Search

Nervous necrosis virus (NNV) which mainly infects sevenband grouper (Hyporthodus Septemfasciatus) is considered a potential threat to the grouper aquaculture industry. The gills being one of the portal of entry and an active site of replication of fish viruses emphasises its role as a key region to study the metabolomic changes caused by viral reprograming and hijacking of metabolic pathways associated with immunity of the host. In the present study, liquid chromatography mass spectrometry (LC-MS) was used to detect changes of endogenous compounds of the grouper after NNV infection. A total of 75 metabolites of ten different pathways were identified. The metabolites were mainly associated with fatty acids, lipids, amino acids and nucleotides. The virus reprogramming lead to the downregulation of majority of the metabolites in their pathways. Arachidonic acid (AA), tryptophan, kynurenine and methandriol were selected as representative metabolites and challenge studies with NNV confirmed the fact that, metabolites controlled the replication of virus in a dose dependent manner. Immune gene expression studies also confirmed the effect of metabolites by upregulated expression of interleukins, cytokines and TLRs which are part of cellular immune response. This study shows the viral reprogramming of NNV in grouper gill cells resulting in alterations in basic metabolic pathways associated with normal functioning of the organism., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The California sea hare (Aplysia californica) is a model for age associated cognitive decline. Recent researched identified a novel nidovirus, Aplysia Abyssovirus 1, with broad tropism enriched in the Aplysia nervous system. This virus is ubiquitous in wild and maricultured, young and old animals without obvious pathology. Here we re-evaluated gene expression data from several previous studies to investigate differential expression in the nervous system and gill in response to virus and aging as well as the mutational spectrum observed in the viral sequences obtained from these datasets. Viral load and age were highly correlated, indicating persistent infection. Upregulated genes in response to virus were enriched for immune genes and signatures of ER and proteostatic stress, while downregulated genes were enriched for mitochondrial metabolism. Differential expression with respect to age suggested increased iron accumulation and decreased glycolysis, fatty acid metabolism, and proteasome function. Interaction of gene expression trends associated with viral infection and aging suggest that viral infection likely plays a role in aging in the Aplysia nervous system. Mutation analysis of viral RNA identified signatures suggesting ADAR and AID/APOBEC like deaminase act as part of Aplysia anti-viral defense., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The immune system of bony fish closely resembles that of mammals, comprising both specific (adaptive) and non-specific (innate) components. Notably, the mucosa-associated lymphoid tissue (MALT) serves as the first line of defense within the non-specific immune system, playing a critical role in protecting these aquatic organisms against invading pathogens. MALT encompasses a network of immune cells strategically distributed throughout the gills and intestines, forming an integral part of the mucosal barrier that interfaces directly with the surrounding aquatic environment. Spring Viremia of Carp Virus（SVCV）, a highly pathogenic agent causing substantial harm to common carp populations, has been designated as a Class 2 animal disease by the Ministry of Agriculture and Rural Affairs of China. Utilizing a comprehensive array of research techniques, including Hematoxylin and Eosin (HE)、Alcian Blue Periodic Acid-Schiff (AB-PAS)、transcriptome analysis for global gene expression profiling and Reverse Transcription-Polymerase Chain Reaction (RT-qPCR), this study uncovered several key findings: SVCV is capable of compromising the mucosal architecture in the gill and intestinal tissues of carp, and stimulate the proliferation of mucous cells both in gill and intestinal tissues. Critically, the study revealed that SVCV's invasion elicits a robust response from the carp's mucosal immune system, demonstrating the organism's capacity to resist SVCV invasion despite the challenges posed by the pathogen., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plastic waste has emerged as a major environmental concern in recent years. As plastic waste discharged into the marine environment, it undergoes a breakdown process, eventually accumulating in aquatic organisms in the form of microplastics (MPs). To date, reduced food intake, nutritional absorption, and impaired immune system are known adverse effects of MPs-exposed aquatic organisms. This study aims to investigate whether MP exposure accelerated white spot syndrome virus (WSSV) infection in Pacific white shrimp ( Penaeus vannamei ) via laboratory tests. Briefly, experimental shrimp were divided into four groups; WSSV (group 1); MP (group 2); WSSV + MP (group 3); and Control (group 4). No mortality was observed in group 2, group 4, and even in group 1. However, group 3 showed a cumulative mortality of 50% during the experimental period. The PCR assay results showed no WSSV in the other three groups (groups 1, 2, and 4), but the dead and alive shrimp collected from group 3 were confirmed to be infected with the virus. Histopathological examination revealed normal structures in the hepatopancreas, gill, and muscle tissues of group 4, whereas numerous abnormally shaped nuclei were detected in the gill tissue of group 2. Moreover, group 1 showed minor WSSV-related lesions with few basophilic inclusion bodies in the gills, interestingly, group 3 exhibited severe lesions with numerous basophilic inclusion bodies in the gills. In conclusion, this study confirmed the correlation between the viral disease of shrimp and MPs, which can cause significant economic losses to the shrimp aquaculture industry.

