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Ranaviruses are responsible for mass die offs of wood frog (Lithobates sylvaticus) tadpoles. What happens in between epidemics is less clear, but juvenile (metamorphosed) stages are hypothesized to move Ranaviruses among wetlands and introduce or reintroduce these viruses into wetlands, initiating new outbreaks. A key question is under what circumstances can juvenile L. sylvaticus infect susceptible conspecifics. We examined Ranavirus transmission between juvenile L. sylvaticus in two settings: first, we measured transmission from a Ranavirus-infected frog to a co-housed susceptible frog via cohabitation over a range of exposure periods. Second, we measured indirect transmission to susceptible frogs from a contaminated environment after a range of waiting times (i.e., from when the infected frog was removed to when the susceptible frog was exposed to the environment). We present evidence that juvenile frogs directly transmitted Ranavirus to susceptible frogs in all exposure periods (99.2% infected), with as little as 1 h of co-housing resulting in 95.8% of susceptibles infected. Indirectly, 96.8% of susceptible frogs became infected after as long as 48 h waiting times. Neither exposure period nor wait times influenced the probability of infection, because these probabilities are already high. In our linear regression models, susceptible frog viral load was significantly correlated with exposure period and cohort for the cohabitation experiment, while wait time was significantly correlated with viral load for the sequential habitation experiment. Collectively, our results suggest that Ranavirus transmission readily occurs between recently metamorphosed wood frogs and via terrestrial environmental exposure., Competing Interests: Declarations. Conflict of interest: The authors declare that there are no conflicts of interest. Ethical approval: Animal research occurred under UConn IACUC protocol #A20-017. Consent to participate: Not applicable. Consent for publication: Not applicable., (© 2025. The Author(s).)

Background: Tilapia lake virus (TiLV) is an emerging threat to the global tilapia industry. In early 2017, FAO declared that Bangladesh was in a high-risk zone of TiLV disease (TiLVD) spread., Objectives: Considering the current scenario, the present work was designed to investigate the occurrence of TiLV in cultured Nile tilapia (Oreochromis niloticus) causing TiLVD in Bangladesh., Methods: In this study, several unusual tilapia mortality incidences were investigated. A total of 102 pooled organ samples of 102 tilapia fish were collected from 34 ponds of 25 farms in the regions of 12 Upazilas in Bangladesh from May to October 2019. As a part of the classical approach, histopathological analysis was performed. For molecular identification, both conventional and real-time PCR were used as diagnostic tools to identify TiLV in farm-raised Nile tilapia., Results: A total of 12 representative organ samples did not show any pathognomonic lesions related to TiLV infection in histopathological analysis. Conventional and real-time PCR assays yielded negative results for TiLV., Conclusions: In our investigation, no trace of TiLV was detected in the studied samples. A large-scale study involving a broader range of samples collected across Bangladesh, along with the application of a viral metagenomics approach, could provide valuable insights into the unusual mortalities of tilapia in the country., (© 2025 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Fermentation of Astragalus by Lactobacillus plantarum and Bacillus coagulans can increase the release of active components and degrade its macromolecular substances. This study investigated the effect of fermentation products (Astragalus + L. plantarum + B. coagulans, ALB) on largemouth bass. We specifically focused on growth performance, serum biochemical indices, intestinal microbial diversity, intestinal enzyme activity, immune gene expression and resistance to infections by Aeromonas hydrophila and largemouth bass ranavirus (LMBRaV). The largemouth bass were divided into five groups based on the amount of ALB added to the feed as following, (1) ALB0 (no ALB, ALB0.5 [0.5% addition of ALB], ALB1 [1% addition of ALB], ALB3 [3% addition of ALB], ALB5 [5% addition of ALB]). The feeding trial spanned 28 days. Comprehensively comparing the feeding results of different ALB concentration, the ALB0.5 group showed the best effect. The ALB0.5 group had significantly increased weight gain rate, alkaline phosphatase, total protein, albumin, digestive enzymes activities of lipase, trypsin and increased intestinal villi and thickness of muscularis propria. And it decreased feed conversion ratio, aspartate aminotransferase and alanine aminotransferase of largemouth bass. Furthermore, the ALB0.5 group improved the richness and diversity of the intestinal microbiota. Increased abundance of dominant phylum and genus in the intestine of largemouth bass included Fusobacteria and Cetobacterium, which promoted the growth and immune performance of largemouth bass. After infection with A. hydrophila and LMBRaV, the survival rates were higher in ALB addition experimental groups than in the ALB0 group, respectively. And the survival rate of ALB0.5 group was higher than other groups. Meanwhile, the ALB added to the feed could regulated the immune gene expression (Mx, IRF-3, TNF-α, IL-1β and IL-10), which also promoted the largemouth bass resistance to disease. In summary, adding 0.5% ALB to the diet of largemouth bass can boost its growth performance, immune genes expression, intestinal health and disease resistance., (© 2024 John Wiley & Sons Ltd.)

Captive, pelagic red crabs (Pleuroncodes planipes) had shortened lifespans and cutaneous black spots. Histologically, epidermal and mesenchymal cells had 3-5 µm diameter intranuclear inclusions, necrosis, vacuolation, and/or hyperplasia. The remaining organs were histologically unremarkable. Ultrastructurally, nuclei contained rod-shaped, 70-100nmx250-350 nm virions with a double membrane. PCR on 32 animals was negative for WSSV, and suggestive of a novel virus. The genomic sequence of the virus could not be retrieved due to the low amount of DNA obtained from the paraffin embedded tissue. This is consistent with a novel viral infection, and is the first description of a viral disease in pelagic red crabs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

