presence of only limited cell culture system for many fish viruses, like RSIV, making large-scale screening efforts challenging. Phycodnaviridae is a family of large double-stranded DNA viruses that infect marine or freshwater eukaryotic algae. It belongs to the group of Nucleocytoplasmic Large DNA Viruses (NCLDVs), along with RSIV, and shares genetic and structural similarities with other members of this group. In this study, we used a Chlorella-infecting virus, PKV2, a member of the Phycodnaviridae, that had been isolated in Korea as a surrogate model for high-risk DNA viruses for the mass screening of antiviral candidates. A screening system in which a mixture of PKV2 and its host, Chlorella variabilis NC64A, was distributed to 96-well plates containing screening material. After 4 days of incubation, wells containing intact Chlorella cells were selected as primary candidates and subjected to spot and plaque reduction assay for secondary screening. Among a total of 19,365 compounds and extracts, 120 and 17 substances were selected from the primary and secondary screening, respectively. To validate the surrogate virus system, in vitro tests were performed using the Dwarf Gourami Fin (DGF) cells, a permissive host cell line for RSIV, treated with selected substances. Cytotoxicity (CC₅₀) was assessed using the WST assay, while antiviral efficacy (IC₅₀) was evaluated based on viral infectivity measured by the TCID₅₀ method. This surrogate virus system is expected to provide foundational data for the discovery and development of antiviral agents targeting high-risk DNA viruses.

1. Jang, SuJin, PUKYONG NATIONAL UNIVERSITY, Author
2. Jang, SuJin, PUKYONG NATIONAL UNIVERSITY, Presenter
3. Kim, Su Yeon, PUKYONG NATIONAL UNIVERSITY, Author
4. Shin, jiwoo, PUKYONG NATIONAL UNIVERSITY, Author
5. Choi, Tae-Jin, PUKYONG NATIONAL UNIVERSITY, Author
6. Ahn, Heejin, PUKYONG NATIONAL UNIVERSITY, Author
7. Sung, Min-Kwan, PUKYONG NATIONAL UNIVERSITY, Author
8. Jo, So hyeon, PUKYONG NATIONAL UNIVERSITY, Author

Outbreaks of Infection Haemopoietic Necrosis (IHN) in Denmark

On the 18th of May 2021, Denmark declared its first outbreak of infectious hematopoietic necrosis (IHN) ever. Denmark was previously declared free from infection with IHN virus and had surveillance in place according to EU Regulation 2021/620. By the end of September, 11 cases of IHNV were declared – eight in fish farms producing rainbow trout and three in put&take lakes. The presence of IHNV in Denmark was also confirmed in 2022 and 2023.

In order to trace the origin of the Danish IHNV isolates and the links among the infected farms and lakes, a detailed molecular investigation of the different isolates present in Denmark was carried out.

For this, the full genome was sequenced for all positive cases of IHNV in Denmark from its introduction in 2021 to the last positive case detected in 2023.

Phylogenetic analyses were conducted based on 1) a large dataset including the full G-gene sequences of all currently available IHNV isolates, and 2) the full genome of all Danish cases and all other IHNV isolates genogroup E available in public databases.

Phylogenetic analyses show no evidence of multiple introductions of IHNV into the country nor of its presence in the country previous to its detection in 2021. Instead, our analyses indicate that, after IHNV was introduced in Denmark, it rapidly spread infecting geographically distant fish farms. Full genome analysis has allowed the establishment of possible transmission routes that, for the cases detected in 2021, were confirmed by an epidemiological investigation done by the Danish authorities.

1. Cuenca, Argelia, DTU AQUA, Presenter
2. Vendramin, Niccolò, DTU AQUA, Author
3. Olesen, Niels Jørgen, DTU AQUA, Author