Recirculating aquaculture systems (RAS) have become more attractive due to reduced water consumption and effluent discharge. However, intensification of production increases the risk of introducing pathogens at farming sites. The emergence of uncultivable pathogens and RAS pathobiome diversity shifts the traditional disease paradigm from "one pathogen, one disease" to complex multiple-pathogen disease cases. Piscine orthoreovirus genotype 3 (PRV-3) is an excellent example, as it is capable of inducing anemia and heart pathology resembling heart and skeletal muscle inflammation under experimental conditions, and is associated with increased mortality in association with other pathogens in the field. The aim of this study was to develop a method for detection of multiple pathogens and putative pathogens, as co-infections are common in aquaculture. To do this, in the pilot study, we mapped the pathobiome of RAS-farmed rainbow trout (Oncorhynchus mykiss) (commercial RAS, farm A) using both standard diagnostic methods and metabarcording (16S rRNA) to investigate the gill microbiome. During this study, we observed infections with multiple pathogens, and detected two putative gill pathogens Candidatus Branchiomonas cysticola and Candidatus Piscichlamydia salmonis, both of which have been linked with complex gill disease in Atlantic salmon (Salmo salar). Based on the pilot study, we developed and tested a high throughput qPCR (HT-qPCR) chip targeting 22 viral and bacterial pathogens and putative pathogens, followed by a surveillance of a fish cohort in a commercial RAS farm during production (farm B). Co-infection with PRV-3 and Ca. B. cysticola combined with stress inducing management practices may explain the severe disease outbreak observed (37% mortality). The time course study sets the base for a future screening scheme for disease prediction and addresses limitations of the method when testing environmental DNA/RNA., (© 2024. The Author(s).)

A 2D primary gill cell culture system of the sevenband grouper (Hyporthodus septemfasciatus) was established to validate the pathogenesis of nervous necrosis virus (NNV) as observed in previous studies. This system, developed using the double-seeded insert (DSI) technique, yielded confluent cell layers. Upon challenge with NNV in a setup containing both autoclaved salt water and L15 media in the apical compartment, viral replication akin to that anticipated based on previous studies was observed. Consequently, we advocate for the utilization of primary gill cell culture as a viable alternative to conventional methodologies for investigating host pathogen interactions., 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.)

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea., (© 2024. The Author(s).)

Viruses that infect fish are understudied, yet they provide important evolutionary context to the viruses that infect terrestrial vertebrates. We surveyed gill tissue meta-transcriptomes collected from two species of native freshwater fish from Aotearoa New Zealand- Retropinna retropinna and Gobiomorphus cotidianus . A total of 64 fish were used for gill tissue meta-transcriptomic sequencing, from populations with contrasting life histories-landlocked (i.e., lacustrine) and diadromous-on the South Island and Chatham Islands. We observed that both viral richness and taxonomic diversity were significantly associated with life history and host species, with lacustrine R. retropinna characterised by higher viral alpha diversity than diadromous R. retropinna . Additionally, we observed transcripts of fish viruses from 12 vertebrate host-associated virus families, and phylogenetically placed eight novel RNA viruses and three novel DNA viruses in the Astroviridae , Paramyxoviridae , Orthomyxoviridae , Rhabdoviridae , Totiviridae , Poxviridae , Alloherpesviridae, and Adintoviridae in their evolutionary contexts. These results represent an important survey of the viruses that infect two widespread native fish species in New Zealand, and provide insight useful for future fish virus surveys.