During viral infection, RIG-I-like receptors (RLRs) are cytoplasmic pattern recognition receptors that recognize and bind to viral RNA components, initiating the transcription of interferon-related genes, inflammatory cytokines and other factors, thereby triggering the cellular production of an antiviral innate immune response. The protein inhibitor of activated signal transducer and activator of transcription (STAT) (PIAS) protein family has become a hot research topic due to its extensive involvement in the regulation of cytokines, inflammatory factors and innate immune signaling pathways. In the present study, we investigated the role of fish PIASy in Singapore grouper iridovirus (SGIV) and red spotted grouper nervous necrosis virus (RGNNV) infections. The homologous sequence of orange-spotted grouper (Epinephelus coioides) PIASy gene (EcPIASy) was cloned and characterized, which encoded a 498-amino acid protein with 99.20 % homology to Plectropomus leopardus. EcPIASy is expressed mainly in gills, blood, and liver. Subcellular localization showed that EcPIASy was uniformly distributed in the nucleus. Overexpression of EcPIASy promoted SGIV and RGNNV replication, and inhibited the expression of interferon related genes and pro-inflammatory factors induced by viruses. In addition, EcPIASy interacts with RLR signaling pathway-related genes EcMDA5, EcIRF3 and EcIRF7, whereas the interaction between EcPIASy and EcIRF3 does not depend on any specific structural domain of EcPIASy. The results provide a better understanding of the relationship between PIASy and viral infection in fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reports in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Nuclear factor-κB (NF-κB)/p65, a vital signaling molecule in the NF-κB pathway, participates in diverse physiological functions and host-virus interactions. However, the involvement of NF-κB/p65 in fish virus infection remains poorly understood. In this study, we explored the role of the p65 in virus infection and its impact on IL-18 regulation in largemouth bass (Micropterus salmoides). Bioinformatics analysis showed that the ORF sequence of Msp65 spanned 1941 bp, encoding 646 amino acids with two conserved functional domains, including RHD and IPT domain. Msp65 mRNA was presented in various tissues, with higher levels detected in the liver and gill. After exposure to largemouth bass virus (LMBV), red grouper nervous necrosis virus, lipopolysaccharide and poly (I:C), Msp65 expression was activated in vivo. In addition, the antiviral role of Msp65 were explored. In vitro, Msp65 overexpression hindered LMBV replication and formation of viral assembly site. In vivo, we found that disruption of Msp65 by using maslinic acid (MA) notably promoted the infectivity of LMBV, indicating its antiviral capabilities in largemouth bass. Besides, the downregulation of Msp65 suppressed the expression of inflammatory and interferon signaling molecules. Conversely, Msp65 overexpression boosted the activities of IFN-I, IFN-III and ISRE promoters, suggesting the positive regulation of Msp65 on interferon immune pathway. Furthermore, to unveil the regulatory role of Msp65 on MsIL-18, a promoter investigation was conducted. The luciferase reporter assay demonstrated that Msp65 positively influenced the expression of MsIL-18. Subsequent analysis suggested that the putative binding sites for MsIL-18 could potentially reside within the -228 to -203 bp of the MsIL-18 promoter. These findings illustrated that Msp65 involved in LMBV infection by modulating immune responses, presenting a novel insight into the antiviral mechanisms of p65 in bony fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Infectious spleen and kidney necrosis virus (ISKNV) and nervous necrosis virus (NNV) are two common and important causative agents in marine-cultured fish. However, high viral loads of both ISKNV and NNV in the same clinical case is unusual. In this study, a mass mortality event of Asian seabass Lates calcarifer juveniles occurred in Zhuhai, the main Asian seabass cultured area in mainland China. The fish samples were pooled for pathogen identification and both high viral loads of ISKNV and NNV were detected by real-time microfluidic quantitative PCR and conventional PCR. Immunohistochemistry and immunofluorescence showed that strong ISKNV signals were detected in spleen and liver, while strong NNV signals were detected in brain and eye. The tissue homogenates were inoculated into MFF-1 cell and SSN-1 cell, respectively. After several viral passages, both individual ISKNV and NNV were purely isolated from each other, and designated as ASB-ISKNV-23 and ASB-NNV-23, respectively. The whole genome sequences of ASB-ISKNV-23 and ASB-NNV-23 were determined and annotated. The result showed that ASB-ISKNV-23 and ASB-NNV-23 are composed of 112,236 bp and 4441 bp, respectively. Phylogeny analysis showed that ASB-ISKNV-23 belongs to ISKNV-II sub-genotype and ASB-NNV-23 belongs to RGNNV genotype. Collectedly, coinfection of ISKNV-II and RGNNV were firstly documented in mass mortality of Asian seabass in mainland China. Importantly, both individual ISKNV-II and RGNNV were purely isolated using two different permissive cell lines. Our study provides useful information for better understanding the complex pathogenesis regarding the coinfection with ISKNV and NNV in farmed fish., (© 2024 John Wiley & Sons Ltd.)

Frog virus 3-like ranaviruses (FV3-like viruses), particularly FV3 (Frog virus 3), represent typical species within the genus Ranavirus, primarily infecting amphibians and reptiles, thereby posing serious threats to aquaculture and biodiversity conservation. We designed a pair of universal primers and a probe targeting the conserved region of the major capsid protein (MCP) genes of FV3-like viruses. By integrating recombinase-aided amplification (RAA) with lateral flow dipstick (LFD) technology and real-time fluorescence (RF) modification, we established RAA-LFD and RF-RAA assays. The limit of detection (LoD) of RAA-LFD was determined to be 35.4 copies/μL under optimized amplification conditions at 35 °C for 15 min. Similarly, the RF-RAA assay exhibited high specificity with a satisfactory LoD of 3.54 × 10 2 copies/μL at 39 °C over a duration of 17-20 min. In diagnosing 53 clinical samples infected by an isolated strain of FV3-like viruses, both assays demonstrated consistency with results obtained from real-time fluorescence PCR assay. These experiments indicate that both methods serve as promising alternatives for point-of care testing (POST), adaptable to various scenarios. This study represents the first establishment of RAA-LFD and RF-RAA assays for FV3-likes viruses, enabling rapid and intuitive assessment of detection results while fulfilling on-site testing requirements, thus contributing positively to swift diagnosis and long-term monitoring in aquaculture., Competing Interests: Declaration of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Ltd.)

Iridovirus SGIV is a highly pathogenic virus of fish that can cause more than 90% mortality in Epinephelus coioides, a marine farmed fish in South China. miRNAs can be involved in regulating the development of virus-induced diseases. In this study, SGIV infection could significantly inhibit the expression of E. coioides miR-21. And, overexpressing miR-21 could inhibit the expressions of viral key genes (ICP18, VP19, LITAF and MCP), SGIV-induced CPE, and viral titers. Overexpression of miR-21 promoted the promoter activity of AP-1/NF-κB, SGIV-induced apoptosis, and activities of caspase 3/9. Inhibiting miR-21 could produce the opposite results. E. Coioides PDCD4 is a targeting gene of miR-21, and we speculate that PDCD4 downregulation may, at least in part, explain the observed antiviral effects. These studies indicate that miR-21 could inhibit the infection and replication of SGIV, which might provide a molecular basis for further exploring the mechanism of SGIV invasion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: The large yellow croaker iridovirus (LYCIV) poses a significant threat to the aquaculture industry of Larimichthys crocea . Understanding the host defense response to LYCIV infection is crucial for developing effective strategies to mitigate its impact., Methods: In this study, an epigenetic approach was employed to investigate dynamic changes in chromatin accessibility using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Additionally, RNA sequencing (RNA-seq) was used to analyze the expression pattern of immune response genes upon LYCIV infection., Results: Substantial alterations in chromatin accessibility were observed, particularly in the regulatory regions of key immune-related genes. Significant changes in the expression of AP-1 transcription factors, including the Batf gene, were noted. CUT&Tag results revealed that AP-1 was significantly enriched in the open chromatin regions of cytokine genes, with Batf potentially regulating the cytokine genes LIF and CLCF1 ., Discussion: These findings suggest that AP-1 may play a crucial role in the defense response against viral infection by modulating inflammatory cytokines and contributing to cellular inflammatory responses. This study provides a comprehensive analysis of the epigenomic landscape and gene expression regulation during iridovirus infection in L. crocea , offering valuable insights for breeding programs aimed at combating iridovirus infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Song, Huang, Han and Wang.)