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID 50 /ml of 10 6.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further., (© 2021 John Wiley & Sons Ltd.)

The use of lumpfish ( Cyclopterus lumpus ) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon ( Salmo salar L. ) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma ( IFNγ ) and granzyme A ( GzmA ) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA -containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD., Competing Interests: ET was employed by Patogen. The remaining 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 © 2021 Amundsen, Tartor, Andersen, Sveinsson, Thoen, Gjessing and Dahle.)

Outbreaks of white spot syndrome virus (WSSV) have caused serious damage to penaeid shrimp aquaculture worldwide. Despite great efforts to characterize the virus, the conditions that lead to infection and the infection mechanisms, there is still a lack of understanding regarding these complex virus-host interactions, which is needed to develop consistent and effective treatment methods for WSSV. In this study, we used a gas chromatography - mass spectrometry (GC-MS)-based metabolomics approach to compare the metabolite profiles of gills, haemolymph and hepatopancreas from whiteleg shrimp (Penaeus vannamei) exposed to WSSV and corresponding controls. The results revealed clear discriminations between metabolite profiles of WSSV-challenged shrimp and controlled shrimp in each tissue. The responses of shrimp gills to WSSV infection were characterized by increases of many fatty acids and amino acids in WSSV-challenged shrimp compared to the controls. Changes in haemolymph metabolite profiles include the increased levels of itaconic acid, energy-related metabolites, metabolites in glutathione cycle and decrease of amino acids. The WSSV challenge led to the decreases of several fatty acids and amino acids and increases of other amino acids, lactic acid and other organic compounds (levulinic acid, malonic acid and putrescine) in hepatopancreas. These alterations of shrimp metabolites suggest several immune responses of shrimp to WSSV in a tissue-specific manner, including upregulation of osmoregulation, antimicrobial activity, metabolic rate, gluconeogenesis, glutathione pathway in control of oxidative stress and shift from aerobic to anaerobic metabolism in shrimp which indicates the Warburg effect. The findings from this study provide a better understanding of molecular process of shrimp response against WSSV invasion which may be useful for development of disease management strategies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon., Competing Interests: The authors have declared that no competing interests exist.

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post-smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus-contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral-infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host-pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention., (© 2020 John Wiley & Sons Ltd.)

The presence of carp edema virus (CEV) was confirmed in imported ornamental koi in Chiang Mai province, Thailand. The koi showed lethargy, loss of swimming activity, were lying at the bottom of the pond, and gasping at the water's surface. Some clinical signs such as skin hemorrhages and ulcers, swelling of the primary gill lamella, and necrosis of gill tissue, presented. Clinical examination showed co-infection by opportunistic pathogens including Dactylogyrus sp., Gyrodactylus sp. and Saprolegnia sp. on the skin and gills. Histopathologically, the gill of infected fish showed severe necrosis of epithelial cells and infiltrating of eosinophilic granular cells. Electron microscope examination detected few numbers of virions were present in the cytoplasm of gill tissue which showed an electron dense core with surface membranes worn by surface globular units. Molecular detection of CEV DNA from gill samples of fish was performed by polymerase chain reaction (PCR) and confirmed by nested-PCR. Phylogenetic analyses revealed that CEV isolate had 99.8% homology with the CEV isolated from South Korea (KY946715) and Germany (KY550420), and was assigned to genogroup IIa. In conclusion, this report confirmed the presence of CEV infection of koi Cyprinus carpio in Chiang Mai province, Thailand using pathological and molecular approaches.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections., (© 2020 John Wiley & Sons Ltd.)

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain., (© 2020 John Wiley & Sons Ltd.)

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity., Competing Interests: Ivo Bertalan is an employee of Milenia Biotech GmbH. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV., (Copyright © 2020 Gjessing, Krasnov, Timmerhaus, Brun, Afanasyev, Dale and Dahle.)