Immunoglobulin M (IgM) and IgM + B cells are key components of the humoral immune system, providing defense against pathogen invasion. While the role of IgM in the systemic and mucosal immune responses of fish to parasites and bacteria has been partially investigated, its function in viral infections remains underexplored. This study successfully developed a largemouth bass ( Micropterus salmoides ) model for ranavirus immersion infection. Our findings revealed that viral infection caused significant pathological changes in the gill and head kidney tissues, along with a marked upregulation of adaptive immune gene expression. Interestingly, fish that survived an initial viral infection exhibited minimal mortality and low viral loads in the gill and head kidney tissues when exposed to a higher viral concentration. Notably, in these fish with secondary infections, there was a significant increase in IgM protein levels in both the blood and gill mucus, as well as a pronounced accumulation of IgM + B cells in the gill and head kidney tissues. Additionally, the serum contained high levels of virus-specific IgM, which demonstrated the ability to neutralize the virus. These findings highlight the crucial role of IgM in the immune response to viral infections in largemouth bass and suggest its potential as a target for enhancing viral resistance in aquaculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Huang, Liu, Xue, Xu, Fan, Meng, Jiang, Li, Liu, He and Zhou.)

Mandarin fish ranavirus (MRV) is widely spread in China and causes huge economic losses to the mandarin fish (Siniperca chuatsi) aquaculture. However, the pathogenesis of MRV is still unclear. In the present study, mandarin fish were artificially infected with MRV, and then different gut compartments from diseased fish were subjected to histologic analysis by H&E staining, quantification of proinflammatory genes and MRV copies by qPCR. MRV-MCP protein expression was assessed using indirect fluorescence assay (IFA) and immunohistochemistry. Proliferation of IgM + B cells was evaluated by indirect fluorescence assay (IFA). Then, we found that MRV infection caused serious histologic lesions along with inflammatory cell infiltration, especially in the foregut. A significant accumulation of IgM + B cells was detected in the foregut (~6.5-fold) and hindgut (~3.3-fold), respectively. The expression of inflammation-related genes such as IL-1β, IL-6, IL-8, TNF-α, CSF1r and NCF1 was significantly upregulated in the foregut, varying from ~2.8-fold to ~11.9-fold. In addition, MRV exhibited foregut tropism, according to the investigation of viral loads and MCP protein expression. Overall, our findings indicated that MRV-induced hyperinflammation in the gut eventually led to enteritis. This study provided new insights into uncovering the pathogenesis of MRV infection., (© 2024 John Wiley & Sons Ltd.)

We detected an emerging human-associated gemykibivirus-2 (HuGkV-2) in rectal swab sample from Molossus molossus bat from Brazil. Phylogenetic analysis further revealed well-supported relationships between our sequence and those associated with human infections. This study underscores the necessity of ongoing monitoring of HuGkV-2 to elucidate potential spillback events, its role in human infections, and its public health implications., (© 2024 Wiley Periodicals LLC.)

To address a gap in our understanding of viral infections in epidemiologically important rat species, we aimed to detect DNA viruses from the tissues of free-ranging rat populations in Hungary. DNA viruses were identified from the parenchymal organs of 230 Rattus norvegicus and Rattus rattus , using family-specific pan-PCR assays followed by sequencing of the PCR products. Adeno-, herpes-, circo-, and polyomaviruses were detected, while irido-, pox-, and dependoparvoviruses were not. Adenovirus DNA was present in 6.5% of the samples, herpesvirus and polyomavirus DNA in 12.2%, and circovirus DNA in 1.7%. All detected herpesviruses belonged to the β and γ subfamilies, with a majority being β herpesviruses. Some adenovirus and herpesvirus sequences were novel, while only the known Rattus norvegicus polyomavirus 1 was detected for polyomaviruses. The rare circovirus-positive samples revealed the presence of both rodent and bird circoviruses, indicating the ability of circoviruses to cross species barriers. Our findings show that rats host a variety of DNA viruses, many of which were previously uncharacterized, highlighting the need for further diagnostic studies.

Stimulator of interferon genes (STING) is a key connector protein in interferon (IFN) signaling, crucial for IFN induction during the activation of antiviral innate immunity. In mammals, ring finger protein 5 (RNF5) functions as an E3 ubiquitin ligase, mediating STING regulation through K150 ubiquitylation to prevent excessive IFN production. However, the mechanisms underlying RNF5's regulation of STING in teleost fish remain unknown. This study investigated the regulatory role of the mandarin fish ( Siniperca chuatsi ) RNF5 ( sc RNF5) in the STING-mediated antiviral immune response and identified the specific regulatory sites on sc STING. Furthermore, an examination of sc RNF5 expression patterns in virus-infected cells revealed its responsiveness to mandarin fish ranavirus (MRV) infection. The ectopic expression of sc RNF5 suppressed sc STING-mediated IFN signaling and facilitated MRV replication. Co-immunoprecipitation experiments indicated an interaction between sc RNF5 and sc STING. The further experiments demonstrated that sc RNF5 exerted its inhibitory effect by promoting the degradation of sc STING, which was observed to be blocked by MG132 treatment. Ubiquitination assays with various sc STING mutants showed that sc RNF5 catalyzed the ubiquitination of sc STING at K135 and K155 residues. Furthermore, we provided evidence that sc RNF5 significantly attenuated sc STING-dependent antiviral immunity by targeting negative regulators within the sc STING signaling cascade. This study underscored that RNF5 negatively regulated the STING-mediated IFN signaling pathway in mandarin fish, attenuated STING's antiviral activity, and facilitated STING degradation via the ubiquitin-proteasome pathway at two novel lysine sites (K135 and K155). Our work offered valuable insights into the regulatory mechanisms of STING-mediated signaling in teleost fish, paving the way for further research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qin, Li, Liang, Qian, You, Weng, He and Guo.)

Mandarin fish ranavirus (MRV) is a distinctive member among the genus Ranavirus of the family Iridoviridae . The persistently covert infection of MRV was previously observed in a natural outbreak of MRV, but the underlying mechanism remains unclear. Here, we show that mandarin fish peripheral B lymphocytes are implemented as viral reservoirs to maintain the persistent infection. When mandarin fish were infected with a sublethal dosage of MRV under a nonpermissive temperature (19 °C) and a permissive temperature (26 °C), all of the fish in the 19 °C group survived and entered the persistent phase of infection, characterized by a very low viral load in white blood cells, whereas some of the fish died of MRV infection in the 26 °C group, and the survival fish then initiated a persistent infection status. Raising the temperature, vaccination and dexamethasone treatment can reactivate the quiescent MRV to replicate and result in partial mortality. The viral reservoir investigation showed that IgM + -labeled B lymphocytes, but not CD3Δ + -labeled T lymphocytes and MRC-1 + -labeled macrophages, are target cells for the persistent infection of MRV. Moreover, the reactivation of the quiescent MRV was confirmed through a non-TLR5 signal pathway manner. Collectively, we demonstrate the presence of the B cell-dependent persistent infection of ranavirus, and provide a new clue for better understanding the complex infection mechanism of vertebrate iridovirus.

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a highly damaging viral pathogen that causes widespread mortality in cultured largemouth bass and poses a significant threat to the farming industry in China. However, there is a paucity of effective preventive methods for this disease by LMBRaV. Research has established that herbal extracts possess antiviral activity, potentially exerting a controlling effect on viruses in aquaculture. Therefore, the identification of simple and structurally safe herbal extract immunomodulators is critical for largemouth bass health and development. Rhein, an herbal compound, exhibits a broad spectrum of pharmacological effects in herbal medicine. This study aimed to evaluate the properties and effects of rhein against LMBRaV in epithelioma papilloma cyprinid (EPC) and largemouth bass. In vitro CCK-8 assays were performed to determine the highest safe concentrations. Significantly, 50 mg/L rhein effectively inhibited cytopathic effects (CPE) in EPC induced by LMBRaV, resulting in a 97 % reduction in viral replication rates. Furthermore, a six-point dose-response experiment revealed that rhein doses as low as 6.4 mg/L inhibited LMBRaV and suppressed LMBRaV infection with a dose-dependent manner. Subsequent experiments demonstrated that sustained rhein activity effectively alleviated CPE and nuclear damage caused by LMBRaV infection. Additionally, rhein exerted protective effects against declines in LMBRaV-induced mitochondrial membrane potential. In vivo experiments validated that largemouth bass diets supplemented with 0.075 % rhein exerted a protective effect against LMBRaV infection. Moreover, LMBRaV MCP gene expression levels were lower in several organs of fish in the rhein supplementation group. Immunogene analysis determined that rhein upregulated relevant gene expression levels in multiple organs of largemouth bass, with higher activities observed for the Mx, INF-γ, and IRF3 genes. Histopathological results displayed that a supplemental dosage of 0.075 % rhein significantly mitigated LMBRaV infection-induced pathological damage in the liver, spleen, and kidney. Taken together, these in vivo and in vitro results support the effective use of rhein as a promising candidate for the development of antiviral treatment against LMBRaV infection, highlighting its role in aquatic animal husbandry as a wide-spectrum antiviral and immune response booster., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could appear to have influenced the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Largemouth bass virus (LMBV) and infectious spleen and kidney necrosis virus (ISKNV) are both belong to Iridoviridae that cause considerable economic losses in the fish industry. There is no reported literature that can detect these two viruses simultaneously. In this study, we established a multiplex quantitative polymerase chain reaction (qPCR) assay that can specifically and simultaneously detect both LMBV and ISKNV in fish samples. The specificity experiment showed that the method only amplified LMBV and ISKNV but not the other 10 common fish viruses. The slope (m), efficiency (E) and linearity (R 2 ) determined from the generated standard curve were all within the optimal range of qPCR values. The detection limit of the multiplex qPCR assay was as low as 4 copies/μL for LMBV DNA and 7 copies/μL for ISKNV DNA, respectively. The established method exhibited adequate repeatability and reproducibility, and the intra- and inter-assay coefficients of variation were both less than 3 %. The accuracy of the multiplex qPCR method was validated using 229 fish samples and was more precise than that of the conventional PCR assay. In summary, the established multiplex qPCR assay can simultaneously detect LMBV and ISKNV to monitor the risk of infection LMBV and ISKNV and control the disease early., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Largemouth bass virus (LMBV) is an infectious pathogen that causes high mortality rates in largemouth bass, and outbreaks of this virus can significantly harm the aquaculture industry. Currently, no vaccine has been developed that can effectively prevent the transmission of LMBV. In this study, we constructed a recombinant Lactobacillus plantarum (L. plantarum) strain capable of expressing the MCP gene of LMBV and displaying this protein on its surface; then, we evaluated the immunoprotective effect of this recombinant bacterium on largemouth bass. Western blotting, immunofluorescence, and flow cytometry confirmed that MCP was successfully expressed and anchored on the surfaces of NC8 cells. Immunization of largemouth bass with NC8-pSIP409-pgsA'-MCP via the oral feeding route induced CD4, CD8, IL-1β, and IL-6 gene expression. In addition, NC8-pSIP409-pgsA'-MCP at different CFUs increased the survival of largemouth bass after LMBV infection; in particular, NC8-pSIP409-pgsA'-MCP (10 9  CFU) resulted in approximately 30 % survival. NC8-pSIP409-pgsA'-MCP immunization alleviated the pathological changes in the liver and spleen, exerting a more advantageous protective effect. These data suggest that the recombinant L. plantarum strain NC8-pSIP409-pgsA'-MCP can increase the resistance of largemouth bass to LMBV infection and that this strain is a promising candidate oral vaccine for the prevention of LMBV infection., Competing Interests: Declaration of competing interest No conflict of interest exists with this paper submission, and all authors have approved the publication of this manuscript. All or any part of the study was not published and was not considered for publication elsewhere., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The genus Megalocytivirus, belonging to the family Iridoviridae, is one of the most detrimental virus groups to fish aquaculture. Megalocytivirus creates a virus-mock basement membrane (VMBM) on the surface of infected cells. This membrane provides attachment sites for lymphatic endothelial cells (LECs), disrupting fish's endothelial cell-extracellular matrix system. This disruption triggers injury to the vascular system and can result in death. Exploring the VMBM-cell interaction mechanism is crucial for uncovering the pathogenesis of Megalocytivirus and identifying therapeutic targets. Claudins, a class of tetra transmembrane proteins, play a key role in creating tight junctions between endothelial or epithelial cells. In this study, we demonstrated that the expression of Claudin2, a member of the Claudin family in fish, was significantly up-regulated by Megalocytivirus infection. Claudin2 was found in LECs attached to the surface of infected cells. It interacted with the VMBM viral components VP23R, VP08R, and VP33L at multiple binding sites through its two extracellular loops. However, it did not interact with the host basement membrane's nidogen. Therefore, Claudin2 is involved in the interaction of LEC with VMBM and plays a role in the disturbed distribution of extracellular matrix and endothelial cells in Megalocytivirus-infected fish tissues. This study aims to uncover the molecular mechanisms by which Megalocytivirus infection leads to pathological changes in the vascular system., (© 2024. The Author(s).)