In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder ( Paralichthys olivaceus ) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

Several investigations have recently assessed the ability of some aquatic invertebrates to act as tools for avian influenza A virus (IAV) surveillance as well as their potential role(s) in IAV ecology. Because of this, as well as the high IAV seroprevalence rates noted in select mesocarnivores that commonly inhabit aquatic and semi-aquatic habitats, we evaluated the effects that freshwater crayfish have on IAV in water at three dose levels and monitored for the presence of IAV in crayfish tissues (gill and green gland) and haemolymph at multiple time points. At relatively high, medium and low (approximately 10 4 , 10 3 and 10 2  EID 50 /ml, respectively) doses, mesocosms containing crayfish (Orconectes sp.) had less detectable IAV RNA present when final water samples were assayed (9 days post-contact [DPC]). In general, containers without crayfish present had nearly three-fold greater quantities of viral RNA at 9 DPC. A varying number of RNA positive samples were detected for the three crayfish sample types collected. Gill tissue produced the largest number of positive non-water samples (n = 26), with the highest quantities detected from crayfish sampled on 1 and 4 DPC (10 3.5  EID 50 equivalent/ml). On a few occasions, gill (n = 8) and haemolymph samples (n = 1) produced higher quantities of viral RNA than their respective water samples or water samples collected 1-2 DPC earlier, but these differences were typically minor. Based upon water samples, statistical models indicated that the interaction of dose and crayfish exposure days explained most of the variation in these data. Future efforts should address if crayfish exposed to IAV-laden water have the capacity to successfully transmit IAVs to mammals and birds which frequently prey upon them., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

At the end of October 2018, a mass fish mortality occurred in Iraq, involving thousands of tons of cultured and wild common carp (Cyprinus carpio) along Euphrates and Tigris rivers. Fish were found dead or moribund along rivers coasts, showing lethargy, dyspnoea and flared gills. At necropsy, discoloration patches were noticed on the gills. Wet preparations showed rare metacercariae and Dactylogyrus spp. Samples were subjected to bacteriological tests and virological investigation through real-time PCR and nested PCR. Both were positive for koi herpesvirus (KHV) and carp oedema virus. Results obtained were confirmed by the OIE reference laboratory of KHV disease (KHVD) at Cefas (UK) and by sequence analysis. This is the first report on the detection of both viruses in Iraq., (© 2019 Blackwell Verlag GmbH.)

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥10 8 ) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1-7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Molecular tools, such as high-throughput quantitative polymerase chain reaction (HT-qPCR), are useful for monitoring multiple infectious agents in wild animal populations (i.e., broad-based screening). If destructive tissue samples cannot be obtained due to experimental design requirements (e.g., bio-telemetry; holding with repeated biopsy) or the conservation status of host species, then nonlethally sampled tissues can be substituted. However, infection profiles have been found to differ between nonlethally and destructively sampled tissues. We present a comparative analysis of nonlethal (gill and blood) and destructive (pool of internal and external tissue) approaches for broad-based infectious agent screening of adult Chinook Salmon Oncorhynchus tshawytscha. Of a possible 47 agents, 16 were detected overall by nonlethal and destructive methods. Our results indicated moderate differences in infection profiles among tissues, with limitations of each tissue type dependent on the ecology of each agent. The gill was the most comprehensive screening tissue, as more infectious agents were detected overall in gill (n = 16) than in blood (n = 12) or multi-tissue pools (n = 15). The agreement in the estimated agent prevalence between tissue types ranged from poor to excellent, while overall agent community structure (the combined prevalence of all agents) showed low agreement between tissue types. Two agents occurred at 100% prevalence in all tissue types. Nine agents, including types of bacteria and gill parasites, were more prevalent in gill than in blood, while five agents, including one virus and several microparasites, were more prevalent in blood. Future studies should pair microscopy and histopathology with HT-qPCR to better characterize host health and disease development relative to molecular detection of agents across tissue types., (© 2019 American Fisheries Society.)

One of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non-lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (10 7 TCID 50 /ml and 10 6 TCID 50 /ml, respectively), and samples collected 7, 15 and 30 days post-infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non-lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT-qPCR). The presence of virus in non-lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular-infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme-linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus-specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection., (© 2019 John Wiley & Sons Ltd.)