Largemouth bass virus (LMBV) is a highly pathogenic pathogen that often causes high mortality of affected largemouth bass and significant financial losses. Type I interferon as an effective and broad spectrum tool has been successfully used for therapeutic or prophylactic treatment some viral infections. However, the implementation of immunotherapies based on interferon administration to combat LMBV infections has not been reported. And Lactic Acid Bacteria (LAB) are a powerful vehicle for expressing cytokines or immunostimulant peptides at the gastrointestinal level after oral administration. In this study, Lactococcus lactis (L. lactis) expression system with lactose as a screening marker was utilized to express the Micropterus salmoides interferon a3 (IFNa3) protein and orally administered to largemouth bass. The genetically engineered strain pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 was successfully constructed, and its potential to elicit immune protection response by oral administration was evaluated. After orally administration, the recombinant L. lactis was detected in guts of experimental fish and remained detectable for 72 h. Additionally, IFNa3 was able to enhance the test fish's immune response, as determined by the relatively increased mRNA relative expression of immune-related genes in the liver, spleen, and kidney tissues, including IFN-γ, TNF-α, IL-1β, IL-8, IgM and IgT. Following LMBV challenge, the experiment group of pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 exhibited a 70 % survival rate, while survival rate were 15 % in the PBS control group, 45 % in the pNZ8149/L. lactis NZ3900 group. Furthermore, the viral load in the surviving fish was significantly lower than that of the control groups. These findings suggest that oral administration of recombinant L. lactis producing IFNa3 induces largemouth bass immune responses at a systemic level to effective prevent and combat of LMBV infection., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Largemouth bass ranavirus (LMBV) infection results in huge economic losses in largemouth bass (Micropterus salmoides) industry. Nanopeptide C-I20 and anthocyanins have a positive effect on promoting immune responses and antioxidant mechanisms in several aquatic organisms, and are therefore used to inhibit LMBV infection. In this study, we developed an LMBV immersion challenge model using three different viral concentrations (1 × 10 4 copies/mL, 1 × 10 5 copies/mL, and 1 × 10 6 copies/mL) to infect largemouth bass, and LMBV-MCP mRNA expression was detected in infected fish. Following infection, the fish exhibited severe external ulceration, redness swelling, and darkening of the skin. Histopathological examination revealed significant necrosis and inflammation in muscle tissue, epithelial cell shedding in renal tubules, macrophage aggregation centers and cellular vacuolization in spleen and head kidney, and cellular hypertrophy in liver. To mitigate LMBV infection, we explored the protective effects of a combined treatment strategy involving C-I20 and anthocyanin. Overall, the combination of anthocyanin and C-I20 demonstrated the highest protective efficacy, significantly reducing viral loads in muscle, liver, spleen, and head kidney. Moreover, this treatment regimen enhanced antioxidant enzyme activities (T-AOC, TSOD, GSH-Px, CAT) and modulated important immune genes (IL-1, IL-8, TNF-α, IL-10, Mx, and IgM) expression. In conclusion, the synergistic application of anthocyanin and C-I20 demonstrates significant efficacy in mitigating LMBV infection. This research introduces a novel and promising approach to managing infectious diseases in aquaculture settings., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The threespine stickleback iridovirus (TSIV), a double-stranded DNA virus, was the first megalocytivirus detected in wild North American fishes. We report a second occurrence of TSIV in threespine stickleback ( Gasterosteus aculeatus ) from Stormy Lake, Alaska, and assemble a nearly complete genome of TSIV. The 115-kilobase TSIV genome contains 94 open reading frames (ORFs), with 91 that share homology with other known iridoviruses. We identify three ORFs that likely originate from recent lateral gene transfers from a eukaryotic host and one ORF with homology to B22 poxvirus proteins that likely originated from a lateral gene transfer between viruses. Phylogenetic analysis of 24 iridovirus core genes and pairwise sequence identity analysis support TSIV as a divergent sister taxon to other megalocytiviruses and a candidate for a novel species designation. Screening of stickleback collected from Stormy Lake before and after a 2012 rotenone treatment to eliminate invasive fish shows 100% positivity for TSIV in the two years before treatment (95% confidence interval: 89-100% prevalence) and 0% positivity for TSIV in 2024 after treatment (95% confidence interval: 0 to 3.7% prevalence), suggesting that the rotenone treatment and subsequent crash and reestablishment of the stickleback population is associated with loss of TSIV.

Introduction: In clinical diagnosis of porcine diseases, co-infection with multiple viruses often leads to similar clinical symptoms. Postweaning multisystemic wasting syndrome (PMWS) can be caused by infections with TTSuV or PCV2, while PCV2, PRV, and PPV can cause respiratory and reproductive disorders in pigs. The overlapping clinical and pathological features of these infections necessitate the development of a rapid and specific method for differentiating and detecting these four DNA viruses., Methods: In this study, four pairs of primers and TaqMan probes were designed targeting the conserved sequence of TTSuV, the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV. After optimizing reaction conditions, including annealing temperature, primer concentration, and probe concentration, a quadruplex real-time PCR method was developed., Results: This method can specifically detect TTSuV1, PCV2, PRV, and PPV simultaneously, with no cross-reactivity with ASFV, CSFV, PRRSV, PEDV, PSV, and TGEV. The minimum detection limit for each virus was 10 copies/μl, and the inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%. Subsequently, 150 clinical samples were tested to evaluate the practical applicability of this method. The positive rates for TTSuV1, PCV2, PRV, and PPV were 8.6% (13/150), 10.67% (16/150), 14% (21/150), and 11.33% (17/150), respectively., Discussion: The results indicate that the established quadruplex real-time PCR method can assist in the accurate and rapid diagnosis of TTSuV1, PCV2, PRV, and PPV in clinical settings, providing robust support for the prevention and control of these infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Quan, Geng, Wu, Jiang, Li and Yu.)

Lateolabrax maculatus iridovirus (LMIV) is a variant strain of red sea bream iridovirus (RSIV), causing serious economic losses in aquaculture. Claudins (CLDNs) are major components of tight junctions (TJs) forming an important line of defense against pathogens. Our pilot miRNA-mRNA joint analysis indicated the degradation of CLDN3, as well as its interaction with miR-181a during LMIV infection. To elucidate the miR-181a/CLDN3/LMIV interactions, in vitro assays were carried out on LMB-L cells. We first confirmed that LMIV infection could decrease the expression of CLDN3, accompanied by the enhancement of permeability, suggesting the dysfunction of TJs. Contrary to the inhibition of CLDN3, the activation of miR-181a was proved, presenting a negative correlation between miR-181a and CLDN3 (Pearson r = -0.773 and p < 0.01). In addition, the influence of CLDN3 on LMIV replication was analyzed by knockdown and over-expression of CLDN3. When CLDN3 was silenced in LMB-L cells with siCLDN3-623 at 9 days post transfection (dpt), LMIV copies and titers were significantly up-regulated by 1.59-fold and 13.87-fold, respectively. By contrast, LMIV replication in LMB-L cells was reduced by 60% and 71%, post transfection with pcDNA3.1-CLDN3 over-expressed plasmid at 6 dpt and 9 dpt, respectively. Ultimately, the regulatory relationship between miR-181a and CLDN3 was further validated by dual luciferase reporter assays. Taking into account the above-described results, we proposed a "miR-181a/CLDN3/LMIV" regulatory relationship. This study provides a new insight for understanding the mechanism of LMIV replication.

Novel pollutants nanoplastics (NPs) are widely distributed in aquatic environments and may pose a health threat to aquatic organisms. Notably, the contribution of NPs to the occurrence of viral diseases in aquatic animals remains largely uncertain. In this study, the effects of polystyrene nanoplastics (PS-NPs) on Largemouth bass ranavirus (LMBV)-infected MsF cells were investigated. MsF cells took up PS-NPs in a time- and dose-dependent manner and significantly affect cell viability at an exposure concentration of 500 μg/mL. Western blot and qPCR assays indicated that exposure to PS-NPs accelerated LMBV replication in MsF cells. PS-NPs act synergistically with LMBV to disrupt the cellular antioxidant system, as evidenced by increased ROS production and decreased mRNA levels of antioxidant-associated genes. Furthermore, PS-NPs was found to exacerbate LMBV-induced inflammatory responses, as demonstrated by disturbed expression of inflammation-related factors. In addition, our results suggest that PS-NPs reduce IFN production by inhibiting the expression of molecules related to the cGAS-STING signaling pathway, thereby promoting viral replication. Collectively, our findings suggest the potential threat of NPs to infectious diseases caused by freshwater fish viruses and provide new insights for fish disease prevention and control., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection., (© Wildlife Disease Association 2024.)

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 μm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1β, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rock bream (Oplegnathus fasciatus) is one of the highly priced cultured marine fish in Korea. Rock bream iridovirus (RBIV) outbreaks in aquaculture farms may involve environmental factors, co-infection with other pathogenic microorganisms and grounded (raw) fish feed. This study evaluated the effects of RBIV-containing tissue intake on mortality and oral transmission in rock bream. Virus-containing tissues administered to rock bream [50 mg (1.53 × 10 8 /major capsid protein, MCP gene copies) to 2400 mg (7.34 × 10 9 )] held at 23 °C lead to 100 % mortality by 27 days post administration. Interestingly, the mortality rates were not viral dose- or concentration dependent. Further, high MCP gene copy numbers were observed in the gill, liver, intestine, stomach, spleen, heart, kidney, brain and muscle tissues (viral load range of 3.03 × 10 6 to 4.01 × 10 7 /mg, average viral load 1.70 × 10 7 /mg) of dead rock bream. Moreover, a high viral load was detected in the intestine and stomach, where the virus was directly administered. This indicated that the intake of RBIV-containing tissue feed weakens the intestinal mucosal immunity and increases viral load in the intestine. Moreover, the levels of complete blood cell count (CBC) indicators, such as red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) significantly decreased from 15 dpi with red blood cell distribution width (RDW), and white blood cells (lymphocyte, monocyte and granulocyte) significantly increased from the initial to later stage of infection. These results highlight the significance of blood-mediated indicators against RBIV infection in rock bream. We demonstrate the existence of an oral transmission route for RBIV in rock bream. Our findings indicate that pathogen-containing feed is an important risk factor for disease outbreaks in rock bream., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Singapore grouper iridovirus (SGIV) always causes high transmission efficiency and mortality in the larval and juvenile stages of grouper in aquaculture industry. Although inactivated virus and recombinant DNA vaccines administered via intraperitoneal injection have shown efficacy in protection against SGIV, their potential applications in field testing were limited due to the vaccine delivery methods. Here, we developed an immersion vaccine containing inactivated virus and Montanide IMS 1312 adjuvant (IMS 1312) and evaluated its protective efficacy against SGIV infection. Compared to the PBS group, fish vaccinated with immersion inactivated vaccine with or without IMS 1312 were significantly protected against SGIV, with a relative percent survival (RPS) of 57.69 % and 38.47 %, respectively. Furthermore, the transcripts of viral core genes were reduced, and the histopathological severity caused by SGIV were relatively mild in multiple tissues of the IMS + V group. The immersion vaccine activated the AKP and ACP activities and increased the mRNA levels of IFN and inflammation-associated genes. The transcriptome analysis showed that a total of 731 and 492 genes were significantly regulated in the spleen and kidney from the IMS + V group compared to the PBS group, respectively. Among them, 129 DEGs were co-regulated, and enriched in the KEGG pathways related to immune and cell proliferation, including MAPK signaling, JAK-STAT signaling and PI3K-Akt signaling pathways. Similarly, the DEGs specially regulated in the kidney and spleen upon vaccine immunization were significantly enriched in the KEGG pathways related to interferon and inflammation response. Together, our results elucidated that the immersion vaccine of inactivated SGIV with IMS 1312 induced a protective immune response of grouper against SGIV., (Copyright © 2024. Published by Elsevier Ltd.)

The emergence of Singapore grouper iridovirus (SGIV) has caused huge losses to grouper farming. SGIV is a DNA virus and belongs to the genus Ranavirus . Groupers infected with SGIV showed haemorrhaging and swelling of the spleen, with a mortality rate of more than 90% within a week. Therefore, it is of great significance to study the escape mechanism of SGIV from host innate immunity for the prevention and treatment of viral diseases in grouper. In this study, the viral proteins that interact with EccGAS were identified by mass spectrometry, and the SGIV VP12 protein that inhibits cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated antiviral innate immunity was screened by the dual-luciferase reporter gene assay. VP12 belongs to the late gene of the virus. The immunofluorescence analysis demonstrated that VP12 was aggregated and distributed in the cytoplasm during the early stage of virus infection and translocated into the nucleus at the late stage of virus infection. VP12 inhibited the activation of IFN3, ISRE and NF-κB promoter activities mediated by cGAS-STING, EcTBK1 and EcIRF3. Quantitative real-time PCR analysis showed that VP12 inhibited the expression of interferon-related genes, including those mediated by cGAS-STING. VP12 enhanced the inhibition of IFN3, ISRE and NF-κB promoter activity by EccGAS, EccGAS-mab-21 and EccGAS-delete-mab21. The interaction between VP12 and EccGAS was found to be domain independent. The immunoprecipitation results demonstrated that VP12 interacted and co-localized with EccGAS, EcTBK1 and EcIRF3. VP12 degraded the protein levels of EcTBK1 and EcIRF3 and degraded EcIRF3 through the protease pathway. These results suggest that SGIV VP12 protein escapes the cGAS-STING signalling pathway and degrades EcIRF3 protein expression through the protease pathway.