During a PCR-based CEV survey in Poland in 2015-2017, the virus was detected in many farms both in clinical and asymptomatic cases and in common as well as in koi carp (Cyprinus carpio). In order to evaluate the potential carrier role of fish species that share the same habitats with carp, an experimental trial was performed. Investigations carried out on specimens of bleak (Alburnus alburnus), crucian carp (Carassius carassius), European perch (Perca fluviatilis), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) cohabited with CEV-infected carp yielded positive results. These species of fish were experimentally cohabited with CEV-infected common carp at a temperature of 16°C ± 1. Material from the brain, gills, spleen, kidneys, intestine and skin was investigated for the presence of CEV DNA. Similar investigations were performed with uninfected fish designated controls. Samples were tested for CEV by qPCR., (© 2019 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

Bivalve molluscan shellfish, such as oysters, clams, and cockles, are well-recognized as vectors that concentrate foodborne pathogens by filter feeding. The objective of this study was to investigate the distribution and persistence of hepatitis A virus (HAV) in experimentally contaminated oysters that were either fed or not fed with algae. Oysters were experimentally contaminated with HAV and maintained in depuration conditions. qRT-PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) were performed on oyster samples collected at 0, 1, 3, 5, and 7 days post-inoculation. When HAV-contaminated oysters were depurated for 7 days, HAV was detected in 91.1-97.8% of the digestive glands and gills. While the high viral load in the digestive glands in oysters did not change significantly regardless of algae-feeding, the viral load of the gills gradually decreased in both groups during the depuration. HAV antigen and RNA were detected in the digestive diverticula and connective tissues by both IHC and ISH. HAV was detected in the stomach, intestine, and gills by only ISH. The distribution of HAV in various oyster tissues may explain the persistence of contamination in oysters during the depuration process., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl 2 , 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus -1 , -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R 2  = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases., (Copyright © 2018. Published by Elsevier B.V.)

Toll-like receptors (TLRs) are well-known pattern recognition receptors that play key immunological roles in a diverse range of organisms. In this study, two novel invertebrate TLRs from disk abalone (designated as AbTLR-A and AbTLR-B) were identified and functionally characterized for the first time. AbTLR-A and AbTLR-B comprised the typical TLR domain architecture containing an extracellular leucine-rich repeat domain, transmembrane domain, and Toll/interleukin-1 receptor domain. Expressional analysis revealed that both TLRs were constitutively expressed at all the early embryonic stages of disk abalone analyzed, with the highest level of AbTLR-A found at the 16-cell stage and AbTLR-B at the trochophore stage. According to tissue distribution analysis, prominent mRNA expression of AbTLR-A and AbTLR-B was detected in the hemocytes and gills, respectively. AbTLR-A and AbTLR-B mRNAs were significantly up-regulated in response to Gram-negative Vibrio parahemolyticus, Gram-positive Listeria monocytogenes, and viral hemorrhagic septicemia virus injections in abalone hemocytes and gills. Overexpression of AbTLR-A and AbTLR-B in HEK293T cells directly activated nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) responsive reporters. Neither TLRs showed a high response to pathogen-associated molecular patterns in vitro. Co-expression of AbTLR-A and AbTLR-B with AbMyD88-2 and AbMyD88-X activated NF-κB-responsive reporters in a synergetic manner. These findings demonstrate the involvement of AbTLR-A and AbTLR-B in abalone innate immunity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are important posttranscriptional regulators. They play an important role in the antiviral innate immunity of invertebrates. In the present study, high-throughput small RNAs Illumina sequencing systems were carried out to identify differentially expressed miRNAs (DEMs) in the gills of Procambarus clarkii, which was challenged with white spot syndrome virus (WSSV). Our results identified 11,617 known and 6 novel miRNAs in normal group (NG) and WSSV-challenged group (WG) small RNA libraries. Additionally, 27 DEMs were shown to participate in the antiviral innate immunity of P. clarkii and were significantly upregulated or downregulated. In addition, the results of the KEGG pathway prediction of the DEMs target genes showed that putative target genes of these 27 DEMs were related mainly to the RNA transport pathway, tight junction pathway, mRNA surveillance pathway, regulation actin cytoskeleton pathway, focal adhesion pathway, and MAPK signaling pathway. These results provide important information for future studies about the antiviral innate immunity of crustaceans., (Copyright © 2018. Published by Elsevier Ltd.)