Largemouth bass virus (LMBV) infections has resulted in high mortality and economic losses to the global largemouth bass industry and has seriously restricted the healthy development of the bass aquaculture industry. There are currently no antiviral therapies available for the control of this disease. In this study, we developed three types of vaccine against LMBV; whole virus inactivated vaccine (I), a subunit vaccine composed of the major viral capsid protein MCP (S) as well as an MCP DNA vaccine(D), These were employed using differing immunization and booster strategies spaced 2 weeks apart as follows: II, SS, DD and DS. We found that all vaccine groups induced humoral and cellular immune responses and protected largemouth bass from a lethal LMBV challenge to varying degrees and DD produced the best overall effect. Specifically, the levels of specific IgM in serum in all immunized groups were elevated and significantly higher than those in the control group. Moreover, the expression of humoral immunity (CD4 and IgM) and cellular immunity (MHCI-α) as well as cytokines (IL-1β) was increased, and the activity of immunity-related enzymes ACP, AKP, LZM, and T-SOD in the serum was significantly enhanced. In addition, the relative percent survival of fish following an LMBV lethal challenge 4 weeks after the initial immunizations were high for each group: DD(89.5 %),DS(63.2 %),SS(50 %) and II (44.7 %). These results indicated that the MCP DNA vaccine is the most suitable and promising vaccine candidate for the effective control of LMBV disease., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Largemouth bass ranavirus (LMBV) causes disease outbreaks and high mortality at all stages of largemouth bass farming. Therefore, live vaccine development is critical for largemouth bass prevention against LMBV by immersion immunization. Herein, an attenuated LMBV strain with good immunogenicity, designated as LMBV-2007136, was screened from the natural LMBV strains bank through challenge assay and immersion immunization experiment. After determing the safe concentration range of LMBV-2007136, the minimum immunizing dose of immersion immunization was verified. When largemouth bass were vaccinated by immersion at the lowest concentration of 10 2.0 TCID 50 /mL, all of fish were survival post virulent LMBV challenge, and the relative percent survival (RPS) was 100 %. And the immune gene expression levels of IL-10, IL-12, IFN-γ, and IgM in the spleen and kidney post-vaccination were significantly up-regulated compared to the control group, but TNF-α expression showed no significant changes. The safety and efficacy of LMBV-2007136 at passages P8, P13, and P18 were futher assessed, and no death of largemouth bass was observed within 21 days post-immunization and RPS of three vaccination groups was 100 %, suggesting that the safety and efficacy of the attenuated strain at different passages was stable. Furthermore, in the virulence reversion test, the attenuated strain was propagated through 5 times in largemouth bass by intraperitoneal injection and no abnormality and mortality were observed, further proving the attenuated vaccine candidate LMBV-2007136 was safe. These results proved that LMBV-2007136 could be a promising candidate for a live vaccine to protect largemouth bass from LMBV disease., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The orange-spotted grouper ( Epinephelus coioides ) is an important mariculture fish in China. However, in recent years, with the rapid development of aquaculture activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing severe economic losses. In the present study, we isolated and identified a virus from diseased, orange-spotted groupers from an aquaculture farm in Hainan Province, China. The isolated virus was identified as orange-spotted grouper iridovirus, hence named the orange-spotted grouper iridovirus Hainan strain (OSGIV-HN-2018-001). OSGIV-HN-2018-001 induces a cytopathic effect after the infection of mandarin fish ( Siniperca chuatsi ) brain clonal passage (SBC) cells. In addition, the cytoplasm of the OSGIV-HN-2018-001-infected SBC cells was found to contain a large number of hexagonal virus particles with a diameter of approximately 134 nm. Using the Illumina NovaSeq system, we assembled the sequence data and annotated the complete genome of OSGIV-HN-2018-001 (GenBank accession number: PP974677), which consisted of 110,699 bp and contained 122 open reading frames (ORFs). Phylogenetic tree analysis showed that OSGIV-HN-2018-001 was most closely related to ISKNV-ASB-23. The cumulative mortality rate of groupers infected with OSGIV-HN-2018-001 reached 100% on day 8. The spleens were enlarged and blackened after the dissection of the dying groupers. These results contribute to the understanding of the molecular regulatory mechanism of the iridovirus infection and provide a basis for iridovirus prevention.

The disease caused by Largemouth bass ranavirus (LMBV) is one of the most severe viral diseases in largemouth bass ( Micropterus salmoides ). It is crucial to evaluate the genetic resistance of largemouth bass to LMBV and develop markers for disease-resistance breeding. In this study, 100 individuals (45 resistant and 55 susceptible) were sequenced and evaluated for resistance to LMBV and a total of 2,579,770 variant sites (SNPs-single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels)) were identified. A total of 2348 SNPs-InDels and 1018 putative candidate genes associated with LMBV resistance were identified by genome-wide association analyses (GWAS). Furthermore, GO and KEGG analyses revealed that the 10 candidate genes ( MHC II , p38 MAPK , AMPK , SGK1 , FOXO3 , FOXO6 , S1PR1 , IL7R , RBL2 , and GADD45 ) were related to intestinal immune network for IgA production pathway and FoxO signaling pathway. The acquisition of candidate genes related to resistance will help to explore the molecular mechanism of resistance to LMBV in largemouth bass. The potential polymorphic markers identified in this study are important molecular markers for disease resistance breeding in largemouth bass.

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi ) VHL ( sc VHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of sc VHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that sc VHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of sc VHL may be mediated through sc IKKα, sc IKKβ, sc IκBα, or sc p65. Co-immunoprecipitation (Co-IP) analysis identified sc IκBα as a novel target protein of sc VHL. Moreover, sc VHL targeted sc IκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that sc VHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of sc VHL in NF-κB signaling and viral infection in fish.

Background: Temperature is a crucial environmental determinant for the vitality and development of teleost fish, yet the underlying mechanisms by which they sense temperature fluctuations remain largely unexplored. Transient receptor potential (TRP) proteins, renowned for their involvement in temperature sensing, have not been characterized in teleost fish, especially regarding their temperature-sensing capabilities., Results: In this study, a genome-wide analysis was conducted, identifying a total of 28 TRP genes in the mandarin fish Siniperca chuatsi. These genes were categorized into the families of TRPA, TRPC, TRPP, TRPM, TRPML, and TRPV. Despite notable variations in conserved motifs across different subfamilies, TRP family members shared common structural features, including ankyrin repeats and the TRP domain. Tissue expression analysis showed that each of these TRP genes exhibited a unique expression pattern. Furthermore, examination of the tissue expression patterns of ten selected TRP genes following exposure to both high and low temperature stress indicated the expression of TRP genes were responsive to temperatures changes. Moreover, the expression profiles of TRP genes in response to mandarin fish virus infections showed significant upregulation for most genes after Siniperca chuatsi rhabdovirus, mandarin fish iridovirus and infectious spleen and kidney necrosis virus infection., Conclusions: This study characterized the TRP family genes in mandarin fish genome-wide, and explored their expression patterns in response to temperature stress and virus infections. Our work will enhance the overall understanding of fish TRP channels and their possible functions., (© 2024. The Author(s).)

Members of the Iridoviridae family, genus Ranavirus , represent a group of globally emerging pathogens of ecological and economic importance. In 2017, an amphibian die-off of wood frogs ( Rana sylvatica ) and boreal chorus frogs ( Pseudacris maculata ) was reported in Wood Buffalo National Park, Canada. Isolation and complete genomic sequencing of the tissues of a wood frog revealed the presence of a frog virus 3 (FV3)-like isolate, Rana sylvatica ranavirus (RSR), with a genome size of 105,895 base pairs, 97 predicted open reading frames (ORFs) bearing sequence similarity to FV3 (99.98%) and a FV3-like isolate from a spotted salamander in Maine (SSME; 99.64%). Despite high sequence similarity, RSR had a unique genomic composition containing ORFs specific to either FV3 or SSME. In addition, RSR had a unique 13 amino acid insertion in ORF 49/50L. No differences were found in the in vitro growth kinetics of FV3, SSME, and RSR; however, genomic differences between these isolates were in non-core genes, implicated in nucleic acid metabolism and immune evasion. This study highlights the importance of viral isolation and complete genomic analysis as these not only provide information on ranavirus spatial distribution but may elucidate genomic factors contributing to host tropism and pathogenicity.

Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response., (Copyright © 2024. Published by Elsevier Ltd.)

Singapore grouper iridovirus (SGIV) belongs to the family Iridoviridae and the genus Ranavirus, which is a large cytoplasmic DNA virus. Infection of grouper with SGIV can cause hemorrhage and swelling of the spleen of the fish. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. In the present study, the protein encoded by SGIV ORF128 (VP128) was identified. VP128 is predominantly localized within the endoplasmic reticulum (ER). Overexpression of VP128 significantly promoted SGIV replication. VP128 inhibited the interferon (IFN)-3 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), and TANK-binding kinase 1 (EcTBK1). Moreover, VP128 interacted with EcSTING and EcTBK1. The interaction between VP128 and EcSTING was independent of any specific structural domain of EcSTING. Together, our results demonstrated that SGIV VP128 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reports in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Small plastic debris (0.1 μm-5 mm) or microplastics (MPs) have become major pollutants of aquatic ecosystems worldwide and studies suggest that MPs exposure can pose serious threats to human and wildlife health. However, to date the potential biological impacts of MPs accumulating in low amount in tissues during early life remains unclear. Here, for a more realistic assessment, we have used environmentally representative, mildly weathered, polyethylene terephthalate microplastics (PET MPs), cryomilled (1-100 μm) and fluorescently labelled. We leveraged the amphibian Xenopus laevis tadpoles as an animal model to define the biodistribution of PET MPs and determine whether exposure to PET MPs induce perturbations of antiviral immunity. Exposure to PET MPs for 1-14 days resulted in detectable PET MPs biodistribution in intestine, gills, liver, and kidney as determined by fluorescence microscopy on whole mount tissues. PET MPs accumulation rate in tissues was further evaluated via a novel in situ enzymatic digestion and subsequent filtration using silicon nanomembranes, which shows that PET MPs rapidly accumulate in tadpole intestine, liver and kidneys and persist over a week. Longer exposure (1 month) of tadpoles to relatively low concentration of PET MPs (25 μg/ml) significantly increased susceptibility to viral infection and altered innate antiviral immunity without inducing overt inflammation. This study provides evidence that exposure to MPs negatively impact immune defenses of aquatic vertebrates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: James L. McGrath reports a relationship with SiMPore, Inc that includes: board membership and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Understanding the epidemiology and transmission dynamics of transboundary animal diseases (TADs) among wild pigs ( Sus scrofa ) will aid in preventing the introduction or containment of TADs among wild populations. Given the challenges associated with studying TADs in free-ranging populations, a surrogate pathogen system may predict how pathogens may circulate and be maintained within wild free-ranging swine populations, how they may spill over into domestic populations, and how management actions may impact transmission. We assessed the suitability of Torque teno sus virus 1 (TTSuV1) to serve as a surrogate pathogen for molecular epidemiological studies in wild pigs by investigating the prevalence, persistence, correlation with host health status and genetic variability at two study areas: Archbold's Buck Island Ranch in Florida and Savannah River Site in South Carolina. We then conducted a molecular epidemiological case study within Archbold's Buck Island Ranch site to determine how analysis of this pathogen could inform transmission dynamics of a directly transmitted virus. Prevalence was high in both study areas (40%, n = 190), and phylogenetic analyses revealed high levels of genetic variability within and between study areas. Our case study showed that pairwise host relatedness and geographic distance were highly correlated to pairwise viral genetic similarity. Molecular epidemiological analyses revealed a distinct pattern of direct transmission from pig to pig occurring within and between family groups. Our results suggest that TTSuV1 is highly suitable for molecular epidemiological analyses and will be useful for future studies of transmission dynamics in wild free-ranging pigs.

The high mortality rate of Singapore grouper iridovirus (SGIV) posing a serious threat to the grouper aquaculture industry and causing significant economic losses. Therefore, finding effective drugs against SGIV is of great significance. Eugenol (C 10 H 12 O 2 ) is a phenolic aromatic compound, has been widely studied for its anti-inflammatory, antioxidant and antiviral capacity. In this study, we explored the effect of eugenol on SGIV infection and its possible mechanisms using grouper spleen cells (GS) as an in vitro model. We found that treatment of GS cells with 100 μM eugenol for 4 h exhibited the optimal inhibitory effect on SGIV. Eugenol was able to reduce the expression level of inflammatory factors by inhibiting the activation of MAPK pathway and also inhibited the activity of NF-κB and AP-1 promoter. On the other hand, eugenol attenuated cellular oxidative stress by reducing intracellular ROS and promoted the expression of interferon-related genes. Therefore, we conclude that eugenol inhibits SGIV infection by enhancing cellular immunity through its anti-inflammatory and antioxidant functions., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Each year, due to climate change, an increasing number of new pathogens are being discovered and studied, leading to an increase in the number of known diseases affecting various fish species in different regions of the world. Viruses from the family Iridoviridae, which consist of the genera Megalocytivirus, Lymphocystivirus, and Ranavirus, cause epizootic outbreaks in farmed and wild, marine, and freshwater fish species (including ornamental fish). Diseases caused by fish viruses of the family Iridoviridae have a significant economic impact, especially in the aquaculture sector. Consequently, vaccines have been developed in recent decades, and their administration methods have improved. To date, various types of vaccines are available to control and prevent Iridoviridae infections in fish populations. Notably, two vaccines, specifically targeting Red Sea bream iridoviral disease and iridoviruses (formalin-killed vaccine and AQUAVAC ® IridoV, respectively), are commercially available. In addition to exploring these themes, this review examines the immune responses in fish following viral infections or vaccination procedures. In general, the evasion mechanisms observed in iridovirus infections are characterised by a systemic absence of inflammatory responses and a reduction in the expression of genes associated with the adaptive immune response. Finally, this review also explores prophylactic procedure trends in fish vaccination strategies, focusing on future advances in the field., (© 2024. The Author(s).)

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia ( Oreochromis niloticus ) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities., Competing Interests: The authors declare there are no competing interests., (©2024 Alathari et al.)

Ranaviruses are pathogens of ectothermic vertebrates (fish, amphibians, and reptiles). Turtles are the most common group of reptiles reported with ranaviral infections. However, there have been no surveys for wild ranaviral infection in any turtles from the suborder Pleurodira, despite ranaviral distributions and experimentally susceptible pleurodiran turtle populations overlapping in several areas, including Australia. We assayed 397 pooled blood samples from six Australian freshwater turtle species collected from five different sites in northern Australia between 2014 and 2019. Historical serologic surveys in the area had found antiranaviral antibodies; however, we did not detect any ranaviral DNA in our samples. Discrepancies between historical serologic and our molecular results may be explained by low viral prevalence during the years that these samples were collected, survivorship bias, or possibly an age class bias in sampling., (© Wildlife Disease Association 2024.)

In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types., Competing Interests: Declaration of competing interest The authors have no financial conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The lymphocystis disease (LCD), caused by Lymphocystis disease virus (LCDV), is a benign and self-limiting disease described in a many freshwater and marine fish species. Hypertrophic fibroblasts and extensive aggregation of inflammatory cells are characteristics of LCD. In the present study, small animal imaging and ultrastructural investigations were carried out on the lymphocystis nodules of black rockfish (Sebastes schlegelii) naturally infected with lymphocystis iridovirus, to assess pathology, and the exudate with particular attention to the formation of extracellular traps (ETs) in vivo. Ex vivo were examined by nodules sections and primary cells stimulation. By histopathological analysis, the nodules contained infiltrated inflammatory cells and extensive basophilic fibrillar filaments at the periphery of the hypertrophied fibroblasts. ETs were assessed in nodules samples using indirect immunofluorescence to detect DNA and myeloperoxidase. Moreover, LCDV was able to infect peritoneal cells of black rockfish in vitro and induce the formation of ETs within 4 h. In summary, this study proved that ETs are involved in the response to LCDV infection and may be involved in formation of lymphoid nodules. Taken together, the findings provide a new perspective to determine the impact factors on the growth of nodules., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)