Plasmolipin has been characterized as a cell entry receptor for mouse endogenous retrovirus. In black tiger shrimp, two isoforms of plasmolipin genes, PmPLP1 and PmPLP2, have been identified from the Penaeus monodon EST database. The PmPLP1 is highly up-regulated in yellow head virus (YHV)-infected shrimp. Herein, the function of PmPLP1 is shown to be involved in YHV infection. The immunoblotting and immunolocalization showed that the PmPLP1 protein was highly expressed and located at the plasma membrane of gills from YHV-infected shrimp. Moreover, the PmPLP1 expressed in the Sf9 insect cells resided at the cell membrane rendering the cells more susceptible to YHV infection. Using the ELISA binding and mortality assays, the synthetic external loop of PmPLP1 was shown to bind the purified YHV and neutralize the virus resulting in the decrease in YHV infection. Our results suggested that the PmPLP1 was likely a receptor of YHV in shrimp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Koi sleepy disease (KSD) is a disease with increasing importance in global common carp aquaculture. Despite the fact that carp edema virus (CEV) is most likely the causative agent of KSD, the disease often presents itself as multifactorial with several parasites and bacteria species present on gills, skin or in internal organs. Therefore, in this study, we analysed and presented initial results on an interaction of flavobacteria and CEV in the development of clinical KSD in carp suffering from proliferative gill disease. We examined selected field samples from Germany and Hungary and confirmed the presence of CEV and flavobacteria co-infections in subset of the samples. In several infection experiments, we studied the transfer and dynamics of both infections. Furthermore, we analysed which Flavobacterium species could be isolated from KSD-affected fish and concluded that Flavobacterium branchiophilum is a possible copathogen. Antibiotic treatment experiments showed that CEV seems to be the primary pathogen causing an insult to the gills of carp and by these enabling other pathogens, including F. branchiophilum, to establish co-infections. Despite the fact that F. branchiophilum co-infection is not required for the development of clinical KSD, it could contribute to the pathological changes recorded during the outbreaks., (© 2018 John Wiley & Sons Ltd.)

Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3-4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the skin or the oral route, and, to a minor extent, through the gills, the SJNNV strain seems to enter fish only through the gills and the RGNNV strain could use all tissues indistinctly. Taken together, all these results support the hypothesis that reassortment has improved betanodavirus infectivity for sole., (© 2018 John Wiley & Sons Ltd.)

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV⁻host interaction and might be promising inhibitors of LCDV infection in fish.

Gill diseases cause serious losses in farming of Atlantic salmon and the number of agents involved increases. Salmon gill poxvirus (SGPV) and the gill disease in causes where SGPV apparently was the only disease-causing agent were initially characterized. Recently, it was further shown that SGPV can be a common denominator in widely different multifactorial gill diseases. Here, we present the challenge of diagnosing gill disease with SGPV in salmon fry of 0,3-5 grams. Apoptosis of gill lamellar epithelial cells and hemophagocytosis was also observed in fry similar to findings in smolts and grow-out fish. Using our newly developed immunohistochemistry method, we further demonstrate that some of the apoptotic epithelial cells covering the oral cavity were positive for SGPV. Thus, SGPV is not restricted to respiratory epithelium alone and may infect the fish at very early life stages. Furthermore, as the cases examined here are from Norway, Faroe Island and Scotland, we show that SGPV is more widespread than previously reported., (© 2018 John Wiley & Sons Ltd.)

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Twenty-five koi (Cyprinus carpio haematopterus) bought from a wholesale fish market in Korea, showed lethargic behaviour and 100% mortality within 20 days. Carp oedema virus (CEV) was detected by PCR in all 25 koi. Results of detailed histopathological and clinical examinations of 17 koi indicated loss of body balance, severe infiltration of inflammatory cells into the inter-lamellar spaces of the gills and vacuolization and inclusion bodies in gill epithelial cells. Sequence analysis of PCR products of these koi showed up to 99% identity to the previously reported sequences, suggesting that the observed mass mortality resulted from koi sleepy disease (KSD) due to CEV infection. To the best of our knowledge, this study is the first report of KSD in the Republic of Korea. Partial sequences of 4a protein from the virus indicated that the present CEV detected in koi from Korea is more closely related to that from the UK and Poland than from Japan. The present findings indicate that the prevalence and spread of KSD must be closely monitored in both European and Asian countries to avoid potential economic losses to the global koi industry., (© 2017 Blackwell Verlag GmbH.)

In 2016, the Norwegian health monitoring programme for wild salmonids conducted a real-time PCR-based screening for salmon gill poxvirus (SGPV) in anadromous Arctic char (Salvelinus alpinus L.), anadromous and non-anadromous Atlantic salmon (Salmo salar L.) and trout (Salmo trutta L.). SGPV was widely distributed in wild Atlantic salmon returning from marine migration. In addition, characteristic gill lesions, including apoptosis, were detected in this species. A low amount of SGPV DNA, as indicated by high Ct-values, was detected in anadromous trout, but only in fish cohabiting with SGPV-positive salmon. SGPV was not detected in trout and salmon from non-anadromous water courses, and thus seems to be primarily linked to the marine environment. This could indicate that trout are not a natural host for the virus. SGPV was not detected in Arctic char but, due to a low sample size, these results are inconclusive. The use of freshwater from anadromous water sources may constitute a risk of introducing SGPV to aquaculture facilities. Moreover, SGPV-infected Atlantic salmon farms will hold considerable potential for virus propagation and spillback to wild populations. This interaction should therefore be further investigated., (© 2017 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

Gill diseases cause considerable losses in Norwegian salmon farming. In 2015, we characterized salmon gill poxvirus (SGPV) and associated gill disease. Using newly developed diagnostic tools, we show here that SGPV infection is more widely distributed than previously assumed. We present seven cases of complex gill disease in Atlantic salmon farmed in seawater and freshwater from different parts of Norway. Apoptosis, the hallmark of acute SGPV infection, was not easily observed in these cases, and qPCR analysis was critical for identification of the presence of SGPV. Several other agents including Costia-like parasites, gill amoebas, Saprolegnia spp. and bacteria were observed. The studied populations experienced significant mortalities, which increased to extreme levels when severe SGPV infections coincided with smoltification. SGPV infection appears to affect the smoltification process directly by affecting the gills and chloride cells in particular. SGPV may be considered a primary pathogen as it was often found prior to identification of complex gill disease. It is hypothesized that SGPV-induced gill damage may impair innate immunity and allow invasion of secondary invaders. The distinct possibility that SGPV has been widely overlooked as a primary pathogen calls for extended use of SGPV qPCR in Atlantic salmon gill health management., (© 2017 John Wiley & Sons Ltd.)

An aquabirnavirus was isolated from diseased marbled eels (Anguilla marmorata; MEIPNV1310) with gill haemorrhages and associated mortality. Its genome segment sequences were obtained through next-generation sequencing and compared with published aquabirnavirus sequences. The results indicated that the genome sequence of MEIPNV1310 contains segment A (3099 nucleotides) and segment B (2789 nucleotides). Phylogenetic analysis showed that MEIPNV1310 is closely related to the infectious pancreatic necrosis Ab strain within genogroup II. This genome sequence is beneficial for studying the geographic distribution and evolution of aquabirnaviruses.

Although herpesviruses such as cyprinid herpesvirus 3 (CyHV-3) can establish lifelong latent infections, little is known about latency conditions in farmed koi populations in China. We used nested polymerase chain reaction targeting the TK gene and an indirect antibody ELISA to screen asymptomatic fish obtained from southern China for evidence of CyHV-3 infection. CyHV-3 DNA could be detected either in peripheral blood leukocytes or from gills of asymptomatic koi. Most koi sera did not contain anti-CyHV-3 antibodies; however, 5 samples were ELISA positive, providing evidence of prior CyHV-3 infections. These findings suggest that koi may survive CyHV-3 infections and become virus carriers.

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E 1/2 ), -0.4 V vs Ag/AgCl and mediated H 2 O 2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10 -3 to 1.36 × 10 7 copies μL -1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

Outbreaks of koi sleepy disease (KSD) caused by carp edema virus (CEV) may seriously affect populations of farmed common carp, one of the most important fish species for global food production. The present study shows further evidence for the involvement of CEV in outbreaks of KSD among carp and koi populations: in a series of infection experiments, CEV from two different genogroups could be transmitted to several strains of naïve common carp via cohabitation with fish infected with CEV. In recipient fish, clinical signs of KSD were induced. The virus load and viral gene expression results confirm gills as the target organ for CEV replication. Gill explants also allowed for a limited virus replication in vitro. The in vivo infection experiments revealed differences in the virulence of the two CEV genogroups which were associated with infections in koi or in common carp, with higher virulence towards the same fish variety as the donor fish. When the susceptibility of different carp strains to a CEV infection and the development of KSD were experimentally investigated, Amur wild carp showed to be relatively more resistant to the infection and did not develop clinical signs for KSD. However, the resistance could not be related to a higher magnitude of type I IFN responses of affected tissues. Despite not having a mechanistic explanation for the resistance of Amur wild carp to KSD, we recommend using this carp strain in breeding programs to limit potential losses caused by CEV in aquaculture.

Background: White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses., Methods/principal Findings: Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR., Conclusion: The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry., Competing Interests: The authors have declared that no competing interests exist.

A disease outbreak was reported in adult koi, Cyprinus carpio koi, from a fish farm in Kerala, India, during June 2015. The clinical signs were observed only in recently introduced adult koi, and an existing population of fish did not show any clinical signs or mortality. Microscopic examination of wet mounts from the gills of affected koi revealed minor infestation of Dactylogyrus sp. in a few koi. In bacteriological studies, only opportunistic bacteria were isolated from the gills of affected fish. The histopathological examination of the affected fish revealed necrotic changes in gills and, importantly, virus particles were demonstrated in cytoplasm of gill epithelial cells in transmission electron microscopy. The tissue samples from affected koi were negative for common viruses reported from koi viz. cyprinid herpesvirus 3, spring viraemia of carp virus, koi ranavirus and red sea bream iridovirus in PCR screening. However, gill tissue from affected koi carp was positive for carp edema virus (CEV) in the first step of nested PCR, and sequencing of PCR amplicons confirmed infection with CEV. No cytopathic effect was observed in six fish cell lines following inoculation of filtered tissue homogenate prepared from gills of affected fish. In bioassay, the symptoms could be reproduced by inoculation of naive koi with filtrate from gill tissue homogenate of CEV-positive fish. Subsequently, screening of koi showing clinical signs similar to koi sleepy disease from different locations revealed that CEV infection was widespread. To our knowledge, this is the first report of infection with CEV in koi from India.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression., (© 2016 John Wiley & Sons Ltd.)

Carp edema virus (CEV), the causative agent of 'koi sleepy disease' (KSD), appears to be spreading worldwide and to be responsible for losses in koi, ornamental varieties of the common carp Cyprinus carpio. Clinical signs of KSD include lethargic behaviour, swollen gills, sunken eyes and skin alterations and can easily be mistaken for other diseases, such as infection with cyprinid herpesvirus 3 (CyHV-3). To improve the future diagnosis of CEV infection and to provide a tool to better explore the relationship between viral load and clinical disease, we developed a specific quantitative PCR (qPCR) for strains of the virus known to infect koi carp. In samples from several clinically affected koi, CEV-specific DNA was present in a range from 1 to 2,046,000 copies, with a mean of 129,982 copies and a median of 45 copies per 250 ng of isolated DNA, but virus DNA could not be detected in all clinically affected koi. A comparison of the newly developed qPCR, which is based on a dual-labelled probe, to an existing end-point PCR procedure revealed higher specificity and sensitivity of the qPCR and demonstrated that the new protocol could improve CEV detection in koi. In addition to improved diagnosis, the newly developed qPCR test would be a useful research tool. For example, studies on the pathobiology of CEV could employ controlled infection experiments in which the development of clinical signs could be examined in parallel with a quantitative determination of virus load.

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.